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At least 253 records · Page 14

Model study of ATP and ADP buffering, transport of Ca(2+) and Mg(2+), and regulation of ion pumps in ventricular myocyte

We extended the model of the ventricular myocyte by Winslow et al. (Circ. Res 1999, 84:571-586) by incorporating equations for Ca(2+) and Mg(2+) buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na(+)-K(+) pump, sarcolemmal and sarcoplasmic Ca(2+) pumps). The results indicate that, under normal conditions, Ca(2+) binding by low-affinity ATP and diffusion of CaATP may affect the amplitude and time course of intracellular Ca(2+) signals. The model also suggests that a fall in ATP/ADP ratio significantly reduces sarcoplasmic Ca(2+) content, increases diastolic Ca(2+), lowers systolic Ca(2+), increases Ca(2+) influx through L-type channels, and decreases the efficiency of the Na(+)/Ca(2+) exchanger in extruding Ca(2+) during periodic voltage-clamp stimulation. The analysis suggests that the most important reason for these changes during metabolic inhibition is the down-regulation of the sarcoplasmic Ca(2+)-ATPase pump by reduced diastolic MgATP levels. High Ca(2+) concentrations developed near the membrane might have a greater influence on Mg(2+), ATP, and ADP concentrations than that of the lower Ca(2+) concentrations in the bulk myoplasm. The model predictions are in general agreement with experimental observations measured under normal and pathological conditions.

NASA Discipline Cardiopulmonary↗

A Method for Preparation, Storage and Activation of Large Populations of Immotile Sea Urchin Sperm

Reversible protein phosphorylation is associated with initiation and modulation of sperm flagellar motility. Many studies aimed at examining the signal transduction mechanisms underlying the expression of motility have relied on detergent-permeabilized sperm reactivated with exogenous 32 P-ATP. However, the reactivation conditions allow variable levels of motility to be expressed and phosphorylation of many proteins that appear to be unrelated to sperm motility. Thus, identification of the few relevant proteins is difficult. We have developed a method to collect and keep sperm immotile until reactivated for analysis to normal motility levels. Artificial sea water (ASW) buffered with 5 mM 2-[N-morpholino]ethanesulfonic acid at pH 6.0 and containing 50 mM KCI, allows collection and storage of immotile sea urchin sperm for up to 96 h at 4-5 C. Motility under these conditions is essentially zero, but sperm is rapidly reactivated to normal motility by diluting with ASW to standard pH (8.0) and KCI concentration (10 mM).

Bracho, Geracimo E.↗

Enzyme-based CO2 capture for advanced life support

Elevated CO2 levels in air can lead to impaired functioning and even death to humans. Control of CO2 is critical in confined spaces that have little physical or biological buffering capacity (e.g., spacecraft, submarines, or aircraft). A novel enzyme-based contained liquid membrane bioreactor was designed for CO2 capture and certain application cases are reported in this article. The results show that the liquid layer accounts for the major transport resistance. With addition of carbonic anhydrase, the transport resistance decreased by 71%. Volatile organic compounds of the type and concentration expected to be present in either the crew cabin or a plant growth chamber did not influence carbonic anhydrase activity or reactor operation during 1-day operation. Alternative sweep method studies, examined as a means of eliminating consumables, showed that the feed gas could be used successfully in a bypass mode when combined with medium vacuum pressure (-85 kPa) to achieve CO2 separation comparable to that with an inert sweep gas. The reactor exhibited a selectivity for CO2 versus N2 of 1400:1 and CO2 versus O2 is 866:1. The CO2 permeance was 1.44 x 10(-7) mol m-2 Pa-1 s-1 (4.3 x 10(-4) cm3 cm-2 s-1 cmHg-1) at a feed concentration of 0.1% CO2. These data show that the enzyme-based contained liquid membrane is a promising candidate technology that may be suitable for NASA applications to control CO2 in the crew or plant chambers.

NASA Discipline Life Support Systems↗

Nanosecond Breakdown Characteristics of C 4 F 7 N and Various Mixtures at Pressures Above 1 Atmosphere in Comparison with SF 6

This report evaluates the pulsed breakdown performance of C 4 F 7 N under a 6.8 kV/ns voltage excitation. The pulsed dielectric strength of C 4 F 7 N is compared to SF 6 in the same experimental setup, and it is found that C 4 F 7 N concentrations of 50% or greater are required to achieve a dielectric strength greater than or equal to SF 6 . Pure C 4 F 7 N demonstrated higher electric field hold-off for longer time periods and less statistical variance under pulsed conditions when compared to SF 6 . Mixtures of 50%C 4 F 7 N with N 2 or CO 2 as buffer gases showed no appreciable difference in pulsed dielectric strength.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

OH in the Tropical Upper Troposhere and Its Relationships to Solar Radiation and Reactive Nitrogen

In situ measurements of [OH], [HO2] (square brackets denote species concentrations), and other chemical species were made in the tropical upper troposphere (TUT). [OH] showed a robust correlation with solar zenith angle. Beyond this dependence, however, [HOx] ([OH] + [HO2]) only weakly responds to variations in its source and sink species. For example, at a given SZA, [HOx] was broadly independent of the product of [O3] and [H2O]. This suggests that [OH] is heavily buffered in the TUT. One important exception to this result is found in regions with very low [O3], [NO], and [NOy], where [OH] is highly suppressed, pointing to the critical role of NO in sustaining OH in the TUT.

Tropical upper troposphere↗

Continuous flow electrophoretic separation of proteins and cells from mammalian tissues

This paper describes an apparatus for continuous flow electrophoresis (CFE), designed to separate macromolecules and cells at conditions of microgravity. In this CFE, buffer flows upward in a 120-cm long flow chamber, which is 16-cm wide x 3.0-mm thick in the microgravity version (and 6-cm wide x 1.5-mm thick in the unit-gravity laboratory version). Ovalbumin and rat serum albumin were separated in space (flight STS-4) with the same resolution of the two proteins achieved at 25 percent total w/v concentration that was obtained in the laboratory at 0.2 percent w/v concentration. Rat anterior pituitary cells, cultured human embryonic kidney cells, and canine Langerhans cells were separated into subpopulations (flight STS-8) more effectively than in unit gravity, with comparable resolution having been achieved at 100 times the concentration possible on earth.

Hymer, W. C.↗

A Novel Protocol for Decoating and Permeabilizing Bacterial Spores for Epifluorescent Microscopy

Based on previously reported procedures for permeabilizing vegetative bacterial cells, and numerous trial-and-error attempts with bacterial endospores, a protocol was developed for effectively permeabilizing bacterial spores, which facilitated the applicability of fluorescent in situ hybridization (FISH) microscopy. Bacterial endospores were first purified from overgrown, sporulated suspensions of B. pumilus SAFR-032. Purified spores at a concentration of approx equals 10 million spores/mL then underwent proteinase-K treatment, in a solution of 468.5 μL of 100 mM Tris-HCl, 30 μL of 10% SDS, and 1.5 microL of 20 mg/mL proteinase-K for ten minutes at 35 ºC. Spores were then harvested by centrifugation (15,000 g for 15 minutes) and washed twice with sterile phosphate-buffered saline (PBS) solution. This washing process consisted of resuspending the spore pellets in 0.5 mL of PBS, vortexing momentarily, and harvesting again by centrifugation. Treated and washed spore pellets were then resuspended in 0.5 mL of decoating solution, which consisted of 4.8 g urea, 3 mL Milli-Q water, 1 mL 0.5M Tris, 1 mL 1M dithiothreitol (DTT), and 2 mL 10% sodium-dodecylsulfate (SDS), and were incubated at 65 ºC for 15 minutes while being shaken at 165 rpm. Decoated spores were then, once again, washed twice with sterile PBS, and subjected to lysozyme/mutanolysin treatment (7 mg/mL lysozyme and 7U mutanolysin) for 15 minutes at 35 C. Spores were again washed twice with sterile PBS, and spore pellets were resuspended in 1-mL of 2% SDS. This treatment, facilitating inner membrane permeabilization, lasted for ten minutes at room temperature. Permeabilized spores were washed two final times with PBS, and were resuspended in 200 mkcroL of sterile PBS. At this point, the spores were permeable and ready for downstream processing, such as oligonucleotideprobe infiltration, hybridization, and microscopic evaluation. FISH-microscopic imagery confirmed the effective and efficient (≈50% successful permeabilization and recovery) permeabilization of numerous spore preparations. The novelty of the technology developed here is in its applicability to bacterial endospores. While protocols abound for the effective permeabilization of bacterial, archaeal, and eukaryotic vegetative cells, there are no such reliable methods for decoating and permeabilizing bacterial endospores in a manner that is amenable to downstream FISH microscopic analyses. This innovation enables the direct visualization and enumeration of spores via FISH-based microscopic techniques, circumventing the complications that accompany previously required germination regimes. The synergistic enzymatic weakening of the many spore layers facilitates a structural compromise that is just enough to render the spores permeable without degrading the spore to a level, which precludes it from recognition.

LaDuc, Myron T.↗

Catalysis of hydrolysis and nucleophilic substitution at the P-N bond of phosphoimidazolide-activated nucleotides in phosphate buffers

Phosphoimidazolide-activated derivatives of guanosine and cytidine 5'-monophosphates, henceforth called ImpN's, exhibit enhanced rates of degradation in the presence of aqueous inorganic phosphate in the range 4.0 < or = pH < or = 8.6. This degradation is been attributed to (i) nucleophilic substitution of the imidazolide and (ii) catalysis of the P-N bond hydrolysis by phosphate. The first reaction results in the formation of nucleoside 5'-diphosphate and the second in nucleoside 5'-monophosphate. Analysis of the observed rates as well as the product ratios as a function of pH and phosphate concentration allow distinction between various mechanistic possibilities. The results show that both H2PO4- and HPO4(2-) participate in both hydrolysis and nucleophilic substitution. Statistically corrected biomolecular rate constants indicate that the dianion is 4 times more effective as a general base than the monoanion, and 8 times more effective as nucleophile. The low Bronsted value beta = 0.15 calculated for these phosphate species, presumed to act as general bases in facilitating water attack, is consistent with the fact that catalysis of the hydrolysis of the P-N bond in ImpN's has not been detected before. The beta nuc = 0.35 calculated for water, H2PO4-, HPO4(2-), and hydroxide acting as nucleophiles indicates a more associative transition state for nucleotidyl (O2POR- with R = nucleoside) transfers than that observed for phosphoryl (PO3(2-)) transfers (beta nuc = 0.25). With respect to the stability/reactivity of ImpN's under prebiotic conditions, our study shows that these materials would not suffer additional degradation due to inorganic phosphate, assuming the concentrations of phosphate, Pi, on prebiotic Earth were similar to those in the present oceans ([Pi] approximately 2.25 micromoles).

NASA Discipline Number 52-20↗

A microprocessor based communications multiplexer/concentrator

In the past communications concentrators have been designed using mini-computers. In this work a design for a high speed concentrator (in the megabits range) is developed using the 3000 series bit slice microprocessor. The proposed concentrator realizes the functions of multiplexing of the data arriving on the low speed lines, demultiplexing of the data arriving on the high speed line, including canned responses and code conversion. The basic system hardware configuration and the principles of operation of the multiplexing and the demultiplexing subsystems are presented. The software for these two functions are also presented. Using queueing theory, an estimate of the required buffer size is provided. Possible areas of further improvement are also indicated.

Brown, D. W.↗

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

Non-NASA Center↗

EPMA-Based Mass Balance Method for Quantitative Fission Product Distribution Comparison between TRISO Particles

Two irradiated AGR-2 TRISO particles were chosen to demonstrate a recently developed mass balance technique in which EPMA-generated concentration data was used to determine fission product mass on a layer-by-layer basis in TRISO particles. EPMA-calculated fission product masses for most fission products in the two particles were within +/- 20% of their ORIGEN-modelled masses. Results show that Sr, Ba, and Eu accumulate preferentially in the carbon-rich kernel periphery on the particles’ side that lacks a gap between the buffer and IPyC. In addition, the more mobile elements--Cs, Sr, and Pd, accumulate in greater quantity in the outer layers of particle AGR2-223-RS34 compared to particle AGR2-223-RS06, which has relatively more of those elements’ mass located in the kernel and kernel periphery, suggesting enhanced fission product transport in particle AGR2-223-RS34. This model can be used better understand and test fission product transport in TRISO particles.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

Progress in the Development of Metamorphic Multi-Junction III-V Space-Solar Cells at Essential Research Incorporated

Theoretical calculations have shown that highest efficiency III-V multi-junction solar cells require alloy structures that cannot be grown on a lattice-matched substrate. Ever since the first demonstration of high efficiency metamorphic single junction 1.1 eV and 1.2 eV InGaAs solar cells by Essential Research Incorporated (ERI), interest has grown in the development of multi-junction cells of this type using graded buffer layer technology. ERI is currently developing a dual-junction 1.6 eV InGaP/1.1 eV InGaAs tandem cell (projected practical air-mass zero (AM0), one-sun efficiency of 28%, and 100-sun efficiency of 37.5%) under a Ballistic Missile Defense Command (BMDO) SBIR Phase II program. A second ongoing research effort at ERI involves the development of a 2.1 eV AlGaInP/1.6 eV InGaAsP/1.2 eV InGaAs triple-junction concentrator tandem cell (projected practical AM0 efficiency of 36.5% under 100 suns) under a SBIR Phase II program funded by the Air Force. We are in the process of optimizing the dual-junction cell performance. In case of the triple-junction cell, we have developed the bottom and the middle cell, and are in the process of developing the layer structures needed for the top cell. A progress report is presented in this paper.

Sinharoy, Samar↗

Fluorescence Studies of Protein Crystallization Interactions

We are investigating protein-protein interactions in under- and over-saturated crystallization solution conditions using fluorescence methods. The use of fluorescence requires fluorescent derivatives where the probe does not markedly affect the crystal packing. A number of chicken egg white lysozyme (CEWL) derivatives have been prepared, with the probes covalently attached to one of two different sites on the protein molecule; the side chain carboxyl of ASP 101, within the active site cleft, and the N-terminal amine. The ASP 101 derivatives crystallize while the N-terminal amine derivatives do not. However, the N-terminal amine is part of the contact region between adjacent 43 helix chains, and blocking this site does would not interfere with formation of these structures in solution. Preliminary FRET data have been obtained at pH 4.6, 0.1M NaAc buffer, at 5 and 7% NaCl, 4 C, using the N-terminal bound pyrene acetic acid (PAA, Ex 340 nm, Em 376 nm) and ASP 101 bound Lucifer Yellow (LY, Ex 425 nm, Em 525 nm) probe combination. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10 (exp 5) M respectively), and all experiments were carried out at approximately Csat or lower total protein concentration. The data at both salt concentrations show a consistent trend of decreasing fluorescence yield of the donor species (PAA) with increasing total protein concentration. This decrease is apparently more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations (reflected in the lower solubility). The estimated average distance between protein molecules at 5 x 10 (exp 6) M is approximately 70 nm, well beyond the range where any FRET can be expected. The calculated RO, where 50% of the donor energy is transferred to the acceptor, for the PAA-CEWL * LY-CEWL system is 3.28 nm, based upon a PAA-CEWL quantum efficiency of 0.41.

Pusey, Marc L.↗

Weak influence of anthropogenic emissions on aerosol, cloud, and rain in the wet season of the Amazon rainforest

Anthropogenic emissions have been shown to affect new particle formation, aerosol concentrations, and clouds. Such effects vary with region, environmental conditions, and cloud types. In the wet season of Amazonia, anthropogenic emissions emitted from Manaus, Brazil, can significantly increase the cloud condensation nuclei (CCN) concentrations compared to the background of mainly natural aerosols. However, the regional response of cloud and rain to anthropogenic emissions in Amazonia remains very uncertain. Here, we aim to quantify how aerosol concentration, cloud, and rain respond to changes in anthropogenic emissions through parameterized new particle formation and primary aerosol emission in the Manaus region and to understand the underlying mechanisms. We ran the atmosphere-only configuration of the HadGEM3 climate model with a nested regional domain that covers most of the rainforest region (720 km by 1200 km with 3 km resolution) under scaled regional emissions. The 7 d simulations show that, in the areas that are affected by anthropogenic emissions, when aerosol and precursor gas emissions are doubled from the baseline emission inventories, aerosol number concentrations increase by 13 %. The nucleation rate that involves sulfuric acid and biogenic compounds generally increases with pollution levels. However, nucleation is suppressed very close to the pollution source, resulting in lower nucleation and soluble Aitken mode aerosol number concentrations. We also found that doubling the anthropogenic emission can increase the cloud droplet number concentrations ( N d ) by 9 %, but cloud water and rain mass mixing ratios do not change significantly. Even very strong reductions in aerosol number concentrations by a factor of 4, which is an unrealistic condition, cause only a 4 % increase in rain over the domain. If we assume our simulation has a fine enough grid resolution and an accurate representation of the relevant atmospheric processes, the simulated weak and non-linear response of cloud and rain properties to linearly scaled anthropogenic emissions suggests that the interactions among aerosol, cloud, and precipitation in the Amazonian convective environment are buffered by microphysical processes. It also implies that the convective environment is resilient to the changes in Nd that occur in response to localized anthropogenic aerosol perturbations.

Wang, Xuemei [University of Leeds (United Kingdom)↗

Investigation of the free flow electrophoretic process. Volume 2: Technical analysis

The effect of gravity on the free flow electrophoretic process was investigated. The demonstrated effects were then compared with predictions made by mathematical models. Results show that the carrier buffer flow was affected by gravity induced thermal convection and that the movement of the separating particle streams was affected by gravity induced buoyant forces. It was determined that if gravity induced buoyant forces were included in the mathematical models, then effective predictions of electrophoresis chamber separation performance were possible. The results of tests performed using various methods of electrophoresis using supportive media show that the mobility and the ability to separate were essentially independent of concentration, providing promise of being able to perform electrophoresis with higher inlet concentrations in space.

Weiss, R. A.↗

Computer simulation of immobilized pH gradients at acidic and alkaline extremes - A quest for extended pH intervals

Computer simulations of the concentration profiles of simple biprotic ampholytes with Delta pKs 1, 2, and 3, on immobilized pH gradients (IPG) at extreme pH values (pH 3-4 and pH 10-11) show markedly skewed steady-state profiles with increasing kurtosis at higher Delta pK values. Across neutrality, all the peaks are symmetric irrespective of their Delta pK values, but they show very high contribution to the conductivity of the background gel and significant alteration of the local buffering capacity. The problems of skewness, due to the exponential conductivity profiles at low and high pHs, and of gel burning due to a strong electroosmotic flow generated by the net charges in the gel matrix, also at low and high pHs, are solved by incorporating in the IPG gel a strong viscosity gradient. This is generated by a gradient of linear polyacrylamide which is trapped in the gel by the polymerization process.

Mosher, Richard A.↗

Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape

Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS).

Non-NASA Center↗

Progress Toward a Compact, Highly Stable Ion Clock

There was an update on the subject of two previous NASA Tech Briefs articles: Compact, Highly Stable Ion Clock (NPO-43075), Vol. 32, No. 5 (May 2008), page 63; and Neon as a Buffer Gas for a Mercury-Ion Clock (NPO-42919), Vol. 32, No. 7 (July 2008), page 62. To recapitulate: A developmental miniature mercury-ion clock has stability comparable to that of a hydrogen-maser clock. The ion-handling components are housed in a sealed vacuum tube, wherein a getter pump maintains the partial vacuum, and the evacuated tube is backfilled with mercury vapor in a neon buffer gas. There was progress in the development of the clock, with emphasis on the design, fabrication, pump-down, and bake-out of the vacuum tube (based on established practice in the travelingwave- tube-amplifier industry) and the ability of the tube to retain a vacuum after a year of operation. Other developments include some aspects of the operation of mercury-vapor source (a small appendage oven containing HgO) so as to maintain the optimum low concentration of mercury vapor, and further efforts to miniaturize the vacuum and optical subsystems to fit within a volume of 2 L.

Prestage, John↗