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At least 505 records · Page 28

Crystallization and preliminary X-ray analysis of recombinant human acid beta-glucocerebrosidase, a treatment for Gaucher's disease

Acid beta-glucocerebrosidase (N-acylsphingosyl-1-O-beta-D-glucoside:glucohydrolase) is a lysosomal glycoprotein that catalyzes the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide. Inadequate levels of this enzyme underly the pathophysiology of Gaucher's disease. Cerezyme (Genzyme Corporation, Cambridge, MA, USA) is a partially deglycosylated form of recombinant human acid beta-glucocerebrosidase that is used in the treatment of Gaucher patients. Although acid beta-glucocerebrosidase belongs to a large family of glycosidases, relatively little is known regarding its structural biology. Here, the crystallization and the initial diffraction analysis of Cerezyme are reported. The crystals are C-centered orthorhombic, with unit-cell parameters a = 285.0, b = 110.2, c = 91.7 A. A 99.9% complete data set has been collected to 2.75 A with an R(sym) of 8.8%.

Glucosylceramidase/chemistry↗

Assignment of fatty acid-beta-oxidizing syntrophic bacteria to Syntrophomonadaceae fam. nov. on the basis of 16S rRNA sequence analyses

After enrichment from Chinese rural anaerobic digestor sludge, anaerobic, sporing and nonsporing, saturated fatty acid-beta-oxidizing syntrophic bacteria were isolated as cocultures with H2- and formate-utilizing Methanospirillum hungatei or Desulfovibrio sp. strain G-11. The syntrophs degraded C4 to C8 saturated fatty acids, including isobutyrate and 2-methylbutyrate. They were adapted to grow on crotonate and were isolated as pure cultures. The crotonate-grown pure cultures alone did not grow on butyrate in either the presence or the absence of some common electron acceptors. However, when they were reconstituted with M. hungatei, growth on butyrate again occurred. In contrast, crotonate-grown Clostridium kluyveri and Clostridium sticklandii, as well as Clostridium sporogenes, failed to grow on butyrate when these organisms were cocultured with M. hungatei. The crotonate-grown pure subcultures of the syntrophs described above were subjected to 16S rRNA sequence analysis. Several previously documented fatty acid-beta-oxidizing syntrophs grown in pure cultures with crotonate were also subjected to comparative sequence analyses. The sequence analyses revealed that the new sporing and nonsporing isolates and other syntrophs that we sequenced, which had either gram-negative or gram-positive cell wall ultrastructure, all belonged to the phylogenetically gram-positive phylum. They were not closely related to any of the previously known subdivisions in the gram-positive phylum with which they were compared, but were closely related to each other, forming a new subdivision in the phylum. We recommend that this group be designated Syntrophomonadaceae fam. nov.; a description is given.

NASA Discipline Number 52-30↗

Microfabricated capillary electrophoresis amino acid chirality analyzer for extraterrestrial exploration

Chiral separations of fluorescein isothiocyanate-labeled amino acids have been performed on a microfabricated capillary electrophoresis chip to explore the feasibility of using such devices to analyze for extinct or extant life signs in extraterrestrial environments. The test system consists of a folded electrophoresis channel (19.0 cm long x 150 microns wide x 20 microns deep) that was photolithographically fabricated in a 10-cm-diameter glass wafer sandwich, coupled to a laser-excited confocal fluorescence detection apparatus providing subattomole sensitivity. Using a sodium dodecyl sulfate/gamma-cyclodextrin pH 10.0 carbonate electrophoresis buffer and a separation voltage of 550 V/cm at 10 degrees C, baseline resolution was observed for Val, Ala, Glu, and Asp enantiomers and Gly in only 4 min. Enantiomeric ratios were determined for amino acids extracted from the Murchison meteorite, and these values closely matched values determined by HPLC. These results demonstrate the feasibility of using microfabricated lab-on-a-chip systems to analyze extraterrestrial samples for amino acids.

NASA Discipline Life Sciences Technologies↗

Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms

Strain SBT is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SBT produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SBT in coculture with Desulfovibrio strain G11. Strain SBT grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 +/- 1 g cell dry mass per mol of crotonate. Strain SBT did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SBT was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SBT in the delta-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SBT was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SBT and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.

Non-NASA Center↗

Energetics of amino acid synthesis in hydrothermal ecosystems

Thermodynamic calculations showed that the autotrophic synthesis of all 20 protein-forming amino acids was energetically favored in hot (100 degrees C), moderately reduced, submarine hydrothermal solutions relative to the synthesis in cold (18 degrees C), oxidized, surface seawater. The net synthesis reactions of 11 amino acids were exergonic in the hydrothermal solution, but all were endergonic in surface seawater. The synthesis of the requisite amino acids of nine thermophilic and hyperthermophilic proteins in a 100 degreesC hydrothermal solution yielded between 600 and 8000 kilojoules per mole of protein, which is energy that is available to drive the intracellular synthesis of enzymes and other biopolymers in hyperthermophiles thriving in these ecosystems.

NASA Discipline Exobiology↗

A search for endogenous amino acids in martian meteorite ALH84001

Trace amounts of glycine, serine, and alanine were detected in the carbonate component of the martian meteorite ALH84001 by high-performance liquid chromatography. The detected amino acids were not uniformly distributed in the carbonate component and ranged in concentration from 0.1 to 7 parts per million. Although the detected alanine consists primarily of the L enantiomer, low concentrations (<0.1 parts per million) of endogenous D-alanine may be present in the ALH84001 carbonates. The amino acids present in this sample of ALH84001 appear to be terrestrial in origin and similar to those in Allan Hills ice, although the possibility cannot be ruled out that minute amounts of some amino acids such as D-alanine are preserved in the meteorite.

NASA Discipline Exobiology↗

Quantification of urinary uric acid in the presence of thymol and thimerosal by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed as an alternative to automated enzymatic analysis of uric acid in human urine preserved with thymol and/or thimerosal. Uric acid (tR = 10 min) and creatinine (tR = 5 min) were separated and quantified during isocratic elution (0.025 M acetate buffer, pH 4.5) from a mu Bondapak C18 column. The uric-acid peak was identified chemically by incubating urine samples with uricase. The thymol/thimerosal peak appeared at 31 min during the washing step and did not interfere with the analysis. We validated the high-performance liquid chromatographic method for linearity, precision and accuracy, and the results were found to be excellent.

NASA Discipline Regulatory Physiology↗

Pyridoxic acid excretion during low vitamin B-6 intake, total fasting, and bed rest

Vitamin B-6 metabolism in 10 volunteers during 21 d of total fasting was compared with results from 10 men consuming a diet low only in vitamin B-6 (1.76 mumol/d) and with men consuming a normal diet during bed rest. At the end of the fast mean plasma concentrations of vitamin B-6 metabolites and urinary excretion of 4-pyridoxic acid tended to be higher in the fasting subjects than in the low-vitamin B-6 group. The fasting subjects lost approximately 10% of their total vitamin B-6 pool and approximately 13% of their body weight. The low-vitamin B-6 group lost only approximately 4% of their vitamin B-6 pool. Compared with baseline, urinary excretion of pyridoxic acid was significantly increased during 17 wk of bed rest. There was no increase in pyridoxic acid excretion during a second 15-d bed rest study. These data suggest the possibility of complex interactions between diet and muscle metabolism that may influence indexes that are frequently used to assess vitamin B-6 status.

NASA Discipline Regulatory Physiology↗

[Genotoxic modification of nucleic acid bases and biological consequences of it. Review and prospects of experimental and computational investigations]

The review is presented of experimental and computational data on the influence of genotoxic modification of bases (deamination, alkylation, oxidation) on the structure and biological functioning of nucleic acids. Pathways are discussed for the influence of modification on coding properties of bases, on possible errors of nucleic acid biosynthesis, and on configurations of nucleotide mispairs. The atomic structure of nucleic acid fragments with modified bases and the role of base damages in mutagenesis and carcinogenesis are considered.

Non-NASA Center↗

Amino Acids in the Antarctic Martian Meteorite MIL03346

The report by McKay et al. that the Martian meteorite ALH84001 contains evidence for life on Mars remains controversial. Of central importance is whether ALH84001 and other Antarctic Martian meteorites contain endogenous organic compounds. In any investigation of organic compounds possibly derived from Mars it is important to focus on compounds that play an essential role in biochemistry as we know it and that have properties such as chirality which can be used to distinguish between biotic versus abiotic origins. Amino acids are one of the few compounds that fulfill these requirements. Previous analyses of the Antarctic Martian meteorites ALH84001 and EETA79001 have shown that these meteorites contain low levels of terrestrial amino acid contamination derived from Antarctic ice meltwater. Here we report preliminary amino acid investigations of a third Antarctic Martian meteorite MIL03346 which was discovered in Antarctica during the 2003-04 ANSMET season. Additional information is included in the original extended abstract

Glavin, D. P.↗

Chemical evolution of RNA under hydrothermal conditions and the role of thermal copolymers of amino acids for the prebiotic degradation and formation of RNA

The roles of thermal copolymers of amino acids (TCAA) were studied for the prebiotic degradation of RNA. A weak catalytic ability of TCAA consisted of Glu, L-Ala, L-Val, L-Glu, L-Asp, and optionally L-His was detected for the cleavage of the ribose phosphodiester bond of a tetranucleotide (5'-dCrCdGdG) in aqueous solution at 80 degees C. The rate constants of the disappearance of 5'-dCrCdGdG were determined in aqueous solutions using different pH buffer and TCAA. The degradation rates were enhanced 1.3-3.0 times in the presence of TCAA at pH 7.5 and 8.0 at 80 degrees C, while the hydrolysis of oligoguanylate (oligo(G)) was accelerated about 1.6 times at pH 8.0. A weak inhibitory activity for the cleavage of oligo(G) was detected in the presence of 0.055 M TCAA-Std. On the other hand, our recent study on the influences of TCAA for the template-directed reaction of oligo(G) on a polycytidylic acid template showed that TCAA has an acceleration activity for the degradation of the activated nucleotide monomer and an acceleration activity for the formation of G5' ppG capped oligo(G). This series of studies suggest that efficient and selective catalytic or inhibitory activities for either the degradation or formation of RNA under hydrothermal conditions could have hardly emerged from the simple thermal condensation products of amino acids. A scenario is going to be deduced on the chemical evolution of enzymatic activities and RNA molecules concerning hydrothermal earth conditions. c2005 COSPAR. Published by Elsevier Ltd. All rights reserved.

Amino Acids/chemistry↗

Soot and Sulfuric Acid from Aircraft: Is There Enough to Cause Detrimental Environmental E-kCTSs?

Aerosol from aircraft can affect the environment in three ways: First, soot aerosol has been implicated to cause Icing-tern ozone depletion at mid-latitudes in the lower stratosphere at a rate of approx. 5% per decade. This effect is in addition and unrelated to the polar ozone holes which are strongly influenced by heterogeneous chemistry on polar stratospheric clouds. Second, the most obvious effect of jet aircraft is the formation of visible contrails in the upper troposphere. The Salt Lake City region experienced an 8% increase in cirrus cloud cover over a 15-year period which covariates with an increase in regional commercial air traffic. If soot particles act as freezing nuclei to cause contrail formation heterogeneously, they would be linked to a secondary effect to cloud modification that very likely is climatologically important. Third, a buildup of soot aerosol could reduce the single scatter albedo of stratospheric aerosol from 0.993+0.004 to 0.98, a critical value that has been postulated to separate stratospheric cooling from warming. Thus arises an important question: Do aircraft emit sufficient amounts of soot to have detrimental effects and warrant emission controls? During the 1996 SUCCESS field campaign, we sampled aerosols in the exhaust wake of a Boeing 757 aircraft and determined emission indices for sulfuric acid (EI(sub H2SO4) = 9.0E-2 and 5.0E-1 g/kg (sub FUEL) for 75 and 675 ppm fuel-sulfur, respectively) and soot aerosol (2.2E-3 less than EI(sub SOOT) = l.lE-2 g/kg (sub FUEL)). The soot particle analysis accounted for their fractal nature, determined electron-microscopically, which enhanced the surface area by a factor of 26 and the volume 11-fold over equivalent-volume spheres. The corresponding fuel-sulfur to H2SO4 conversion efficiency was 10% (for 675 ppmm fuel-S) and 37% (for 75 ppmm fuel-S). Applying the H2SO4 emission index to the 1990 fuel use by the worlds commercial fleets of 1.3E11 kg, a conversion efficiency of 30% of 500 ppmm fuel-S would have led to an annual contribution to the atmospheric sulfur budget by aircraft of 2.E7 kg H2SO4. This is about one part in 1.E4 of anthropogenic sulfate from other sources. The soot emission index given above yielded a 1990 injection of soot aerosol by aircraft of 1.E6 kg. Thus, soot amounts to only five percent of the aerosol generated by aircraft. Its reactivity with ozone would have to be 20 times that of sulfuric acid particles to make it chemically significant. Nevertheless, the findings, of stratospheric soot loadings commensurate with aircraft fuel consumption, based on the emission index given above and the assumption of stratospheric residence times of the order of one year implicate aircraft as stratospheric polluters. A trend similar to soot of H2SO4 aerosol loading could not be deciphered, neither from in situ measurements nor SAGE II satellite extinction, against the "noise" due to volcanic eruptions. Observation of soot particles at 20 km altitude which, if emitted by aircraft were generated at 10-12 km altitude, suggests a displacement of those particles against gravity. Because eddy mixing is virtually absent in the lower stratosphere and isentropic mixing explains lofting to only about 15 km, radiometric forces acting on morphologically and chemically asymmetric soot particles must be considered a possibility. The consequence could be an extended residence time of soot against that of sulfuric acid aerosol that would lower the single scatter albedo with time.

Pueschel, R. F.↗

Proton conducting membrane using a solid acid

A solid acid material is used as a proton conducting membrane in an electrochemical device. The solid acid material can be one of a plurality of different kinds of materials. A binder can be added, and that binder can be either a nonconducting or a conducting binder. Nonconducting binders can be, for example, a polymer or a glass. A conducting binder enables the device to be both proton conducting and electron conducting. The solid acid material has the general form M.sub.a H.sub.b (XO.sub.t).sub.c.

Chisholm, Calum↗

Proteomic Retrieval from Nucleic Acid Depleted Space-Flown Human Cells

Compared to experiments utilizing humans in microgravity, cell-based approaches to questions about subsystems of the human system afford multiple advantages, such as crew safety and the ability to achieve statistical significance. To maximize the science return from flight samples, an optimized method was developed to recover protein from samples depleted of nucleic acid. This technique allows multiple analyses on a single cellular sample and when applied to future cellular investigations could accelerate solutions to significant biomedical barriers to human space exploration. Cell cultures grown in American Fluoroseal bags were treated with an RNA stabilizing agent (RNAlater - Ambion), which enabled both RNA and immunoreactive protein analyses. RNA was purified using an RNAqueous(registered TradeMark) kit (Ambion) and the remaining RNA free supernatant was precipitated with 5% trichloroacetic acid. The precipitate was dissolved in SDS running buffer and tested for protein content using a bicinchoninic acid assay (1) (Sigma). Equal loads of protein were placed on SDS-PAGE gels and either stained with CyproOrange (Amersham) or transferred using Western Blotting techniques (2,3,4). Protein recovered from RNAlater-treated cells and stained with protein stain, was measured using Imagequant volume measurements for rectangles of equal size. BSA treated in this way gave quantitative data over the protein range used (Fig 1). Human renal cortical epithelial (HRCE) cells (5,6,7) grown onboard the International Space Station (ISS) during Increment 3 and in ground control cultures exhibited similar immunoreactivity profiles for antibodies to the Vitamin D receptor (VDR) (Fig 2), the beta isoform of protein kinase C (PKC ) (Fig 3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Fig 4). Parallel immunohistochemical studies on formalin-fixed flight and ground control cultures also showed positive immunostaining for VDR and other biomarkers (Fig 5). These results are consistent with data from additional antigenic recovery experiments performed on human Mullerian tumor cells cultured in microgravity (8).

Hammond, D. K.↗

Enigmatic Isovaline: Investigating the Stability, Racemization, and Formation of a Non-biological Meteoritic Amino Acid

Among the Murchison meteoritic amino acids, isovaline stands out as being both nonbiological (non-protein) and having a relatively high abundance. While approximately equal amounts of D- and L-isovaline have been reported in Murchison and other CM meteorites, the molecule's structure appears to prohibit its racemization in aqueous solutions. We recently have investigated the low-temperature solid-phase chemistry of both isovaline and valine with an eye toward each molecule's formation, stability, and possible interconversions of D and L enantiomers. Ion-irradiated isovaline- and valinecontaining ices were examined by IR spectroscopy and highly-sensitive liquid chromatography/time-of-flight mass spectral methods to assess both amino acid destruction and racemization. Samples were studied in the presence and in the absence of water-ice, and the destruction of both isovaline and valine was measured as a function of radiation dose. In addition, we have undertaken experiments to synthesize isovaline, valine, and their amino acid isomers by solid-phase radiation-chemical pathways other than the oft-invoked Strecker process. This presentation will review and summarize some of our recent findings. -- Our work has been supported by a grant to the Goddard Center for Astrobiology through the NASA Astrobiology Institute. Experiments were performed in the Cosmic Ice Laboratory (RLH, MHM, AL) and the Astrobiology Analytical Laboratory (JPD, DPG) at the NASA Goddard Space Flight Center.

Hudson, Reggie↗

Synthesis of asymmetric tetracarboxylic acids and corresponding dianhydrides

This invention relates to processes for preparing asymmetrical biphenyl tetracarboxylic acids and the corresponding asymmetrical dianhydrides, namely 2,3,3',4'-biphenyl dianhydride (a-BPDA), 2,3,3',4'-benzophenone dianhydride (a-BTDA) and 3,4'-methylenediphthalic anhydride (-MDPA). By cross-coupling reactions of reactive metal substituted o-xylenes or by cross-coupling o-xylene derivatives in the presence of catalysts, this invention specifically produces asymmetrical biphenyl intermediates that are subsequently oxidized or hydrolyzed and oxidized to provide asymmetric biphenyl tetracarboxylic acids in comparatively high yields. These asymmetrical biphenyl tetracarboxylic acids are subsequently converted to the corresponding asymmetrical dianhydrides without contamination by symmetrical biphenyl dianhydrides.

Chuang, Chun-Hua↗

Carbon Isotopic Measurements of Amino Acids in Stardust-Returned Samples

NASA's Stardust spacecraft returned to Earth samples from comet 81P/Wild 2 in January 2006. Preliminary examinations revealed the presence of a suite of organic compounds including several amines and amino acids, but the origin of these compounds could not be identified. Here, we present the carbon isotopic ratios of glycine and e-aminocaproic acid (EACA), the two most abundant amino acids, in Stardust-returned foil samples measured by gas chromatography-combustion-isotope ratio mass spectrometry coupled with quadrupole mass spectrometry (GC-CAMS/IRMS).

Elsila, Jamie↗

A 16-yr Comparison of Fine Particle and Aerosol Strong Acidity at the Interface Zone (1,540 m) and Within (452 m) the Planetary Boundary Layer of the Great Gulf and Presidential-Dry River Class I Wildernesses on the Presidential Range, New Hampshire

Mount Washington, NH in the White Mountain National Forest, is flanked to the north-northeast and south by two Class I Wilderness areas, the Great Gulf and Presidential Range-Dry River Wildernesses, respectively. The Clean Air Act protects Class I area natural resource values from air pollution. Aerosol sulfate, a fine particulate component that is often transported long distances, is a known contributor to visibility degradation and acidic deposition. We examined summertime fine particulate aerosol mass and sulfate, strong acidity and ammonium concentrations from 1988 to 2004 on Mount Washington at two elevations, 452 and 1,540 meters (msl). The former site is within, and the latter at the interface of, the planetary boundary layer. Comparisons of sampling interval durations (10 and 24 hours), site vs. site, and different sampling methods are made. We also examine the extent to which aerosol sulfate is neutralized. Ten hour (daytime) compared to 24 hour samples have higher mass and aerosol sulfate concentrations, however paired samples are well correlated. Fine mass concentrations compared between the 452 m and 1,540 m sites (standard temperature and pressure corrected) show a weak positive linear relationship with the later being approximately 34% lower. We attribute the lack of a strong correlation to the facts that the 1,540 m site is commonly at the interface of and even above the regional planetary boundary layer in summer and that it can intercept different air masses relative to the 452 m site. Sulfate is approximately 19% lower at the higher elevation site, but comprises a greater percentage of total fine mass; 42% compared to 35% for the high and low elevation site, respectively. Aerosol strong acidity was found to increase with increasing sulfate concentrations at both sites. At the high elevation site, elevated mass and sulfate concentrations are associated with westerly and southwesterly regional flows.

Murray, Georgia L.↗