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At least 793 records · Page 44
All sourcesNASA NTRSDOE OSTI
NASA NTRS1993

Stiffening of deployable space booms: Automated Protein Crystal Growth Facility

Part of the curriculum for the seniors at Vanderbilt University in the Mechanical Engineering Program is to take a design class. The purpose of the class is to expose the students to the open ended problems which working engineers are involved with every day. In the past, the students have been asked to work in a variety of projects developed by the professor. This year Vanderbilt was admitted into the Advanced Design Program (ADP) sponsored by the Universities Space Research Association (USRA) and the National Aeronautics and Space Association (NASA). The grant sponsored undergraduate design and research into new and innovative areas in which NASA is involved. The grant sponsors the Teaching Assistant as well as provides monies for travel and other expenses. The design and research of the seniors of the 1992-1993 school year in association with NASA and USRA is documented.

Cruse, Thomas↗
NASA NTRS1994

Automated documentation generator for advanced protein crystal growth

To achieve an environment less dependent on the flow of paper, automated techniques of data storage and retrieval must be utilized. This software system, 'Automated Payload Experiment Tool,' seeks to provide a knowledge-based, hypertext environment for the development of NASA documentation. Once developed, the final system should be able to guide a Principal Investigator through the documentation process in a more timely and efficient manner, while supplying more accurate information to the NASA payload developer. The current system is designed for the development of the Science Requirements Document (SRD), the Experiment Requirements Document (ERD), the Project Plan, and the Safety Requirements Document.

Maddux, Gary A.↗
NASA NTRS1993

Recombinant protein production and insect cell culture and process

A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using the cultured insect cells as host for a virus encoding the described polypeptide such as baculovirus. The insect cells can also be a host for viral production.

Spaulding, Glenn↗
NASA NTRS1995

Convective flow effects on protein crystal growth

During the fifth semi-annual period under this grant we have pursued the following activities: (1) Characterization of the purity and further purification of lysozyme solutions, these efforts are summarized in Section 2; (2) Crystal growth morphology and kinetics studies with tetragonal lysozyme, our observation on the dependence of lysozyme growth kinetics on step sources and impurities has been summarized in a manuscript which was accepted for publication in the Journal of Crystal Growth; (3) Numerical modelling of the interaction between bulk transport and interface kinetics, for a detailed summary of this work see the manuscript which was accepted for publication in the Journal of Crystal Growth; and (4) Light scattering studies, this work has been summarized in a manuscript that has been submitted for publication to the Journal of Chemical Physics.

Rosenberger, Franz↗
NASA NTRS1997

Recombinant Protein Production and Insect Cell Culture and Process

A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

Spaulding, Glenn F.↗
NASA NTRS1997

Suppressed PHA Activation of T Lymphocytes in Simulated Microgravity Is Restored by Direct Activation of Protein Kinase C with Phorbol Ester

Various aspects of spaceflight, including microgravity, cosmic radiation, and physiological stress, may perturb immune function. We sought to understand the impact of microgravity alone on the cellular mechanisms critical to immunity. We utilized clinostatic RWV bioreactors that simulate aspects of microgravity to analyze the response of human PBMC to polyclonal activation. PHA responsiveness in the RWV was almost completely diminished. IL-2 and IFN-gamma secretion was reduced whereas IL- 1 beta and IL-6 secretion was increased, suggesting that monocytes may not be as adversely affected by simulated microgravity as T cells. Activation marker expression (CD25, CD69, CD71) was significantly reduced in RWV cultures. Furthermore, addition of exogenous IL-2 to these cultures did not restore proliferation. Reduced cell-cell and cell-substratum interactions may play a role in the loss of PHA responsiveness. However, PHA activation in Teflon culture bags that limit cell-substratum interactions did not suppress PHA activation. Furthermore, increasing cell density and, therefore, cell-cell interactions in the RWV cultures did not help restore PHA activation. However, placing PBMC within small collagen beads did partially restore PHA responsiveness. Activation of both PBMC and purified T cells with PMA and ionomycin was unaffected by RWV culture, indicating that signaling mechanisms downstream of PKC activation and calcium flux are not sensitive to simulated microgravity. Furthermore, submitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture. Thus, our data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to simulated microgravity.

Cooper, David↗
NASA NTRS2000

Protein Crystals Grow Purer in Space: Physics of Phenomena

This presentation will summarize the quantitative experimental and theoretical results obtained by B.R. Thomas, P.G. Vekilov, D.C. Carter, A.M. Holmes, W.K. Widierow and the Author, the team with expertise in physics, biochemistry, crystallography and engineering. Impurities inhomogeneously trapped by a growing crystal - e.g., producing sectorial structure and/or striations - may induce macroscopic internal stress in it if an impurity molecule has slightly (less than 10%) different shape or volume than the regular one(s) they replace. We tested for the first time plasticity and measured Young modulus E of the triclinic, not cross-linked lysozyme by triple point bending technique. Triclinic lysozyme crystals are purely elastic with E similar or equal to 1/5 (raised dot) 10 (exp 9) partial derivative yn/sq cm. The strength limit, sigma (sub c) similar or equal to 10 (exp -3)E similar or equal to Epsilon (sub c), where sigma (sub c) and epsilon (sub c) are critical stress and strain, respectively. Scaling E and sigma (sub c) with the lattice spacing suggests similar binding stiffness in inorganic and biomolecular crystals. The inhomogeneous internal stress may be resolved in these brittle crystals either by cracking or by creation of misoriented mosaic blocks during, not after growth. If each impurity molecule induces in the lattice elementary strain epsilon (sub 0) similar or equal to 3 (raised dot) 10 (exp -2) (this is maximal elementary strain that can arise at the supersaturation DELTA mu/kT similar or equal to 2 and macroscopic molecular concentration difference between subsequent macrolayers or growth sectors is partial derivativeC similar or equal to 5 (raised dot) 10 (exp -3), the internal strain epsilon similar or equal to epsilon (sub 0) partial derivative C similar or equal to 10 (exp -4). Mosaic misorientation resolving such strain is approximately 30 arcsec. Tenfold increase of impurity concentration may cause cracking. Estimates of stress in an isometric sectorial crystal show that lysozyme crystals can tolerate the stress till the size of 0.5mm. Dissolving mosaic lysozyme crystal shows that the mosaicity, indeed, is absent below that size.

Chernov, Alex A.↗
NASA NTRS2000

End-To-End Risk Assesment: From Genes and Protein to Acceptable Radiation Risks for Mars Exploration

The human exploration of Mars will impose unavoidable health risks from galactic cosmic rays (GCR) and possibly solar particle events (SPE). It is the goal of NASA's Space Radiation Health Program to develop the capability to predict health risks with significant accuracy to ensure that risks are well below acceptable levels and to allow for mitigation approaches to be effective at reasonable costs. End-to-End risk assessment is the approach being followed to understand proton and heavy ion damage at the molecular, cellular, and tissue levels in order to predict the probability of the major health risk including cancer, neurological disorders, hereditary effects, cataracts, and acute radiation sickness and to develop countermeasures for mitigating risks.

Cucinotta, Francis A.↗
NASA NTRS2003

Towards the Structure Determination of a Modulated Protein Crystal: The Semicrystalline State of Profilin:Actin

One of the remaining challenges to structural biology is the solution of modulated structures. While small molecule crystallographers have championed this type of structure, to date, no modulated macromolecular structures have been determined. Modulation of the molecular structures within the crystal can produce satellite reflections or a superlattice of reflections in reciprocal space. We have developed the data collection methods and strategies that are needed to collect and analyze these data. If the macromolecule's crystal lattice is composed of physiologically relevant packing contacts, structural changes induced under physiological conditions can cause distortion relevant to the function and biophysical processes of the molecule making up the crystal. By careful measurement of the distortion, and the corresponding three-dimensional structure of the distorted molecule, we will visualize the motion and mechanism of the biological macromolecule(s). We have measured the modulated diffraction pattern produced by the semicrystalline state of profilin:actin crystals using highly parallel and highly monochromatic synchrotron radiation coupled with fine phi slicing (0.001-0.010 degrees) for structure determination. These crystals present these crystals present a unique opportunity to address an important question in structural biology. The modulation is believed to be due to the formation of actin helical filaments from the actin beta ribbon upon the pH-induced dissociation of profilin. To date, the filamentous state of actin has resisted crystallization and no detailed structures are available. The semicrystalline state profilin:actin crystals provides a unique opportunity to understand the many conformational states of actin. This knowledge is essential for understanding the dynamics underlying shape changes and motility of eukaryotic cells. Many essential processes, such as cytokinesis, phagocytosis, and cellular migration depend upon the capacity of the actin microfilament system to be restructured in a controlled manner via polymerization, depolymerization, severing, cross-linking, and anchorage. The structure the semicrystalline state of profilin:actin will challenge and validate current models of muscle contraction and cell motility. The methodology and theory under development will be easily extendable to other systems.

Borgstahl, G.↗
NASA NTRS2003

Crystal Structure of the Catalytic Domain of a Serine Threonine Protein Phosphatase

Reversible phosphorylation of serine and threonine residues is a well-recognized mechanism in eukaryotic cells for the regulation of cell-cycle progression, cell growth and metabolism. Human serine/threonine phosphatases can be placed into two major families, PPP and PPM. To date the structure on one PPP family member (PPl) has been determined. Here we present the structure of a 323-residue catalytic domain of a second phosphatase belonging to the PPP family of enzyme. catalytic domain of the enzyme has been determined to 1.60Angstrom resolution and refined to R=17.5 and Rfree = 20.8%. The catalytic domain possesses a unique fold consisting of a largely monolithic structure, divisible into closely-associated helical and sheet regions. The catalytic site contains two manganese ions that are involved in substrate binding and catalysis. The enzyme crystallizes as a dimer that completely buries catalytic surfaces of both monomers, Also, the structure shows evidence of some flexibility around the active site cleft that may be related to substrate specificity of this enzyme.

Swinglel, Mark↗
NASA NTRS2004

Step and Kink Dynamics in Inorganic and Protein Crystallization

Behavior of low-kink-density steps in solution growth and consequences for general understanding of spiral crystal growth processes will be overviewed. Also, influence of turbulence on step bunching and possibility to diminish this bunching will be presented.

Chernov, A. A.↗
NASA NTRS2003

Protein turnover in atrophying muscle: from nutritional intervention to microarray expression analysis

PURPOSE OF REVIEW: In response to decreased usage, skeletal muscle undergoes adaptive reductive remodeling due to the decrease in tension on the weight bearing components of the musculo-skeletal system. This response occurs with uncomplicated disuse (e.g. bed rest, space flight), as a secondary consequence of several widely prevalent chronic diseases for which activity is reduced (e.g. chronic obstructive pulmonary disease and chronic heart failure) and is part of the aging process. The problem is therefore one of considerable clinical importance. RECENT FINDINGS: The impaired function and exercise intolerance is related more to the associated muscle wasting rather than to the specific organ system primarily impacted by the disease. Progress has continued in describing the use of anabolic drugs and dietary manipulation. The major advance in the field has been: (i) the discovery of the atrogin-1 gene and (ii) the application of microarray expression analysis and proteomics with the objectives of obtaining comprehensive understanding of the pathways changed with disuse atrophy. SUMMARY: Disuse atrophy is a common clinical problem. There is a need for therapeutic interventions that do not involve exercise. A better understanding of the changes, particularly at the molecular level, could indicate hitherto unsuspected sites for nutritional and pharmacological intervention.

Non-NASA Center↗
NASA NTRS2002

AmphiFoxE4, an amphioxus winged helix/forkhead gene encoding a protein closely related to vertebrate thyroid transcription factor-2: expression during pharyngeal development

The full-length sequence and developmental expression of amphioxus AmphiFoxE4 are described. Transcripts of the gene are first detected in the pharyngeal endoderm, where the club-shaped gland is forming and subsequently in the definitive gland itself. AmphiFoxE4 is closely related to vertebrate genes encoding the thyroid-specific transcription factor-2 (TTF2), which plays an early developmental role in the morphogenesis of the thyroid gland and a later role in hormone-mediated control of thyroid function. In amphioxus, AmphiFoxE4 expression is not thyroid specific because the club-shaped gland, the only structure expressing the gene, is not homologous to the vertebrate thyroid; instead, the thyroid homologue of amphioxus is a specialized region of the pharyngeal endoderm called the endostyle. We propose that (a) the pharynx of an amphioxus-like ancestor of the vertebrates included a club-shaped gland that expressed FoxE4 as well as an endostyle that did not, and (b) the club-shaped gland soon disappeared in the vertebrate line of descent but (c) not before there was a homeogenetic transfer of FoxE4 expression from the club-shaped gland to the nearby endostyle. Such a transfer could have provided part of the genetic program enabling the endostyle to separate from the pharyngeal endoderm and migrate away as the rudiment of the thyroid gland.

Non-NASA Center↗
NASA NTRS2001

Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells of Arabidopsis thaliana transformed with green-fluorescent-protein gene construct GFP-MBD

Arabidopsis thaliana plants were transformed with GFP-MBD (J. Marc et al., Plant Cell 10: 1927-1939, 1998) under the control of a constitutive (35S) or copper-inducible promoter. GFP-specific fluorescence distributions, levels, and persistence were determined and found to vary with age, tissue type, transgenic line, and individual plant. With the exception of an increased frequency of abnormal roots of 35S GFP-MBD plants grown on kanamycin-containing media, expression of GFP-MBD does not appear to affect plant phenotype. The number of leaves, branches, bolts, and siliques as well as overall height, leaf size, and seed set are similar between wild-type and transgenic plants as is the rate of root growth. Thus, we conclude that the transgenic plants can serve as a living model system in which the dynamic behavior of microtubules can be visualized. Confocal microscopy was used to simultaneously monitor growth and microtubule behavior within individual cells as they passed through the elongation zone of the Arabidopsis root. Generally, microtubules reoriented from transverse to oblique or longitudinal orientations as growth declined. Microtubule reorientation initiated at the ends of the cell did not necessarily occur simultaneously in adjacent neighboring cells and did not involve complete disintegration and repolymerization of microtubule arrays. Although growth rates correlated with microtubule reorientation, the two processes were not tightly coupled in terms of their temporal relationships, suggesting that other factor(s) may be involved in regulating both events. Additionally, microtubule orientation was more defined in cells whose growth was accelerating and less stringent in cells whose growth was decelerating, indicating that microtubule-orienting factor(s) may be sensitive to growth acceleration, rather than growth per se.

NASA Discipline Plant Biology↗
NASA NTRS1987

Multistep modeling of protein structure: application towards refinement of tyr-tRNA synthetase

The scope of multistep modeling (MSM) is expanding by adding a least-squares minimization step in the procedure to fit backbone reconstruction consistent with a set of C-alpha coordinates. The analytical solution of Phi and Psi angles, that fits a C-alpha x-ray coordinate is used for tyr-tRNA synthetase. Phi and Psi angles for the region where the above mentioned method fails, are obtained by minimizing the difference in C-alpha distances between the computed model and the crystal structure in a least-squares sense. We present a stepwise application of this part of MSM to the determination of the complete backbone geometry of the 321 N terminal residues of tyrosine tRNA synthetase to a root mean square deviation of 0.47 angstroms from the crystallographic C-alpha coordinates.

NASA Discipline Exobiology↗