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At least 307 records · Page 17

Studies on the "Aerobic" Acetyl-Coenzyme A Synthetase of Saccharomyces Cerevisiae: Purification, Crystallization, and Physical Properties of the Enzyme

A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis. Sedimentation velocity runs revealed a single symmetric peak with an s(sub (20,w)) value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 +/- 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 +/- 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic Saccharomyces cerevisiae is composed of three subunits of identical or nearly identical size.

Satyanarayana, T.↗

Effects of Microheterogeneity in Hen Egg-White Lysozyme Crystallization

In earlier sodium dodecylsulfate polyacylamide gel electrophoresis (SDS-PAGE) studies it has been found that commonly utilized commercial hen egg-white lysozyme (HEWL) preparations contained 0.2-0.4 mol% covalently bound dimers. Here it is shown, using high-performance capillary electrophoresis (HPCE), that HEWL contains, in addition, two differently charged monomers in comparable amounts. To explore the origin of these microheterogeneous contaminants, purified HEWL (PHEWL) has been oxidized with hydrogen peroxide (0.0026-0.88 M) at various pH levels between 4.5 and 12.0. Optical densitometry of oxidized PHEWL (OHEWL) bands in SDS PAGE gels shows that hydrogen peroxide at 0.88 M in acetate buffer pH 4.5 increased the amount of dimers about sixfold over that in commercial HEWL. OHEWL had, in addition to one of the two monomer forms found in HEWL and PHEWL, three other differently charged monomer forms, each of them representing about 25% of the preparation. SDS-PAGE analysis of OHEWL yielded two closely spaced dimer bands with M(sub r) = 28 000 and 27 500. In addition, larger HEWL oligomers with M, = 1.7 million and 320 000 were detected by gel-filtration fast protein liquid chromatography with multiangle laser light scattering detection. Non-dissociating PAGE in large pore size gels at pH 4.5 confirmed the presence of these large oligomers in HEWL and OHEWL. Increased microheterogeneity resulted in substantial effects on crystal growth and nucleation rate. On addition of 10 microgram(exp -1) mg ml(exp -1) OHEWL to 32 mg ml(exp -1) HEWL crystallizing solutions, both the number and size of forming crystals decreased roughly proportionally to the concentration of the added microheterogeneity. The same effect was observed in HEWL solutions on addition of 0.03-9,3 M Hydrogen peroxide. Repartioning of the dimer during crystallzation aat various temperatures between 277 and 293 K was analyzed by SDS-PAGE. The crystals contained <= 25 % weight by volume of the oligomers in the solution, with no apparent temperature dependence of the repartioning.

Thomas, B. R.↗

Aqueous Cleaning and Validation for Space Shuttle Propulsion Hardware at the White Sands Test Facility

The NASA White Sands Test Facility (WSTF) has developed an entirely aqueous final cleaning and verification process to replace the current chlorofluorocarbon (CFC) 113 based process. This process has been accepted for final cleaning and cleanliness verification of WSTF ground support equipment. The aqueous process relies on ultrapure water at 50 C (323 K) and ultrasonic agitation for removal of organic compounds and particulate. The cleanliness is verified bv determining the total organic carbon (TOC) content and filtration with particulate counting. The effectiveness of the aqueous methods for detecting hydrocarbon contamination and particulate was compared to the accepted CFC 113 sampling procedures. Testing with known contaminants, such as hydraulic fluid and cutting and lubricating oils, to establish a correlation between aqueous TOC and CFC 113 nonvolatile residue (NVR) was performed. Particulate sampling on cleaned batches of hardware that were randomly separated and sampled by the two methods was performed. This paper presents the approach and results, and discusses the issues in establishing the equivalence of aqueous sampling to CFC 113 sampling, while describing the approach for implementing aqueous techniques on Space Shuttle Propulsion hardware.

Hornung, Steven D.↗

Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

Malone, Christine C.↗

Optical Metrology for the Filter Set for the Hubble Space Telescope (HST) Advanced Camera for Surveys (ACS)

The Hubble Space Telescope (HST) Advanced Camera for Surveys (ACS) will employ a wide variety of spectral filtration components including narrow band, medium band, wide band, and far ultraviolet (FUV) long pass filters, spatially- variable filters (ramp filters), VIS/IR polarizers, NUV polarizers, FUV prisms, and a grism. These components are spread across ACS's Wide Field, High Resolution, and Solar Blind channels which provide diffraction-limited imaging of astronomical targets using aberration-correcting optics which remove most aberrations from HST's Optical Telescope Assembly (OTA). In order for ACS to be truly advanced, these filters must push the state-of-the-art in performance in a number of key areas at the same time. Important requirements which these filters must meet include outstanding transmitted wavefront, high transmittance, uniform transmittance across each filter, spectrally structure-free bandpasses, exceptionally high out of band rejection, and a high degree of parfocality. These constitute a very stringent set of requirements indeed, especially for filters which are up to 90 mm in diameter. The development of optical metrology stations used to demonstrate that each ACS filter will meet its design specifications is discussed. Of particular note are specially-designed spectral transmissometers and interferometers.

Leviton, Douglas B.↗

Does Velocity Redistribution Really Enhance the HE 304 A Line to Observed Intensities?

Previous work by this group has demonstrated that small-scale nonthermal velocities probably play a significant role in enhancing the intensity of the He II 304 A line above values predicted by the static atmosphere NLTE theories, and more in conformity with Skylab and SOHO observations. This presentation briefly summarizes the evidence for this conclusion, emphasizing SOHO and correlated groundbased observations, of which examples are presented. However, in contrast to the previous studies, the tact taken here is more critical, asking the question "Can velocity redistribution fully explain the observations of the 304 A line, and what counter-indications and problems remain?" The conclusion reached is that, while velocity redistribution plays a significant role in the intensity enhancement, it may not be the whole story. Some other mechanism, associated with velocity filtration, may be at work.

Jordan, Stuart↗

The MECA Payload as a Dust Analysis Laboratory on the MSP 2001 Lander

In a companion abstract, the "Mars Environmental Compatibility Assessment" (MECA) payload for Mars Surveyor Program 2001 (MSP 2001) is described in terms of its capabilities for addressing exobiology on Mars. Here we describe how the same payload elements perform in terms of gathering data about surface dust on the planet. An understanding of the origin and properties of dust is important to both human exploration and planetary geology. The MECA instrument is specifically designed for soil/dust investigations: it is a multifunctional laboratory equipped to assess particulate properties with wet chemistry, camera imagery, optical microscopy (potentially with LTV fluorescence capability), atomic force microscopy (AFM; potentially with mineral-discrimination capabilities), electrometry, active & passive external materials-test panels, mineral hardness testing, and electrostatic & magnetic materials testing. Additionally, evaluation of soil chemical and physical properties as a function of depth down to about 50 cm will be facilitated by the Lander/MECA robot arm on which the camera (RAC) and electrometer are mounted. Types of data being sought for the dust include: (1) general textural and grain-size characterization of the soil as a whole --for example, is the soil essentially dust with other components or is it a clast-supported material in which dust resides only in the clast interstices, (2) size frequency distribution for dust particles in the range 0.01 to 10.00 microns, (3) particle-shape distribution of the soil components and of the fine dust fraction in particular, (4) soil fabric such as grain clustering into clods, aggregates, and cemented/indurated grain amalgamations, as well as related porosity, cohesiveness, and other mechanical soil properties, (5) cohesive relationship that dust has to certain types of rocks and minerals as a clue to which soil materials may be prime hosts for dust "piggybacking", (6) particle, aggregate, and bulk soil electrostatic properties, (7) particle hardness, (8) particle magnetic properties, (9) bulk dust geochemistry (solubility, reactivity, ionic and mineral species). All of these quantities are needed in order for the human exploration program to make assessments of hazards on Mars, and to better enable the production on earth, of soil/dust simulants that can act as realistic test materials in terms of those properties that render dust a contaminant.Such properties include the small grain size that enables penetration of space-suit joints, mechanical interfaces and bearings, seals, etc., and presents difficulty for filtration systems. Size also plays a critical role in the potential for lung disease in long-term habitats. The properties of grain shape and hardness are important parameters in determining the abrasiveness of dust as it enters mechanical systems, or bombards helmet visors and habitat windows in dust-laden winds. Adhesive electrostatic and magnetic properties of dust will be prime causes of contamination of space suits and equipment. Contamination causes mechanical malfunction, tracking of dirt into habitats, "piggybacking" of toxins on dust into habitats, changes in albedo and efficiency of solar arrays and heat exchangers, and changes in electrical conductivity of suit surfaces and other materials that may have specific safety requirements regarding electrical conductivity. Other potentially hazardous properties of dust include the possibility of high solubility of some component grains (rendering them reactive), and toxicity of some materials --grains of superoxidants and heavy metals (there is always the slim, but not inconceivable possibility of biogenic components such as spores). Because Mars has no active surface aqueous regime, volcanic emissions, meteoritic debris, weathering products, and photochemical products of Mars have nowhere to go except reside in the surface; there are few mechanical or chemical (buffering) processes to remove the accumulation of eons. From a planetology perspective, there are many enigmatic issues relating to dust and the aeolian regime in general. MECA will be able to address many questions in this area. For example, if MECA determines a particular particle size distribution (size and sorting values), it will be possible to make inferences about the origin of the dust - - is it all aeolian, or a more primitive residue of weathering, volcanic emissions, and meteoritic gardening? Trenching with the Lander/MECA robot arm will enable local stratigraphy to be determined in terms of depositional rates, amounts and cyclicity in dust storms and/or local aeolian transport. Grain shape will betray the origin of the dust fragments as being the product of recent or ancient weathering, or the comminution products of aeolian transport --the dust-silt ratio might be a measure of aeolian comminution energy. Additional information is contained in the original.

Marshall, J.↗

Development of Paper Products from Dried Sweetpotato Stems and Peanut Shells

One of the goals of NASA's Advanced Life Support Program (ALS) for sustaining human life in space is to achieve a closed system in plant production and usage. That all inedible plant parts should be recycled or used in some way. A Tuskegee University team researching sweetpotato and peanut for ALS has developed paper products from dried sweet-potato stems and peanut shells. In this study, the sweet-potato stems and peanut shells were soaked separately in water for 48 hours. After 48 hours, researchers manually separated the pulp and the unusable parts. To form the paper, 160 g of pulp and water mixture was poured through a 15.1 cm (diameter) filtration funnel and the pulp was trapped on 15 cm (diameter) filter paper. The filter paper and pulp were dried in an air oven, and the filter paper was removed, An examination under a scanning electron microscope showed that the sweet-potato paper was composed of "fibers", whereas the peanut shell paper was composed of "blocks". Results of physical testing showed that the sweet-potato stem paper was stronger than the peanut shell paper. It is anticipated that there may be other uses of these products such as writing paper, bags and packaging material. Because of its biodegradability, it can be incorporated into the resource recycling system at the end of its use.

R McConnell↗

Microbial Impact on Success of Human Exploration Missions

The purpose of this study is to identify microbiological risks associated with space exploration and identify potential countermeasures available. Identification of microbial risks associated with space habitation requires knowledge of the sources and expected types of microbial agents. Crew data along with environmental data from water, surfaces, air, and free condensate are utilized in risk examination. Data from terrestrial models are also used. Microbial risks to crew health include bacteria, fungi, protozoa, and viruses. Adverse effects of microbes include: infections, allergic reactions, toxin production, release of volatiles, food spoilage, plant disease, material degradation, and environmental contamination. Risk is difficult to assess because of unknown potential changes in microbes (e.g., mutation) and the human host (e.g., immune changes). Prevention of adverse microbial impacts is preferred over remediation. Preventative measures include engineering measures (e.g., air filtration), crew microbial screening, acceptability standards, and active verification by onboard monitoring. Microbiological agents are important risks to human health and performance during space flight and risks increase with mission duration. Acceptable risk level must be defined. Prevention must be given high priority. Careful screening of crewmembers and payloads is an important element of any risk mitigation plan. Improved quantitation of microbiological risks is a high priority.

Pierson, Duane L.↗

Renal Function of Rats in Response to 37 Days of Head-Down Tilt

Spaceflight induces changes in human renal function, suggesting similar changes may occur in rats. Since rats continue to be the prime mammalian model for study in space, the effects of chronic microgravity on rat renal function should be clarified. Acute studies in rats using the ground-based microgravity simulation model, head-down tilt (HDT), have shown increases in glomerular filtration rate (GFR), electrolyte excretion, and a diuresis. However, long term effects of HDT have not been studied extensively. This study was performed to elucidate rat renal function following long-term simulated microgravity. Chronic exposure to HDT will cause an increase in GFR and electrolyte excretion in rats, similar to acute exposures, and lead to a decrease in the fractional excretion of filtered electrolytes. Experimental animals (HDT, n=10) were tail-suspended for 37 days and renal function compared to ambulatory controls (AMB, n=10). On day 37 of HDT, GFR, osmolal clearance, and electrolyte excretion were decreased, while plasma osmolality and free water clearance were increased. Urine output remained similar between groups. The fractional excretion of the filtered electrolytes was unchanged except for a decrease in the percentage of filtered calcium excreted. Chronic exposure to HDT results in decreased GFR and electrolyte excretion, but the fractional excretion of filtered electrolytes remained primarily unaffected.

Wang, Tommy J.↗

Water Filters

The Aquaspace H2OME Guardian Water Filter, available through Western Water International, Inc., reduces lead in water supplies. The filter is mounted on the faucet and the filter cartridge is placed in the "dead space" between sink and wall. This filter is one of several new filtration devices using the Aquaspace compound filter media, which combines company developed and NASA technology. Aquaspace filters are used in industrial, commercial, residential, and recreational environments as well as by developing nations where water is highly contaminated.

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Water Filters

Seeking to find a more effective method of filtering potable water that was highly contaminated, Mike Pedersen, founder of Western Water International, learned that NASA had conducted extensive research in methods of purifying water on board manned spacecraft. The key is Aquaspace Compound, a proprietary WWI formula that scientifically blends various types of glandular activated charcoal with other active and inert ingredients. Aquaspace systems remove some substances; chlorine, by atomic adsorption, other types of organic chemicals by mechanical filtration and still others by catalytic reaction. Aquaspace filters are finding wide acceptance in industrial, commercial, residential and recreational applications in the U.S. and abroad.

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Metallic Filters

Filtration technology originated in a mid 1960's NASA study. The results were distributed to the filter industry, an HR Textron responded, using the study as a departure for the development of 421 Filter Media. The HR system is composed of ultrafine steel fibers metallurgically bonded and compressed so that the pore structure is locked in place. The filters are used to filter polyesters, plastics, to remove hydrocarbon streams, etc. Several major companies use the product in chemical applications, pollution control, etc.

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Microgravity and Hypogravity Compatible Methods for the Destruction of Solid Wastes by Magnetically Assisted Gasification

This report summarizes a three-year collaborative effort between researchers at UMPQUA Research Company (URC) and the Chemical Engineering Department at Oregon State University (OSU). The Magnetically Assisted Gasification (MAG) concept was originally conceived as a microgravity and hypogravity compatible means for the decomposition of solid waste materials generated aboard spacecraft, lunar and planetary habitations, and for the recovery of potentially valuable resources. While a number of methods such as supercritical water oxidation (SCW0), fluidized bed incineration, pyrolysis , composting and related biological processes have been demonstrated for the decomposition of solid wastes, none of these methods are particularly well- suited for employment under microgravity or hypogravity conditions. For example, fluidized bed incineration relies upon a balance between drag forces which the flowing gas stream exerts upon the fluidization particles and the opposing force of gravity. In the absence of gravity, conventional fluidization cannot take place. Hypogravity operation can also be problematic for conventional fluidized bed reactors, because the various factors which govern fluidization phenomena do not all scale linearly with gravity. For this reason it may be difficult to design and test fluidized bed reactors in lg, which are intended to operate under different gravitational conditions. However, fluidization can be achieved in microgravity (and hypogravity) if a suitable replacement force to counteract the forces between fluid and particles can be found. Possible alternatives include: centripetal force, electric fields, or magnetic fields. Of these, magnetic forces created by the action of magnetic fields and magnetic field gradients upon ferromagnetic media offer the most practical approach. The goal of this URC-OSU collaborative effort was to develop magnetic hardware and methods to control the degree of fluidization (or conversely consolidation) of granular ferromagnetic media and to employ these innovations in sequential filtration and fluidized bed processes for the segregation and decomposition of solid waste materials, and for the concentration and collection of inorganic residue (ash). This required the development of numerous enabling technologies and tools.

Atwater, James E.↗

Purification, Crystallization, and Preliminary X-ray Analysis of Native Canavalin

The protein canavalin is a 7S vicilin, from the Jack Bean, Canavalis ensfomis. Canavalin is described as a seed storage protein, an energy source for a developing seed, as no other known activity or function has been found. The protein was first isolated and crystallized by Sumner and Howell (J. Biol. Chem. 113, 607-610, 1936). Canavalin spontaneously crystallizes after proteolytic cleavage at neutral pH, which removes residues 1-46, 224-245, and 325-330 and produces peptides of approximately 25, 13, and 12 kDa. Preliminary gel filtration experiments indicated the presence of nucleic acid with the uncleaved protein. We developed a dual column procedure, ion exchange followed by hydroxy apatite chromatography, that effectively removes the nucleic acid and yields an essentially pure uncut canavalin preparation with an OD 280/260 ratio of approximately 1.9-2.0. Standard crystallization screens using this material gave a number of positive results having a common requirement for alcohols and Mg(2+) ion, with crystals typically appearing within a day or less. Optimization experiments to date have shown that we can obtain crystals from pH 6.5 to pH 8.2, using MPD from 5 to 20% and 0.05 to 0.2M Mg(2+) (sulfate or acetate). The crystals are of space group P2(sub 1)2(sub 1)2(sub 1), unit cell dimensions, and a complete data set to 1.5 Angstroms, resolution has now been collected at a synchrotron source. Most importantly, the crystals are not twinned, a persistent problem with the most commonly obtained rhombohedral form of proteolytically cleaved canavalin.

Pusey, Marc↗

Smoke Mask

Smoke inhalation injury from the noxious products of fire combustion accounts for as much as 80 percent of fire-related deaths in the United States. Many of these deaths are preventable. Smoke Mask, Inc. (SMI), of Myrtle Beach, South Carolina, is working to decrease these casualties with its line of life safety devices. The SMI personal escape hood and the Guardian Filtration System provide respiratory protection that enables people to escape from hazardous and unsafe conditions. The breathing filter technology utilized in the products is specifically designed to supply breathable air for 20 minutes. In emergencies, 20 minutes can mean the difference between life and death.

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Purification of the small mechanosensitive channel of Escherichia coli (MscS): the subunit structure, conduction, and gating characteristics in liposomes

The small mechanosensitive channel, MscS, is a part of the turgor-driven solute efflux system that protects bacteria from lysis in the event of osmotic downshift. It has been identified in Escherichia coli as a product of the orphan yggB gene, now called mscS (Levina et al., 1999, EMBO J. 18:1730). Here I show that that the isolated 31-kDa MscS protein is sufficient to form a functional mechanosensitive channel gated directly by tension in the lipid bilayer. MscS-6His complexes purified in the presence of octylglucoside and lipids migrate in a high-resolution gel-filtration column as particles of approximately 200 kDa. Consistent with that, the protein cross-linking patterns predict a hexamer. The channel reconstituted in soybean asolectin liposomes was activated by pressures of 20-60 mm Hg and displayed the same asymmetric I-V curve and slight anionic preference as in situ. At the same time, the single-channel conductance is proportional to the buffer conductivity in a wide range of salt concentrations. The rate of channel activation in response to increasing pressure gradient across the patch was slower than the rate of closure in response to decreasing steps of pressure gradient. Therefore, the open probability curves were recorded with descending series of pressures. Determination of the curvature of patches by video imaging permitted measurements of the channel activity as a function of membrane tension (gamma). Po(gamma) curves had the midpoint at 5.5 +/- 0.1 dyne/cm and gave estimates for the energy of opening DeltaG = 11.4 +/- 0.5 kT, and the transition-related area change DeltaA = 8.4 +/- 0.4 nm(2) when fitted with a two-state Boltzmann model. The correspondence between channel properties in the native and reconstituted systems is discussed.

NASA Discipline Cell Biology↗

Heterogeneous responses of human limbs to infused adrenergic agonists: a gravitational effect?

Unlike quadrupeds, the legs of humans are regularly exposed to elevated pressures relative to the arms. We hypothesized that this "dependent hypertension" would be associated with altered adrenergic responsiveness. Isoproterenol (0.75-24 ng x 100 ml limb volume-1 x min-1) and phenylephrine (0.025-0.8 microg x 100 ml limb volume-1 x min-1) were infused incrementally in the brachial and femoral arteries of 12 normal volunteers; changes in limb blood flow were quantified by using strain-gauge plethysmography. Compared with the forearm, baseline calf vascular resistance was greater (38.8 +/- 2.5 vs. 26.9 +/- 2.0 mmHg x 100 ml x min x ml-1; P < 0.001) and maximal conductance was lower (46.1 +/- 11.9 vs. 59.4 +/- 13.4 ml x ml-1 x min-1 x mmHg-1; P < 0.03). Vascular conductance did not differ between the two limbs during isoproterenol infusions, whereas decreases in vascular conductance were greater in the calf than the forearm during phenylephrine infusions (P < 0.001). With responses normalized to maximal conductance, the half-maximal response for phenylephrine was significantly less for the calf than the forearm (P < 0.001), whereas the half-maximal response for isoproterenol did not differ between limbs. We conclude that alpha1- but not beta-adrenergic-receptor responsiveness in human limbs is nonuniform. The relatively greater response to alpha1-adrenergic-receptor stimulation in the calf may represent an adaptive mechanism that limits blood pooling and capillary filtration in the legs during standing.

Clinical Trial↗