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At least 109 records · Page 6

Mechanisms of isothermal phase transformations in rapidly solidified Ti-24Al-11Nb

A dynamic simulation for the motion of an isolated superdislocation (SD) under an applied stress is presented. The simulation incorporates the concept that cross slip from (111) planes to (010) planes can locally pin the SD. It is determined that the principal effect of cross-slip-pinning is not the reduction in the SD velocity, but rather the exhaustion of the density of mobile SDs.

Hsiung, L. M.↗

Purification and properties of an ATPase from Sulfolobus solfataricus

The paper reports properties of a sulfite-activated ATPase from Sulfolobus solfataricus, purified using ammonium sulfate precipitation, column chromatography on UltraGel and Sepharose 6B, and SDS-PAGE. The 92-fold purified enzyme had a relative molecular mass of 370,000. It could be dissociated into three subunits with respective molecular masses of 63,000, 48,000, and 24,000. The ATPase activity was found to be inhibitable by nitrate, N-ethylmaleimide (which bound predominantly to the largest subunit), and 4-chloro 7-nitrobenzofurazan, but not by azide, quercetin, or vanadate. While the ATPase from S. solfataricus shared a number of properties with the S. acidocaldarius ATPase, there were also significant differences suggesting the existence of several types of archaeal ATPases.

Hochstein, Lawrence I.↗

BMDO materials testing in the EOIM-3 experiment

The NASA Evaluation of Oxygen Interactions with Materials-3 (EOIM-3) experiment served as a testbed for a variety of materials that are candidates for Ballistic Missile Defense Organization (BMDO) space assets. The materials evaluated on this flight experiment were provided by BMDO contractors and technology laboratories. A parallel ground-based exposure evaluation was conducted using the Fast Atom Sample Tester (FAST) atomic-oxygen simulation facility at Physical Sciences, Inc. The EOIM-3 flight materials were exposed to an atomic oxygen fluence of approximately 2.3 x 10(exp 20) atoms/sq cm. The ground-based exposure fluence of 2.0 - 2.5 x 10(exp 20) atoms/sq cm permits direct comparison with that of the flight-exposed specimens. The results from the flight test conducted aboard STS-46 and the correlative ground-based exposure are summarized here. A more detailed correlation study is presented in the JPL Publication 93-31 entitled 'Flight-and Ground-Test Correlation Study of BMDO SDS Materials: Phase 1 Report'. In general, the majority of the materials survived the AO environment with their performance tolerances maintained for the duration of the exposure. Optical materials, baffles, and coatings performed extremely well as did most of the thermal coatings and tribological materials. A few of the candidate radiator, threat shielding, and structural materials showed significant degradation. Many of the coatings designed to protect against AO erosion of sensitive materials performed this function well.

Chung, Shirley Y.↗

BATSE spectroscopy catalog of bright gamma-ray bursts

This paper presents comprehensive results on the spectra of 30 bright gamma ray bursts (GRBs) as observed by the Spectroscopy Detectors (SDs) of the Burst And Transient Source Experiment (BATSE). The data selection was strict in including only spectra that are of high reliability for continuum shape studies. This BATSE Spectroscopy Catalog presents fluences, model fits (for five spectral models for three energy ranges), and photon spectra in a standard manner for each burst. Complete information is provided to describe the data selection and analysis procedures. The catalog results are also presented in electronic format (from the Compton Observatory Science Support Center) and CD-ROM format (AAS CD-ROM series, Vol. 2). These electronic formats also present the count spectra and detector response matrices so as to allow for independent study and fitting by researchers outside the BATSE Team. This BATSE Spectroscopy Catalog complements the catalog from BATSE Large Area Detector (LAD) data by Fishman et al. (1994).

Schaefer, Bradley E.↗

On the determination of the cosmic infrared background radiation from the high-energy spectrum of extragalactic gamma-ray sources

In a recent paper Stecker, De Jager, & Salamon have suggested using the observed approximately MeV to TeV spectra of extragalactic gamma-ray sources as probes of the local density of the cosmic infrared background radiation (CIBR) and have subsequently claimed a first possible measurement of the CIBR from the analysis of the gamma-ray spectrum of Mrk 421 (De Jager, Stecker, & Salamon). The CIBR from normal galaxies consists of two components: a stellar emission component (CIBRs), and a thermal dust emission component (CIBRd). Photons with energies in the approximately 0.1-2 TeV range interact primarily with the CIBRs, whereas interactions with CIBRd dominate the absorption of photons in the approximately 2-100 TeV energy range. SDS 92 and DSS94 considered only the interaction of the gamma-rays with the dust emission component of the CIBR. We present here an improved analysis of the absorption of extragalactic TeV gamma rays by the CIBR, taking the dual nature of its origin into account. Applying the analysis to the observed gamma-ray spectrum of Mrk 421, a BL Lac object at z = 0.031, we find agreement with DSS94 tentative evidence for absorption by the CINRs. Our analysis therefore limits the detection of the CIBR to the approximately 15-40 micron wavelength regime which, considering the uncertainties in the highest energy (greater than 4 TeV) data and ion the possibility of absorption inside the source, many turn out to be an upper limit on its energy density. At shorter wavelengths (lambda approximately = 1-15 microns), where the gamma-ray interactions are dominated by the CIBRs, our analysis definitely yields only an upper limit on the energy density of the CIBR. In contrast, DSS94 have claimed a possible first measurement of the CIBR over the entire 1-120 micron wavelength region. The upper limit on the CIBRs and tentative detection of the CIBRd are consistent with normal galaxies contributing most of the energy to the CIBR, and constrain the contribution of some exotic sources. With careful modeling of infrared foreground emissions, these constraints on the CIBR are above the values measurable by the DIRBE experiment on board the Cosmic Background Explorer (COBE) satellite.

Dwek, Eli↗

BATSE gamma-ray burst line search. 1: Search for narrow lines in spectroscopy detector data

Analysis of data from the Spectroscopy Detectors (SDs) of the Burst and Transient Source Experiment (BATSE) on the Compton Gamma Ray Observatory (GRO) has found no convincing line features in the spectra of gamma-ray bursts (GRBs) in almost 3 years of operation, in contrast to expectations based on results from other experiments. In this Letter we discuss the visual search for narrow lines in the SD data. The search has examined 192 bursts, of which approximately 18 were intense enough that lines similar to those seen by instruments on the Ginga satellite would have been visible between approximately 20 and approximately 100 keV. A simplified calculation shows that the BATSE and Ginga results are consistent at the 13% level.

Palmer, David M.↗

Protein expression in Arabidopsis thaliana after chronic clinorotation

Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

Piastuch, William C.↗

Peculiarities of Crystallization of the Restriction Endonuclease EcoRII

Nucleases interfere with most standard molecular biology procedures. We have purified and crystallized the restriction endonuclease EcoRII, which belongs to the type II of restriction- modification enzyme, to study the protein crystallization process using a "non standard" macromolecule. A procedure for the purification of EcoRII was developed and 99% pure protein as determined by SDS PAGE electrophoresis obtained. Light scattering experiments were performed to assist in screening protein suitable crystallization conditions. The second virial coefficient was determined as a function of precipitating salt concentration, using sodium chloride, ammonium sulfate, and sodium sulfate. Small (maximum size approximately 0.2 mm) well shaped crystals have been obtained. Larger poorly formed crystals (ca 0.5 mm) have also been obtained, but we have been unable to mount them for diff-raction analysis due to their extreme fragility. Crystallization experiments with PEG have shown that using this precipitant, the best crystals are obtained from slightly over-saturated solutions. Use of higher precipitant concentration leads to dendritic crystal formation. EcoRII is difficult to solubilize and meticulous attention must be paid to the presence of reducing agents.

Karpove, Elizaveta↗

Effects of Microgravity on Embryonic Quail Eye Development

Immunohistochemical methods were used to stain neurofilament protein in corneal nerves of Embryonic Day 16 (E16) quail eyes that had been fixed in 4% paraformaldehyde at room temperature for several months. Fixation was according to the methods used by the Mir 21/NASA 2 Avian Developmental Biology Flight Experiments for quail embryos incubated on the Mir Space Station. After fixation, corneas were pretreated to improve immunohistochemical visualization of neurofilaments. A sequential combination of three pretreatments [microwave heating in saline G, followed by extraction with sodium dodecyl sulfate (SDS) at 37 C, followed by digestion with hyaluronidase at 37 C], produced increased antibody staining of corneal nerve neurofilament proteins, compared with corneas subjected to no prior pretreatments. Darker nerve staining and increased numbers of fine branches were observed, together with lower background staining after such pretreatments. In contrast, use of any single pretreatment or pair of pretreatments resulted in only slight and inconsistent enhancement of nerve staining. Only the sequential combination of all three pretreatments resulted in consistently better nerve staining.

Barrett, Joyce E.↗

Interdisciplinary Research Scenario Testing of EOSDIS

During the reporting period, the Principle Investigator (PI) has continued to serve on numerous review panels, task forces and committees with the goal of providing input and guidance for the Earth Observing System Data and Information System (EOSDIS) program at NASA Headquarters and NASA GSFC. In addition, the PI has worked together with personnel at the University of Virginia and the subcontractor (Simpson Weather Associates (SWA)) to continue to evaluate the latest releases of various versions of the user interfaces to the EOSDIS. Finally, as part of the subcontract, SWA has created an on-line Hierarchial Data Format (HDF) tutorial for non-HDF experts, particularly those that will be using EOSDIS and future EOS data products. A summary of these three activities is provided. The topics include: 1) Participation on EODIS Panels and Committees; 2) Evaluation and Tire Kicking of EODIS User Interfaces; and 3) An On-line HDF Tutorial. The report also includes attachments A, B, and C. Attachment A: Report From the May 1999 Science Data Panel. The topics include: 1) Summary of Data Panel Meeting; and 2) Panel's Comments/Recommendations. Attachment B: Survey Requesting Integrated Design Systems (IDS) Teams Input on the Descoping and Rescoping of the EODIS; and Attachment C: An HDF Tutorial for Beginners: EODIS Users and Small Data Providers (HTML Version). The topics include: 1) Tutorial Overview; 2) An introduction to HDF; 3) The HDF Library: Software and Hardware; 4) Methods of Working with HDF Files; 5) Scientific Data API; 6) Attributes and Metadata; 7) Writing a SDS to an HDF file; 8) Obtaining Information on Existing HDF Files; 9) Reading a Scientific Data Set from an HDF file: 10) Example Programs; 11) Browsing and Visualizing HDF Data; and 12) Laboratory (Question and Answer).

Emmitt, G. D.↗

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

Hong, Y. S.↗

Purification of Restriction Endonuclease EcoRII and its Co-Crystallization

Restriction endonuclease EcoRII (EcoRII) is a homodimeric DNA-binding protein. It belongs to the type II family of restriction-modification enzymes (subclass IIe). EcoRII recognizes the nucleotide sequence 5'-CCWGG (W=A or T) and cleaves the phosphodiester bond preceding the first cytosine. Methylation at C5 of the second cytosine inhibits cleavage. The enzyme has a unique ability to search for the presence of two substrate sites before cleavage. To the best of our knowledge no other subclass IIe restriction endonuclease has been crystallized yet, without or with a DNA-substrate. We have recently grown and characterized the crystals of this enzyme (1) Here we report on the result of co-crystallization experiments of EcoRII with an 11 b.p. oligonucleotide substrate. The dissociation constant (Kd) EcoRII: 11 b.p. was determined earlier (unpublished results). The needle-like crystals of oligonucleotide-EcoRII protein complex were obtained with this substrate by the technique of vapor diffusion hanging drops. The crystals obtained were washed and dissolved in an aliquot of 10 mM Tris-HCl buffer, pH=7.5. Running a portion of this solution on the SDS-get indicated the presence of endonuclease in the solution. A UV-spectrophotometric test of a second portion confirmed the presence of DNA. We are now working on improvement of the DNA-EcoRII protein crystals. Results obtained from these and ongoing efforts will be reported.

Karpova, E. A.↗

Carbon Isotope Fractionations Associated with Methanotrophic Growth with the Soluble and Particulate Methane Monooxygenases

Growth experiments with the RuMP-type methanotroph, Methylococcus capsulatus (Bath), have demonstrated that biomass and lipid biomarkers are significantly depleted in C-13 compared to the substrate methane and that the extent of fractionation is dependent on whether cells express the soluble (s) or particulate (p) methane monooxygenase (MMO). The presence or absence of the characteristic sMMO subunits was monitored using SDS-polyacrylamide gels. In M. capsulatus grown with no Cu supplementation, the characteristic sMMO subunits were observed in the soluble fraction throughout the entire growth period and biomass was depleted in C-13 by approximately 14,700 relative to substrate methane. In cells grown with 5uM Cu, no sMMO bands were observed and a greater fractionation of approximately 27,700 in resultant biomass was obtained. Methanol growth experiments with M. capsulatus and with a RuMP methylotroph, Methylophilus methylotrophus, in which biomass measurements yielded depletions in C-13 of 9 and 5%(sub o), respectively, suggest that oxidation of methane is the major fractionation step. Growth of M. capsulatus at a low level of oxygen, approximately 0.5%, had no significant effect on carbon isotope fractionation by either sMMO or pMMO. These observations are significant for identification of molecular biomarkers; and methanotrophic contributions to carbon isotope composition in natural environments.

Jahnke, Linda L.↗

Low-Energy Study of Gamma-Ray Bursts Using Two BATSE Spectroscopy Detectors

Gamma-ray bursts (GRBs) are energetic, short-duration emissions of gamma-rays from astronomical sources typically well beyond our galaxy. The Burst and Transient Source Experiment (BATSE) that was onboard NASA's Compton Gamma-Ray Observatory (CGRO) had detected an unprecedented 2704 GRBs during CGRO's nine-year mission. BATSE consisted of eight detector assemblies located at the corners of CGRO to give full sky coverage. Each assembly consisted of two detectors, a Large Area Detector (LAD) and a Spectroscopy Detector (SD). In determining the detail features of GRBs, the degree to which they possess a low-energy component (approx. 10 keV) is of interest. Preece have developed a method to study the low-energy characteristics and concluded that 14% of the 86 bright GRBs they studied had a definite low-energy component, referred to as a low-energy excess. Their study, and for the present study as well, needed to use SD data, because it extends down to the low-energy range when operating in a high-gain mode. For their study, low-energy data was used from just one SD. To better quantify the low-energy behavior, this study will consider bursts for which two SDs satisfy the same criteria as used by Preece.

Michael J. Pangia↗

Low-Energy Study of Gamma-Ray Bursts Having Spectral Line Features

Gamma-ray bursts (GRBs) are energetic, short-duration emissions of gamma-rays. The Burst and Transient Source Experiment (BATSE) that was onboard NASA s Compton Gamma-Ray Observatory has done much to advance our understanding of GRBs. Perhaps foremost is to establish that GRBs originate from astronomical sources that exist well beyond our galaxy. Another area in which BATSE has been instrumental is to provide high-resolution data that can be used in spectral studies. Before BATSE, there were many reports of GRB spectra containing what appeared to be spectral absorption lines, whereas Briggs, after an extensive computer search of 117 bright BATSE GRBs, reported finding only one case that might be an absorption line and ten cases that might be emission lines. None of the eleven BATSE cases were definitively identified as spectral lines, and Briggs indicated reasons as to why the pre-BATSE reports should not be taken as conclusive. It remains an open question as to what these spectral-like features are, or if they are even real. The purpose of this work is, for the subset of the eleven BATSE GRBs for which low-energy data are available from two BATSE's Spectroscopy Detectors (SDs), to include these data in the spectral analysis. Such a study will provide additional constraints on the model spectral functions to better ascertain the reality of the line features. The spectral analysis program used was RMFIT. Of the six GRBs that met the selection criteria, the analysis was performed on only three of them due to a lack of time.

Pangia, Michael J.↗

Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

Non-NASA Center↗

Characterization of GTP binding and hydrolysis in plasma membranes of zucchini

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.

NASA Discipline Plant Biology↗

Protein expression in Arabidopsis thaliana after chronic clinorotation

Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

NASA Center KSC↗