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At least 55 records · Page 3

A Comparison Study: The New Extended Shelf Life Isopropyl Ester PMR Technology versus The Traditional Methyl Ester PMR Approach

Polymerization of Monomeric Reactants (PMR) monomer solutions and carbon cloth prepregs of PMR II-50 and VCAP-75 were prepared using both the traditional limited shelf life methanol based PMR approach and a novel extended shelf life isopropanol based PMR approach. The methyl ester and isopropyl ester based PMR monomer solutions and PMR prepregs were aged for up to four years at freezer and room temperatures. The aging products formed were monitored using high pressure liquid chromatography (HPLC). The composite processing flow characteristics and volatile contents of the aged prepregs were also correlated versus room temperature storage time. Composite processing cycles were developed and six ply cloth laminates were fabricated with prepregs after various extended room temperature storage times. The composites were then evaluated for glass transition temperature (Tg), thermal decomposition temperature (Td), initial flexural strength (FS) and modulus (FM), long term (1000 hours at 316 C) thermal oxidative stability (TOS), and retention of FS and FM after 1000 hours aging at 316 C. The results for each ester system were comparable. Freezer storage was found to prevent the formation of aging products for both ester systems. Room temperature storage of the novel isopropyl ester system increased PMR monomer solution and PMR prepreg shelf life by at least an order of magnitude while maintaining composite properties.

Alston, William B.↗

Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases

Two protein inhibitors of metalloproteinases (TIMP) were isolated from medium conditioned by the clonal rat osteosarcoma line UMR 106-01. Initial purification of both a 30-kDa inhibitor and a 20-kDa inhibitor was accomplished using heparin-Sepharose chromatography with dextran sulfate elution followed by DEAE-Sepharose and CM-Sepharose chromatography. Purification of the 20-kDa inhibitor to homogeneity was completed with reverse-phase high-performance liquid chromatography. The 20-kDa inhibitor was identified as rat TIMP-2. The 30-kDa inhibitor, although not purified to homogeneity, was identified as rat TIMP-1. Amino terminal amino acid sequence analysis of the 30-kDa inhibitor demonstrated 86% identity to human TIMP-1 for the first 22 amino acids while the sequence of the 20-kDa inhibitor was identical to that of human TIMP-2 for the first 22 residues. Treatment with peptide:N-glycosidase F indicated that the 30-kDa rat inhibitor is glycosylated while the 20-kDa inhibitor is apparently unglycosylated. Inhibition of both rat and human interstitial collagenase by rat TIMP-2 was stoichiometric, with a 1:1 molar ratio required for complete inhibition. Exposure of UMR 106-01 cells to 10(-7) M parathyroid hormone resulted in approximately a 40% increase in total inhibitor production over basal levels.

NASA Discipline Musculoskeletal↗

Distribution of iodine into blood components of the Sprague-Dawley rat differs with the chemical form administered

It has been reported previously that radioactivity derived from iodine distributes differently in the Sprague-Dawley rat depending on the chemical form administered (Thrall and Bull, 1990). In the present communication we report the differential distribution of radioactivity derived from iodine (I2) and iodide (I-) into blood components. Twice as much radioiodine is in the form of I- in the plasma of animals treated with 125I- compared to 125I2-treated rats. No I2 could be detected in the plasma. With an increase in dose, increasing amounts of radioactivity derived from 125I2-treated animals distribute to whole blood compared to equivalent doses of 125I-, reaching a maxima at a dose of 15.8 mumol I/kg body weight. Most of the radioactivity derived from I2 associates with serum proteins and lipids, in particular with albumin and cholesteryl iodide. These data indicate a differential distribution of radioactivity depending on whether it is administered as iodide or iodine. This is inconsistent with the commonly held view that iodine (I2) is reduced to iodide (I-) before it is absorbed systemically from the gastrointestinal tract.

Non-NASA Center↗

New method for estimating bacterial cell abundances in natural samples by use of sublimation

We have developed a new method based on the sublimation of adenine from Escherichia coli to estimate bacterial cell counts in natural samples. To demonstrate this technique, several types of natural samples, including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert, were heated to a temperature of 500 degrees C for several seconds under reduced pressure. The sublimate was collected on a cold finger, and the amount of adenine released from the samples was then determined by high-performance liquid chromatography with UV absorbance detection. Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from approximately 10(5) to 10(9) E. coli cell equivalents per gram. For most of these samples, the sublimation-based cell counts were in agreement with total bacterial counts obtained by traditional DAPI (4,6-diamidino-2-phenylindole) staining.

Evaluation Studies↗

Nitrogen metabolism in Lignifying Pinus taeda cell cultures

The primary metabolic fate of phyenylalanine, following its deamination in plants, is conscription of its carbon skeleton for lignin, suberin, flavonoid, and related metabolite formation. Since this accounts for approximately 30-40% of all organic carbon, an effective means of recycling the liberated ammonium ion must be operative. In order to establish how this occurs, the uptake and metabolism of various 15N-labeled precursors (15N-Phe, 15NH4Cl, 15N-Gln, and 15N-Glu) in lignifying Pinus taeda cell cultures was investigated, using a combination of high performance liquid chromatography, 15N NMR, and gas chromatograph-mass spectrometry analyses. It was found that the ammonium ion released during active phenylpropanoid metabolism was not made available for general amino acid/protein synthesis. Rather it was rapidly recycled back to regenerate phenylalanine, thereby providing an effective means of maintaining active phenylpropanoid metabolism with no additional nitrogen requirement. These results strongly suggest that, in lignifying cells, ammonium ion reassimilation is tightly compartmentalized.

NASA Discipline Number 40-50↗

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↗

Biomass measurement of methane forming bacteria in environmental samples

Methane-forming bacteria contain unusual phytanylglycerol ether phospholipids which can be extracted from the bacteria in sediments and assayed quantitatively by high performance liquid chromatography (HPLC). In this procedure the lipids were extracted, the phospholipids recovered, hydrolyzed, purified by thin layer chromatography, derivatized and assayed by HPLC. Ether lipids were recovered quantitatively from Methanobacterium thermoautotrophicum and sediments at levels as low as 8 x 10(-14) moles. In freshwater and marine sediments the flux of methane to the atmosphere and the methane levels in the pore water reflects the recovery of the phytanyl glycerol ether lipid 'signature'. The proportion of the ether phospholipid to the total recoverable phospholipid was highest in anaerobic digester sewage sludge and deeper subsurface freshwater sediment horizons.

Methanobacterium/metabolism/physiology↗

An assay for intermolecular exchange of alpha crystallin

An affinity column of alpha crystallin linked to cyanogen bromide-activated Sepharose was developed to study the exchange of alpha subunits. Alpha crystallin bound to the Sepharose-alpha complex was dissociated with 8 mol/l urea, followed by quantitation using high-performance reverse-phase liquid chromatography. The time course of binding at 37 degrees C showed a hyperbolic binding pattern reaching equilibrium between 6-18 hr. Under these conditions, binding of beta and gamma crystallins to the same matrix was less than 10% of the alpha values, as was binding of alpha to glycine-coupled Sepharose. This assay was used to demonstrate changes in the subunit exchange of alpha crystallins present in high molecular weight versus lower molecular weight aggregates of the human lens. These results show that this binding procedure was a specific reproducible assay that might be used to study intermolecular interactions of the alpha crystallins.

NASA Discipline Cell Biology↗

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

Non-NASA Center↗

Kinetic preference for the 3'-5'-linked dimer in the reaction of guanosine 5'-phosphorylmorpholinamide with deoxyguanosine 5'-phosphoryl-2-methylimidazolide as a function of poly(C) concentration

The formation of the internucleotide bond in diguanylate synthesis was studied in aqueous solution at pH 8 and 0.2 M Mg2+ in the presence and absence of polycytidylate, poly(C). The investigation was simplified by using guanosine 5'-phosphorylmorpholinamide, mor-pG, which can act only as a nucleophile, and deoxyguanosine 5'-phosphoryl-2-methylimidazolide, 2-MeImpdG, which can act only as an electrophile. The time-dependent product distribution was monitored by high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC/MS). In the absence of poly(C) the reaction between mor-pG and 2-MeImpdG yielded small amounts of the dimer mor-pGpdG with a regioselectivity of 2'-5':3'-5' = 3.5. In the presence of poly(C) dimer yields increased and a reversal in regioselectivity occurred; both effects were in proportion to the concentration of the polymer. The results can be quantitatively explained with the proposition that poly(C), acting as the template, catalyzes the reaction between template-bound monomers by about a factor of 4-5 over the reaction in solution and yields dimers with a regioselectivity of 2'-5':3'-5' approximately 0.33. These findings illustrate the intrinsic preference of guanosine monomers to correctly self-assemble on the appropriate template.

NASA Discipline Exobiology↗

[3H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue

[3H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37 degrees C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as alpha-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.

NASA Program Space Biology↗

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron-exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions "antisense" to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.

Arabidopsis/genetics↗

5' modification of duplex DNA with a ruthenium electron donor-acceptor pair using solid-phase DNA synthesis

Incorporation of metalated nucleosides into DNA through covalent modification is crucial to measurement of thermal electron-transfer rates and the dependence of these rates with structure, distance, and position. Here, we report the first synthesis of an electron donor-acceptor pair of 5' metallonucleosides and their subsequent incorporation into oligonucleotides using solid-phase DNA synthesis techniques. Large-scale syntheses of metal-containing oligonucleotides are achieved using 5' modified phosporamidites containing [Ru(acac)(2)(IMPy)](2+) (acac is acetylacetonato; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (3) and [Ru(bpy)(2)(IMPy)](2+) (bpy is 2,2'-bipyridine; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (4). Duplexes formed with the metal-containing oligonucleotides exhibit thermal stability comparable to the corresponding unmetalated duplexes (T(m) of modified duplex = 49 degrees C vs T(m) of unmodified duplex = 47 degrees C). Electrochemical (3, E(1/2) = -0.04 V vs NHE; 4, E(1/2) = 1.12 V vs NHE), absorption (3, lambda(max) = 568, 369 nm; 4, lambda(max) = 480 nm), and emission (4, lambda(max) = 720 nm, tau = 55 ns, Phi = 1.2 x 10(-)(4)) data for the ruthenium-modified nucleosides and oligonucleotides indicate that incorporation into an oligonucleotide does not perturb the electronic properties of the ruthenium complex or the DNA significantly. In addition, the absence of any change in the emission properties upon metalated duplex formation suggests that the [Ru(bpy)(2)(IMPy)](2+)[Ru(acac)(2)(IMPy)](2+) pair will provide a valuable probe for DNA-mediated electron-transfer studies.

Non-NASA Center↗

HPLC/ESI-quadrupole ion trap mass spectrometry for characterization and direct quantification of amphoteric and nonionic surfactants in aqueous samples

An amphoteric (cocamidopropylbetaine, CAPB) and a nonionic (alcohol polyethoxylate, AE) surfactant were characterized by electrospray ionization quadrupole ion trap mass spectrometry (ESI-MS) as to their homologue distribution and ionization/fragmentation chemistry. Quantitative methods involving reversed-phase gradient HPLC and (+)ESI-MSn were developed to directly determine these surfactants in hydroponic plant growth medium that received simulated graywater. The predominant homologues, 12 C alkyl CAPB and 9 EO AE, were monitored to represent the total amount of the respective surfactants. The methods demonstrated dynamic linear ranges of 0.5-250 ng (r2 > 0.996) for CAPB and 8-560 ng (r2 > 0.998) for AE homologue mixture, corresponding to minimum quantification limits of 25 ppb CAPB and 0.4 ppm AE with 20-microL injections. This translated into an even lower limit for individual components due to the polydispersive nature of the surfactants. The procedure was successfully employed for the assessment of CAPB and AE biodegradation in a hydroponic plant growth system used as a graywater bioreactor.

NASA Discipline Life Support Systems↗

2'-modified nucleosides for site-specific labeling of oligonucleotides

We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.

Non-NASA Center↗

Adenine derivatives as phosphate-activating groups for the regioselective formation of 3',5'-linked oligoadenylates on montmorillonite: possible phosphate-activating groups for the prebiotic synthesis of RNA

Methyladenine and adenine N-phosphoryl derivatives of adenosine 5'-monophosphate (5'-AMP) and uridine 5'-monophosphate (5'-UMP) are synthesized, and their structures are elucidated. The oligomerization reactions of the adenine derivatives of 5'-phosphoramidates of adenosine on montmorillonite are investigated. 1-Methyladenine and 3-methyladenine derivatives on montmorillonite yielded oligoadenylates as long as undecamer, and the 2-methyladenine and adenine derivatives on montmorillonite yielded oligomers up to hexamers and pentamers, respectively. The 1-methyladenine derivative yielded linear, cyclic, and A5'ppA-derived oligonucleotides with a regioselectivity for the 3',5'-phosphodiester linkages averaging 84%. The effect of pKa and amine structure of phosphate-activating groups on the montmorillonite-catalyzed oligomerization of the 5'-phosphoramidate of adenosine are discussed. The binding and reaction of methyladenine and adenine N-phosphoryl derivatives of adenosine are described.

NASA Discipline Exobiology↗

Use of phosphoimidazolide-activated guanosine to investigate the nucleophilicity of spermine and spermidine

Guanosine 5'-phosphate 2-methylimidazolide (2-MeImpG), a labile phosphoimidazolide analog of guanosine triphosphate, was used to test the reactivity of the natural polyamines (PAs), spermine (spm) and spermidine (spd). The products are the guanosine 5'-phosphate-polyamine derivatives (PA-pG: spd-pG and spm-pG) which are quite stable in the range 4 < pH < 11. Our study is the first of which we are aware that reports on the nucleophilicity of these amines. The main findings are as follows. (i) HPLC analysis of the products indicates the formation of only two of the three possible spd products and only one of the two possible spm products. These results can be explained if only the primary amino groups of the two polyamines are reactive, while the secondary amino groups are rendered unreactive by a steric effect. The reactions of 2-MeImpG and other phosphoimidazolide derivatives of nucleosides (ImpNs) with primary and secondary monoamines support this interpretation (Kanavarioti et al. J. Org. Chem. 1995, 60, 632). (ii) The product ratio of the two spd-pG adducts derived from the primary amino groups varies between 2.40 and 0.71 in the range 6.1 < or equal to pH < or equal to 11.9. Such small variation in the product ratio can only be rationalized by the similar, but not identical, basicity of the two primary amino groups and provides strong support for a previously reported model for polyamine ionization (Onasch et. al. Biophys. Chem. 1984, 19, 245). (iii) On the basis of our kinetic determinations conditions at which the nucleophilicity of these amines is at a minimum and at which other interactions with ImpNs could be tested can be chosen.

NASA Discipline Exobiology↗

Synthesis of C60H2 by rhodium-catalyzed hydrogenation of C60

Reduction of C60 with rhodium(0) on alumina and hydrogen in deuterated benzene (C6D6) at ambient temperature and pressure yields a mixture of hydrogenated compounds; C60H2 has been characterized as the major product in 14% yield based on 1H NMR.

Non-NASA Center↗