Search NASA⌕ Search

SEARCH · Search NASA

Results for “activity assay”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
NASA NTRS1992

Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae

A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

NASA Discipline Exobiology↗
NASA NTRS1993

The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear lamins

The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.

Non-NASA Center↗
NASA NTRS2004

Rapid Chemometric Filtering of Spectral Data

A method of rapid, programmable filtering of spectral transmittance, reflectance, or fluorescence data to measure the concentrations of chemical species has been proposed. By programmable is meant that a variety of spectral analyses can readily be performed and modified in software, firmware, and/or electronic hardware, without need to change optical filters or other optical hardware of the associated spectrometers. The method is intended to enable real-time identification of single or multiple target chemical species in applications that involve high-throughput screening of multiple samples. Examples of such applications include (but are not limited to) combinatorial chemistry, flow cytometry, bead assays, testing drugs, remote sensing, and identification of targets. The basic concept of the proposed method is to perform real-time crosscorrelations of a measured spectrum with one or more analytical function(s) of wavelength that could be, for example, the known spectra of target species. Assuming that measured spectral intensities are proportional to concentrations of target species plus background spectral intensities, then after subtraction of background levels, it should be possible to determine target species concentrations from cross-correlation values. Of course, the problem of determining the concentrations is more complex when spectra of different species overlap, but the problem can be solved by use of multiple analytical functions in combination with computational techniques that have been developed previously for analyses of this type. The method is applicable to the design and operation of a spectrometer in which spectrally dispersed light is measured by means of an active-pixel sensor (APS) array. The row or column dimension of such an array is generally chosen to be aligned along the spectral-dispersion dimension, so that each pixel intercepts light in a narrow spectral band centered on a wavelength that is a known function of the pixel position. The proposed method admits of two hardware implementations for computing cross-correlations in real time.

Beaman, Gregory↗
NASA NTRS2001

Evidence for a role in growth and salt resistance of a plasma membrane H+-ATPase in the root endodermis

The plasma membrane of plant cells is energized by an electrochemical gradient produced by P-type H+-ATPases (proton pumps). These pumps are encoded by at least 12 genes in Arabidopsis. Here we provide evidence that isoform AHA4 contributes to solute transport through the root endodermis. AHA4 is expressed most strongly in the root endodermis and flowers, as suggested by promoter-GUS reporter assays. A disruption of this pump (aha4-1) was identified as a T-DNA insertion in the middle of the gene (after VFP(574)). Truncated aha4-1 transcripts accumulate to approximately 50% of the level observed for AHA4 mRNA in wild-type plants. Plants homozygous for aha4-1 (-/-) show a subtle reduction in root and shoot growth compared with wild-type plants when grown under normal conditions. However, a mutant phenotype is very clear in plants grown under salt stress (e.g., 75 or 110 mM NaCl). In leaves of mutant plants subjected to Na stress, the ratio of Na to K increased 4-5-fold. Interestingly, the aha4-1 mutation appears to be semidominant and was only partially complemented by the introduction of additional wild-type copies of AHA4. These results are consistent with the hypothesis that aha4-1 may produce a dominant negative protein or RNA that partially disrupts the activity of other pumps or functions in the root endodermal tissue, thereby compromising the function of this cell layer in controlling ion homeostasis and nutrient transport.

NASA Discipline Plant Biology↗
NASA NTRS2016

GeneLab: Multi-Omics Investigation of Rodent Research-1 Bio-Banked Tissues

NASAs Rodent Research (RR) project is playing a critical role in advancing biomedical research on the physiological effects of space environments. Due to the limited resources for conducting biological experiments aboard the International Space Station (ISS), it is imperative to use crew time efficiently while maximizing high-quality science return. NASAs GeneLab project has as its primary objectives to 1) further increase the value of these experiments using a multi-omics, systems biology-based approach, and 2) disseminate these data without restrictions to the scientific community. The current investigation assessed viability of RNA, DNA, and protein extracted from archived RR-1 tissue samples for epigenomic, transcriptomic, and proteomic assays. During the first RR spaceflight experiment, a variety of tissue types were harvested from subjects, snap-frozen or RNAlater-preserved, and then stored at least a year at -80OC after return to Earth. They were then prioritized for this investigation based on likelihood of significant scientific value for spaceflight research. All tissues were made available to GeneLab through the bio-specimen sharing program managed by the Ames Life Science Data Archive and included mouse adrenal glands, quadriceps, gastrocnemius, tibialis anterior, extensor digitorum longus, soleus, eye, and kidney. We report here protocols for and results of these tissue extractions, and thus, the feasibility and value of these kinds of omics analyses. In addition to providing additional opportunities for investigation of spaceflight effects on the mouse transcriptome and proteome in new kinds of tissues, our results may also be of value to program managers for the prioritization of ISS crew time for rodent research activities. Support from the NASA Space Life and Physical Sciences Division and the International Space Station Program is gratefully acknowledged.

GeneLab↗
NASA NTRS1999

Leukocyte subsets and neutrophil function after short-term spaceflight

Changes in leukocyte subpopulations and function after spaceflight have been observed but the mechanisms underlying these changes are not well defined. This study investigated the effects of short-term spaceflight (8-15 days) on circulating leukocyte subsets, stress hormones, immunoglobulin levels, and neutrophil function. At landing, a 1.5-fold increase in neutrophils was observed compared with preflight values; lymphocytes were slightly decreased, whereas the results were variable for monocytes. No significant changes were observed in plasma levels of immunoglobulins, cortisol, or adrenocorticotropic hormone. In contrast, urinary epinephrine, norepinephrine, and cortisol were significantly elevated at landing. Band neutrophils were observed in 9 of 16 astronauts. Neutrophil chemotactic assays showed a 10-fold decrease in the optimal dose response after landing. Neutrophil adhesion to endothelial cells was increased both before and after spaceflight. At landing, the expression of MAC-1 was significantly decreased while L-selectin was significantly increased. These functional alterations may be of clinical significance on long-duration space missions.

short duration↗
NASA NTRS1997

Changes in gravity inhibit lymphocyte locomotion through type I collagen

Immunity relies on the circulation of lymphocytes through many different tissues including blood vessels, lymphatic channels, and lymphoid organs. The ability of lymphocytes to traverse the interstitium in both nonlymphoid and lymphoid tissues can be determined in vitro by assaying their capacity to locomote through Type I collagen. In an attempt to characterize potential causes of microgravity-induced immunosuppression, we investigated the effects of simulated microgravity on human lymphocyte function in vitro using a specialized rotating-wall vessel culture system developed at the Johnson Space Center. This very low shear culture system randomizes gravitational vectors and provides an in vitro approximation of microgravity. In the randomized gravity of the rotating-wall vessel culture system, peripheral blood lymphocytes did not locomote through Type I collagen, whereas static cultures supported normal movement. Although cells remained viable during the entire culture period, peripheral blood lymphocytes transferred to unit gravity (static culture) after 6 h in the rotating-wall vessel culture system were slow to recover and locomote into collagen matrix. After 72 h in the rotating-wall vessel culture system and an additional 72 h in static culture, peripheral blood lymphocytes did not recover their ability to locomote. Loss of locomotory activity in rotating-wall vessel cultures appears to be related to changes in the activation state of the lymphocytes and the expression of adhesion molecules. Culture in the rotating-wall vessel system blunted the ability of peripheral blood lymphocytes to respond to polyclonal activation with phytohemagglutinin. Locomotory response remained intact when peripheral blood lymphocytes were activated by anti-CD3 antibody and interleukin-2 prior to introduction into the rotating-wall vessel culture system. Thus, in addition to the systemic stress factors that may affect immunity, isolated lymphocytes respond to gravitational changes by ceasing locomotion through model interstitium. These in vitro investigations suggest that microgravity induces non-stress-related changes in cell function that may be critical to immunity. Preliminary analysis of locomotion in true microgravity revealed a substantial inhibition of cellular movement in Type I collagen. Thus, the rotating-wall vessel culture system provides a model for analyzing the microgravity-induced inhibition of lymphocyte locomotion and the investigation of the mechanisms related to lymphocyte movement.

Weightlessness↗
NASA NTRS1992

Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3',5'-monophosphate and requiring protein synthesis

Collagenase is synthesized and secreted by rat osteoblastic cells in response to PTH. We have previously demonstrated that this effect involves a substantial increase in collagenase mRNA via transcription. Northern blots and nuclear run-on assays were performed to further investigate the induction of collagenase by PTH in the rat osteoblastic cell line UMR 106-01. Detectable amounts of collagenase mRNA were not apparent until 2 h of PTH treatment, showed the greatest abundance at 4 h, and declined to approximately 30% of maximum by 8 h. The changes in the rate of transcription of the collagenase gene in response to PTH paralleled and preceded the changes in the steady state mRNA levels. After an initial lag period of about 1 h, collagenase transcription rates increased from very low levels to a maximal response at 2 h, returning to about 50% of maximum by 10 h. The increased transcriptional rate of the collagenase gene was found to be dependent on the concentration of PTH, with a half-maximal response at approximately 7 x 10(-10) M rat PTH-(1-34) and a maximal effect with a dose of 10(-8) M. The PTH-mediated induction of collagenase transcriptional activity was completely abolished by cycloheximide, while transcription of the beta-actin gene was unaffected by the translation inhibitor. These data suggest that a protein factor(s) is required for PTH-mediated transcriptional induction of collagenase. Since PTH increases intracellular levels of several potential second messengers, agents that mimic these substances were employed to determine which signal transduction pathway is predominant in the PTH-mediated stimulation of collagenase transcription.(ABSTRACT TRUNCATED AT 250 WORDS).

NASA Discipline Cell Biology↗
NASA NTRS2017

Coastal California's Fog as a Unique Habitable Niche: Design for Autonomous Sampling and Preliminary Aerobiological Characterization

Just as on the land or in the ocean, atmospheric regions may be more or less hospitable to life. The aerobiosphere, or collection of living things in Earth's atmosphere, is poorly understood due to the small number and ad hoc nature of samples studied. However, we know viable airborne microbes play important roles, such as providing cloud condensation nuclei. Knowing the distribution of such microorganisms and how their activity can alter water, carbon, and other geochemical cycles is key to developing criteria for planetary habitability, particularly for potential habitats with wet atmospheres but little stable surface water. Coastal California has regular, dense fog known to play a major transport role in the local ecosystem. In addition to the significant local (1 km) geographical variation in typical fog, previous studies have found that changes in height above surface of as little as a few meters can yield significant differences in typical concentrations, populations and residence times. No single current sampling platform (ground-based impactors, towers, balloons, aircraft) is capable of accessing all of these regions of interest.A novel passive fog and cloud water sampler, consisting of a lightweight passive impactor suspended from autonomous aerial vehicles (UAVs), is being developed to allow 4D point sampling within a single fog bank, allowing closer study of small-scale (100 m) system dynamics. Fog and cloud droplet water samples from low-altitude aircraft flights in nearby coastal waters were collected and assayed to estimate the required sample volumes, flight times, and sensitivity thresholds of the system under design.125 cloud water samples were collected from 16 flights of the Center for Interdisciplinary Remotely Piloted Aircraft Studies (CIRPAS) instrumented Twin Otter, equipped with a sampling tube collector, occurring between 18 July and 12 August 2016 below 1 km altitude off the central coast. The collector was flushed first with 70 ethanol, then with sterile DI water, between sampling regions. Collected volumes ranged from 100 L to 12 mL. All samples were diluted serially and plated on two different types of agar, nutrient-dense (PCA) and sparse (R-2A). Plates were incubated at room temperature and counted when colonies first appeared and again at 2 weeks.Preliminary results from seven flights are consistent with generally reported colony-forming unit (CFU) values for terrestrial fog water (e.g., [4]). The PCA assay ranged from 400 to 125,000 CFU/mL, R-2A from 700 to 130,000 CFU/mL. PCA and R-2A counts were not significantly different from each other at I^ plus or minus +/- 0.05, although observationally, the R2A plates had more pigmented colonies. CFU counts from the majority of flights were not different from each other in mean at the same level of significance, but about half differed in median, indicating differences in underlying distribution. These results validate the presence of viable microorganisms in coastal California fog at levels that should be easily detectable by our sampling system. The indicated distribution differences underscore the need for small-scale, long-term sampling surveys. Future planned work includes ion chromatography for limiting nutrients, ATP quantification, and qPCR for several microbial classes of interest.

Coastal↗
NASA NTRS1996

Products of Dissociative Recombination in the Ionosphere

SRI International undertook a novel experimental measurement of the product states formed by dissociative recombination (DR) of O2(+), NO(+), and N2(+) as a function of both electron energy and reactant ion vibrational level. For these measurements we used a recently developed experimental technique for measuring dissociation product distributions that allows both the branching ratios to be accurately determined and the electronic and rovibrational state composition of the reactant ions to be specified. DR is the dominant electron loss mechanism in all regions of the ionosphere. In this process, electron attachment to the molecular ion produces an unstable neutral molecule that rapidly dissociates. For a molecular ion such as O2(+), the dissociation recombination reaction is (1) O2(+) + e yields O + O + W. The atomic products of this reaction, in this case two oxygen atoms, can be produced in a variety of excited states and with a variety of kinetic energies, as represented by W in Eq. (1). These atoms are not only active in the neutral chemistry of the ionosphere, but are also especially important because their optical emissions are often used to infer in situ concentrations of the parent molecular ion and ambient electron densities. Many laboratory measurements have been made of DR reaction rates under a wide range of electron temperatures, but very little is known about the actual distributions among the final states of the atomic products. This lack of knowledge seriously limits the validity and effectiveness of efforts to model both natural and man-made ionospheric disturbances. Bates recently identified major deficiencies in the currently accepted branching ratios for O2(+) as they relate to blue and green line emission measurements in the nocturnal F-region. During our two-year effort, we partially satisfied our ambitious goals. We constructed and operated a variable pressure, electron-impact ion source and a high pressure, hollow-cathode discharge ion source for O2(+), NO(+), and N2(+) beams. Translational spectroscopy of the products of dissociative charge transfer in Cs vapor was used to accurately assay the composition of the O2(+) and NO(+) beams and to develop a methodology for the vibrationally controlled preparation of the ground state ion beams. Attempts to assay the N2(+) beam revealed a novel two-electron process in the charge transfer reactions. A coaxial electron gun for the DR measurements was constructed following an extensive numerical design of the fields. Tests of the gun, however, found substantial perturbations of the magnetic fields by the soft iron (CMI-C) assembly containing the Langmuir probe that locates the electron beam. Hydrogen annealing of the iron failed to eliminate the field perturbations, necessitating the removal of the probe assembly. During this work on the coaxial electron gun, we discovered that predissociated high Rydberg states of O2 could be produced by subjecting the molecules to a sudden perturbation by an electromagnetic field. This technique allowed a measurement of the product branching to the atomic limits for the lowest seven vibrational levels of O2(+).

Cosby, Philip↗
NASA NTRS2002

Lipid Biomarkers for Methanogens in Hypersaline Cyanobacterial Mats for Guerrero Negro, Baja California Sur

Analyses of sediments from the vicinity of active methane seeps have uncovered a particular suite of lipid biomarker patterns that characterize methane consuming archaea and their syntrophic, sulfate reducing partners. These isoprenoid biomarkers, largely identified by their anomalously light carbon isotopic signatures, have been a topic of intense research activity and are recorded in numerous methane-rich environments from Holocene to Cenozoic. This phenomenon has implications for depleted kerogens at 2.7 Ga on early Earth (Hinrichs 2002). In contrast, the lipid biosignatures of methane producing archaea are not readily identified through distinct isotopic labels and have received comparably little attention in analyses of archaea in environmental samples. Indeed, environmental analyses generally detect only free archaeal lipids, not the intact, polar molecules found in the membrane of living organisms. As part of the Ames NAI, the 'Early Microbial Ecosystem Research Group' (EMERG) is working to understand microbial processes in the hypersaline cyanobacterial mats growing in the salt evaporation ponds of the Exportadora de Sal at Guerrero Negro, Baja California Sur, Mexico. The aim of this study was to develop methods by which we could identify the organisms responsible for methane generation in this environment. While the ester-bound fatty acids, hopanoids and wax esters provide a means to identify most of the bacterial components of these mats, the archaea which Ere evidently present through genomic assays and the fact of intense methane production (Hoehler et al. 200l), have not been identified through their corresponding lipid signatures. Archaeal core lipids present a number of analytical challenges. The core lipids of methanogens comprise C20, C40 and sometimes C25 isoprenoid chains, linked through ether bonds to glycerol. As well as archaeal (C20), sn-2- and sn-3-hydroxyarchaeol are associated particularly with methylotrophic methanogens. Recently, we have also identified a dihydroxyarchaeol in a hyperthermophilic methanogen (Summons et al. 2002). Additional structural diversity is encoded into the polar head groups that are attached to the glycerol ether cores. The C20 core lipids are readily analyzed by GC-MS as their volatile trimethylsilyl derivatives while compounds with intact polar head groups can only be detected using LC-MS approaches. Our approach was to utilize the alternative of an ether cleavage reagent (BBr3 vs. HI) and a hydride reducing agent to convert all ether lipids to hydrocarbon in order to provide a vertical profile of quantitative information that might be matched to methane fluxes. We have found that while conventional acid hydrolysis and HI treatment will destroy hydroxyarchaeols, molecular information remains intact through use of BBr3 for ether cleavage. This method revealed the presence of traces of biphytane and various ether alkyls associated with some sulfate reducing bacteria within the mat structure. An interesting, and potentially valuable, byproduct of the method utilizing HI was the identification of abundant homohopanoids after superhydride reduction. Evidently present as sulfur-bound diagenetic products these hopanoids are likely cyanobacterial biomarkers in the early stages of diagenetic preservation.

Jahnke, Linda L.↗
NASA NTRS1985

Distribution and metabolism of quaternary amines in salt marshes

Quaternary amines such as glycine betaine (GBT) are common osmotically active solutes in much of the marine biota. GBT is accumulated by various bacteria, algae, higher plants, invertebrates, and vertebrates in response to salinity or water stresses; in some species, GBT occurs at tens to hundreds of millimolar concentrations and can account for a significant fraction of total nitrogen. Initial studies suggest that GBT is readily converted to two potential methane precursors, trimethylamine (TMA) and acetate, in anoxic sediments. TMA is apparently the most important methane precursor in surface sediments containing sulfate reducing bacteria. In salt marshes, the bulk of the methane formed may be due to the metabolism of TMA rather than other substrates. Current research is focussed on testing this hypothesis and on determining the role of quaternary amino osmoregulatory solutes in methane fluxes from marine environments. Preliminary studies have dealt with several problems: (1) determination of GBT concentrations in the dominant flora and fauna of salt marshes; (2) synthesis of radiolabelled GBT for metabolic studies; and (3) determination of fates of BGT in marine sediments using radiotracers. Both GC and HPLC techniques have been used to assay GBT concentrations in plant and animal tissues. S. alterniflora is probably the only significant source of GBT (and indirectly of methane) since the biomass and distribution of most other species is limited. Current estimates suggest that S. alterniflora GBT could account for most of the methane efflux from salt marshes.

King, Gary M.↗
NASA NTRS1991

A serum factor promotes collagenase synthesis by an osteoblastic cell line

Regulation of the synthesis of collagenase was investigated in the osteoblastic cell line, UMR 106-01. The cells were stained by the avidin-biotin-complex technique for the presence of the enzyme. By this method, it was possible to identify cells producing collagenase. Synthesis, but not secretion, was found to be constitutive in these cells with the enzyme located intracellularly in cytoplasmic vesicles and the Golgi apparatus. The amount of collagenase contained within UMR cells and the number of cells synthesizing the enzyme were proportional to the concentration of fetal bovine serum in the incubating medium. When serum was withdrawn from the osteosarcoma cells, the content of collagenase decreased with time and the enzyme became undetectable by 48 h of serum depletion. The decrease in collagenase content could be completely reversed by resupplying serum to the cells. The collagenase promoting activity of serum could not be eliminated by adsorption on activated charcoal but was retained by a dialysis membrane with a 12,000 mol wt cutoff. A range of bone-seeking hormones or agents known to affect collagenase secretion was added to the medium in an attempt to mimic the effect of serum on collagenase accumulation. None of these agonists, including parathyroid hormone, could reproduce the effect of serum on these cells, although parathyroid hormone could act as a collagenase secretagogue in the presence or absence of serum. It is concluded that fetal bovine serum contains a yet unidentified factor or factors greater than 12,000 mol wt responsible for the continued synthesis of collagenase by UMR 106-01 cells.

Non-NASA Center↗
NASA NTRS1994

Effects of biomass burning on summertime nonmethane hydrocarbon concentrations in the Canadian wetlands

Approximately 900 whole air samples were collected and assayed for selected C2-C10 hydrocarbons and seven halocarbons during the 5-week Arctic Boundary Layer Expedition (ABLE) 3B conducted in eastern Canadian wetland areas. In more than half of the 46 vertical profiles flown, enhanced nonmethane hydrocarbon (NMHC) concentrations attributable to plumes from Canadian forest fires were observed. Urban plumes, also enhanced in many NMHCs, were separately identified by their high correlation with elevated levels of perchloroethene. Emission factors relative to ethane were determined for 21 hydrocarbons released from Canadian biomass burning. Using these data for ethane, ethyne, propane, n-butane, and carbon monoxide enhancements from the literature, global emissions of these four NMHCs were estimated. Because of its very short atmospheric lifetime and its below detection limit background mixing ratio, 1,3-butadiene is an excellent indicator of recent combustion. No statistically significant emissions of nitrous oxide, isoprene, or CFC 12 were observed in the biomass-burning plumes encountered during ABLE 3B. The presence of the short-lived biogenically emitted isoprene at altitudes as high as 3000 m implies that mixing within the planetary boundary layer (PBL) was rapid. Although background levels of the longer-lived NMHCs in this Canadian region increase during the fire season, isoprene still dominated local hydroxyl radical photochemistry within the PBL except in the immediate vicinity of active fires. The average biomass-burning emission ratios for hydrocarbons from an active fire sampled within minutes of combustion were, relative to ethane, ethene, 2.45; ethyne 0.57; propane, 0.25; propene, 0.73; propyne, 0.06; n-butane, 0.09; i-butane, 0.01; 1-butene, 0.14; cis-2-butene, 0.02; trans-2-butene, 0.03; i-butylene, 0.07; 1,3-butadiene, 0.12; n-pentane, 0.05; i-pentane, 0.03; 1-pentene, 0.06; n-hexane, 0.05; 1-hexene, 0.07; benzene, 0.37; toluene, 0.16.

Blake, D. R.↗
NASA NTRS1994

Alpha-helical hydrophobic polypeptides form proton-selective channels in lipid bilayers

Proton translocation is important in membrane-mediated processes such as ATP-dependent proton pumps, ATP synthesis, bacteriorhodopsin, and cytochrome oxidase function. The fundamental mechanism, however, is poorly understood. To test the theoretical possibility that bundles of hydrophobic alpha-helices could provide a low energy pathway for ion translocation through the lipid bilayer, polyamino acids were incorporated into extruded liposomes and planar lipid membranes, and proton translocation was measured. Liposomes with incorporated long-chain poly-L-alanine or poly-L-leucine were found to have proton permeability coefficients 5 to 7 times greater than control liposomes, whereas short-chain polyamino acids had relatively little effect. Potassium permeability was not increased markedly by any of the polyamino acids tested. Analytical thin layer chromatography measurements of lipid content and a fluorescamine assay for amino acids showed that there were approximately 135 polyleucine or 65 polyalanine molecules associated with each liposome. Fourier transform infrared spectroscopy indicated that a major fraction of the long-chain hydrophobic peptides existed in an alpha-helical conformation. Single-channel recording in both 0.1 N HCl and 0.1 M KCl was also used to determine whether proton-conducting channels formed in planar lipid membranes (phosphatidylcholine/phosphatidylethanolamine, 1:1). Poly-L-leucine and poly-L-alanine in HCl caused a 10- to 30-fold increase in frequency of conductive events compared to that seen in KCl or by the other polyamino acids in either solution. This finding correlates well with the liposome observations in which these two polyamino acids caused the largest increase in membrane proton permeability but had little effect on potassium permeability. Poly-L-leucine was considerably more conductive than poly-L-alanine due primarily to larger event amplitudes and, to a lesser extent, a higher event frequency. Poly-L-leucine caused two populations of conductive events, one in the 0.1-0.5 pA range, and one in the 1.0-5.0 pA range, whereas nearly all events caused by poly-L-alanine were in the 0.1-0.5 pA range at an applied voltage of +60 mV. The channel-like activity appeared to switch between conductive and nonconductive states, with most open-times in the range of 50-200 ms. We conclude that hydrophobic polyamino acids produce proton-conducting defects in lipid bilayers that may be used to model functional proton channels in biological membranes.

NASA Discipline Exobiology↗
NASA NTRS2003

The dynamics of herpesvirus and polyomavirus reactivation and shedding in healthy adults: a 14-month longitudinal study

Humans are infected with viruses that establish long-term persistent infections. To address whether immunocompetent individuals control virus reactivation globally or independently and to identify patterns of sporadic reactivation, we monitored herpesviruses and polyomaviruses in 30 adults, over 14 months. Epstein-Barr virus (EBV) DNA was quantitated in saliva and peripheral blood mononuclear cells (PBMCs), cytomegalovirus (CMV) was assayed in urine, and JC virus (JCV) and BK virus (BKV) DNAs were assayed in urine and PBMCs. All individuals shed EBV in saliva, whereas 67% had >or=1 blood sample positive for EBV. Levels of EBV varied widely. CMV shedding occurred infrequently but occurred more commonly in younger individuals (P<.03). JCV and BKV virurias were 46.7% and 0%, respectively. JCV shedding was age dependent and occurred commonly in individuals >or=40 years old (P<.03). Seasonal variation was observed in shedding of EBV and JCV, but there was no correlation among shedding of EBV, CMV, and JCV (P>.50). Thus, adults independently control persistent viruses, which display discordant, sporadic reactivations.

Non-NASA Center↗
NASA NTRS2000

Epstein-Barr virus reactivation associated with diminished cell-mediated immunity in antarctic expeditioners

Epstein-Barr virus (EBV) reactivation and cell-mediated immune (CMI) responses were followed in 16 Antarctic expeditioners during winter-over isolation at 2 Australian National Antarctic Research Expedition stations. Delayed-type hypersensitivity (DTH) skin testing was used as an indicator of the CMI response, that was evaluated 2 times before winter isolation and 3 times during isolation. At all 5 evaluation times, 8 or more of the 16 subjects had a diminished CMI response. Diminished DTH was observed on every test occasion in 4/16 subjects; only 2/16 subjects exhibited normal DTH responses for all 5 tests. A polymerase chain reaction (PCR) assay was used to detect EBV DNA in saliva specimens collected before, during, and after the winter isolation. EBV DNA was present in 17% (111/642) of the saliva specimens; all 16 subjects shed EBV in their saliva on at least 1 occasion. The probability of EBV shedding increased (P = 0.013) from 6% before or after winter isolation to 13% during the winter period. EBV appeared in saliva during the winter isolation more frequently (P < 0.0005) when DTH response was diminished than when DTH was normal. The findings indicate that the psychosocial, physical, and other stresses associated with working and living in physical isolation during the Antarctic winter result in diminished CMI and an accompanying increased reactivation and shedding of latent viruses.

NASA Program Biomedical Research and Countermeasur↗
NASA NTRS1993

Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

Non-NASA Center↗