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All sourcesNASA NTRSDOE OSTI
NASA NTRS2003

Crystallization Physics in Biomacromolecular Systems

The crystals are built of molecules of protein, nucleic acid and their complexes, like viruses, approx. 5x10(exp 3)+ 3x10(exp 6) Da in weight and 2 + 20 nm in effective diameter. This size strongly exceeds action range of molecular forces and makes a big difference with inorganic crystals. Intermolecular contacts form patches on the biomacromolecular surface. Each patch may occupy only a small percent of the whole surface and vary from polymorph to polymorph of the same protein. Thus, under different conditions (pH, solution chemistry, temperature, any area on the macromolecular surface may form a contact. The crystal Young moduli, E approx. equals 0.1 + 0.5 GPa are more than 10 times lower than that of inorganics and the biomolecules themselves. Water within biocrystals (30-70%) is unable to flow unless typical deformation time is longer than approx. 10(exp -5)s. This explains the discrepancy between light scattering and static measurements of E. Nucleation and Growth requires typically concentrations exceeding the equilibrium ones up to 100 times - because of the new size scale results in 10 - 10(exp 3) times lower kinetic coefficients than that needed for inorganic solution growth. All phenomena observed in the latter occur with protein crystallization and are even better studied by AFM. Crystals are typically facetted. Among unexpected findings of general significance are - net molecular exchange flux at kinks is much lower than that expected from supersaturation, steps with low (< approx. 10(exp -2)) kink density at steps follow Gibbs-Thomson law only at very low supersaturations, step segment growth rate may be independent of step energy. Crystal perfection is a must of biocrystallization to achieve the major goal to find 3-D atomic structure of biomacromolecules by x-ray diffraction. Poor diffraction resolution (> 3Angstrom) makes crystallization a bottleneck for structural biology. All defects typical of small molecule crystals are found in biocrystals, but the defects responsible for poor resolution are not identified. Conformational changes are one of them. Biocrystallization in microgravity reportedly results in 20% cases of better crystals. The mechanism of how lack of convection can do this is still not clear. Lower supersaturation, self-purification &om preferentially trapped homologous impurities and step bunching are viable hypotheses.

Chernov, A. A.↗
NASA NTRS1990

Immunological characterization of plant ornithine transcarbamylases

Pea (Pisum sativum L.) ornithine transcarbamylase (OTC) antisera were used to investigate the immunological relatedness of several plant and animal OTC enzymes. The antisera immunoprecipitated OTC activity in all monocot and dicot species tested, and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of immunoprecipitated protein revealed monomeric proteins ranging from 35,200 to 36,800 daltons in size. Pea OTC antisera did not recognize mammalian OTC protein. OTC activity and protein levels detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblots from homogenates of green leaf, etiolated epicotyl and cotyledon, and root tissues of pea were poorly correlated. This might result from differences in amounts of enzymatically active OTC protein in the homogenates. Alternatively, the antisera may fail to recognize different isozyme forms of OTC, which have been reported for some plant species. A putative cytosolic precursor OTC (pOTC) polypeptide exhibiting and Mr = 39,500 to 40,000 daltons was immunoprecipitated from in vitro translation mixtures of total pea leaf poly(A)+ RNA. The size of the pOTC polypeptide, as compared with mature OTC monomer (36,000 daltons), suggests that a 4 kilodalton N-terminal leader sequence, like that responsible for mitochondrial targeting of the mammalian enzyme, may be involved in organellar import of the plant enzyme.

NASA Discipline Plant Biology↗
NASA NTRS1993

Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity

This study ascertained the effects of 9 days of zero gravity on the relative (percentage of total) and calculated absolute (mg/muscle) content of isomyosin expressed in both antigravity and locomotor skeletal muscle of ground control (CON) and flight-exposed (FL) rats. Results showed that although there were no differences in body weight between FL and CON animals, a significant reduction in muscle mass occurred in the vastus intermedius (VI) (P < 0.05) but not in the vastus lateralis (VL) or the tibialis anterior. Both total muscle protein and myofibril protein content were not different between the muscle regions examined in the FL and CON groups. In the VI, there were trends for reductions in the relative content of type I and IIa myosin heavy chains (MHCs) that were offset by increases in the relative content of both type IIb and possibly type IIx MHC protein (P > 0.05). mRNA levels were consistent with this pattern (P < 0.05). The same pattern held true for the red region of the VL as examined at both the protein and mRNA level (P < 0.05). When the atrophy process was examined, there were net reductions in the absolute content of both type I and IIa MHCs that were offset by calculated increases in type IIb MHC in both VI and red VL. Collectively, these findings suggest that there are both absolute and relative changes occurring in MHC expression in the "red" regions of antigravity skeletal muscle during exposure to zero gravity that could affect muscle function.

short duration↗
NASA NTRS1989

Potential utilization of algal protein concentrate as a food ingredient in space habitats

Green alga Scenedesmus obliquus was studied as one of the potential sources of macronutrients in a space habitat. Algal protein concentrate (70.5% protein) was incorporated into a variety of food products such as bran muffins, fettuccine (spinach noodle imitation) and chocolate chip cookies. Food products containing 20 to 40% of incorporated algal proteins were considered. In the sensory analysis the greenish color of the bran muffins and cookies was not found to be objectional. The mild spinachy flavor (algae flavor) was less detectable in chocolate chip cookies than in bran muffins. The color and taste of the algae noodles were found to be pleasant and compared well with commercially available spinach noodles. Commercially available spray-dried Spirulina algae was also incorporated so the products can be compared with those containing Scenedesmus obliquus concentrate. Food products containing commercial algae had a dark green color and a "burnt after taste" and were less acceptable to the panelists.

NASA Discipline Life Support Systems↗
NASA NTRSDate not supplied

The membrane bioreactor (MBR): A hybrid technology for bioregenerative wastewater treatment and resource recovery in space

Extraplanetary surface habitat life support systems (LSS) on the Moon and Mars, as well as long-duration space travel, will require novel capabilities to withstand anticipated unique, harsh conditions. In order to provide safe, habitable environments for the crew, water purification and waste processing systems will be required to treat all sources of water (condensate, Sabatier, urine, hygiene, fecal, food waste) in order to achieve the necessary levels of recovery needed to sustain life over the long-duration missions. The ability to recycle organic wastes creates an opportunity to recover critical elements (e.g., C, H, O, N, P) for subsequent food production, water purification, and atmospheric regeneration. Bioregenerative systems mimic functions of nature in engineered systems, or bioreactors, utilizing combination of prokaryotes, eukaryotes and archaea. While these systems are commonly used on Earth for wastewater treatment, bioreactors for space travel face additional challenges. Terrestrial bioreactors often rely on gravitational settling of dense flocs and granules for cell retention. For micro- or partial-gravity environments, density differential alone will not be adequate for cell retention; a gravity-independent means for cell retention is crucial. The membrane bioreactor represents the state of the art in wastewater treatment. This hybrid system combines biological processes with membrane filtration to achieve performance beyond what each can accomplish individually. The complete cell retention in an MBR allows for the decoupling of hydraulic retention time (HRT) and solids retention time (SRT), which result in a high-thruput, compact, treatment system. The Bioregenerative Water Technology Team at NASA Kennedy Space Center and the University of South Florida has developed a bioregenerative platform based on the hybrid MBR technology. The overall architecture is compact, modular, flexible, and adaptable to mission evolutions. The main subsystems of the bioregenerative architecture include: 1) Anaerobic membrane bioreactor (AnMBR): Also termed the Organic processor assembly (OPA), the function of the AnMBR is to treat organic wastes such as fecal and food wastes. These wastes are characterized by a concentration of suspended solids comprised of carbohydrates, proteins and lipids. The assigned function of the AnMBR is to break down and covert suspended solids to biogas (methane, hydrogen and carbon dioxide), reduce effluent chemical oxygen demand (COD), liberate organically-bound nutrients, and remove pathogenic organisms. 2) Phototrophic membrane bioreactor (PMBR): The PMBR is comprised of a co-culture of microalgae and bacteria. The assigned function of the PMBR is to polish the permeate of the AnMBR to further remove dissolved organic carbon, manage nutrients (nitrogen transformation, load dampening), and perform air revitalization. 3) Food processor assembly (FPA): The FPA is a food production platform (prokaryotic or eukaryotic), fueled by outputs from the AnMBR, or PMBR. For the presentation, we will describe each step of the bioregenerative architecture, and present performance data from extended trials treating analog and real metabolic wastes.

Bioreactor↗
NASA NTRS1993

Three distinct cell phenotypes of induced-TNF cytotoxicity and their relationship to apoptosis

We have identified three distinct cell phenotypes with respect to the conditions under which cells became susceptible to TNF-mediated lysis. These conditions include: 1) treatment with the protein synthesis inhibitor, cycloheximide; 2) contact with activated macrophages, and 3) infection with vaccinia virus. Whereas vaccinia virus-infected 3T3 cells became sensitive to soluble TNF, F5b cells required contact with activated macrophages. We showed that the "macrophage-resistant" F5m cells did not become sensitive to TNF or to killing by activated macrophages after infection with vaccinia virus. Therefore, vaccinia infection does not sensitize all cells to TNF. We also determined the pathways of lysis for cells after sensitization. Whereas 3T3, LM929, and F5b cells were killed by the process of necrosis, F5m cells lysis was characterized by the release of low mol wt DNA fragments (apoptosis).

Non-NASA Center↗
NASA NTRS1996

Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

NASA Discipline Cell Biology↗
DOE OSTI2021

Incorporation of Membrane Proteins Into Bicontinuous Microemulsions Through Winsor‐III System‐Based Extraction

Abstract The membrane proteins (MP) α‐synuclein (ASYN) and bacteriorhodopsin (BR) were readily incorporated into bicontinuous microemulsions (BμEs) formed by two microemulsion systems: water/heptane/Aerosol‐OT (AOT)/CK‐2,13 and water/dodecane/sodium dodecyl sulfate (SDS)/1‐pentanol. (CK‐2,13 is an alkyl ethoxylate possessing two alkyl tail groups of carbon chain length 2 and 13 and an average degree of ethoxylation of 5.6.) MP were encapsulated in BμEs through preparation of Winsor‐III systems at optimal salinity, with the anionic surfactants AOT and SDS providing the driving force for extraction. Dissolution of ASYN in BμEs greatly increased the former's α‐helicity, similar to ASYN's behavior in the presence of biomembranes, while BμE‐ and vesicle‐encapsulated BR possessed similar secondary structure. Small‐angle neutron scattering (SANS) results clearly demonstrated the direct interaction of MP with the surfactants, resulting in a decrease of surface area per volume for surfactant monolayers due to decreased surfactant efficiency. The SANS signal for ASYN was isolated through the use of neutron contrast matching for the surfactants through partial deuteration of water and oil, one of the first reports of contrast matching for BμEs in the literature. The SANS results of the contrast‐matched sample reflected similar aggregation for ASYN in BμEs as was reported previously for vesicles and SDS solution. This study demonstrates the potential use of BμEs as MP host systems for conducting biochemical reactions such as the conversion of sunlight into adenosine triphosphate by BR and studying the fundamental behavior of MP, such as the role of ASYN dysfunction in Parkinson's disease, as well as for isolation and purification of MP via Winsor‐III‐based extraction.

Hayes, Douglas G.↗
NASA NTRS1997

[Ribosomal RNA Evolution]

It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modern organisms came to exist by the time of the last common ancestor (as detected by 16S rRNA sequence studies). Third, the ribosomal RNAs themselves likely had a very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.During the past year we have ave conducted a comparative study of four completely sequenced bacterial genoames. We have focused initially on conservation of gene order. The second component of the project continues to build on the model system for studying the validity of variant 5S rRNA sequences in the vicinity of the modern Vibrio proteolyticus 5S rRNA that we established earlier. This system has made it possible to conduct a detailed and extensive analysis of a local portion of the sequence space. These core methods have been used to construct numerous mutants during the last several years. Although it has been a secondary focus, this work has continued over the last year such that we now have in excess of 125 V. proteolyticus derived constructs which have been made and characterized. We have also continued high resolution NMR work on RNA oligomers originally initiated by G. Kenneth Smith who was funded by a NASA Graduate Student Researcher's Fellowship Award until May of 1996. Mr. Smith developed synthesis & purification protocols in order to obtain large quantities of RNA oligomers for NMR studies. He succeeded in obtaining preliminary one dimensional spectra for an analog of the helix II/loop B/helix III domain of 5S rRNAs well as a 29mer which includes helix III and the highly characteristic 13 nucleotide loop C. Significant progress in laying the required groundwork for this work has been accomplished during the past year. A plasmid, pLeuS-1, carrying the E. coli leucine tRNA synthetase gene (LeuRS) has been obtained and used to overexpressed the leucine synthetase. A T7 runoff transcription has been established in our laboratory and successfully used to make both minihelix RNAs and tRNAs. In addition, encapsulation studies have been conducted and we have successfully encapsulated tRNA in lipid vesicles. Initial efforts to obtain peptide bond synthesis will be underway over the next several moths.

Source record↗
NASA NTRS1990

The Chemical Basis for the Origin of the Genetic Code and the Process of Protein Synthesis

A model for the origin of protein synthesis. The essential features of the model are that 5'-AMP and perhaps other monoribonucleotides can serve as catalysts for the selective synthesis of L-based peptides. A unique set of characteristics of 5'-AMP is responsible for the selective catalysts and these characteristics are described in detail. The model involves the formation of diesters as intermediates and selectivity for use of the L-isomer occurs principally at the step of forming the diester. However, in the formation of acetyl phenylalanine-AMP monoester there is a selectivity for esterification by the D-isomer. Data showing this selectivity is presented. This selectivity for D-isomer disappears after the first step. The identity was confirmed of all four of possible diesters of acetyl-D- and -L phenylaline with 5'-AMP by nuclear magnetic resonance (NMR). The data using flourescence and NMR show the Trp ring can associate with the adenine ring more strongly when the D-isomer is in the 2' position than it can when in the 3' position. These same data also suggest a molecular mechanisim for the faster esterificaton of 5'-AMP by acetyl-D-phenylaline. Some new data is also presented on the possible structure of the 2' isomer of acetyl-D-tryptophan-AMP monoester. The HPLC elution times of all four possible acetyl diphenylalanine esters of 5'-AMP were studied, these peptidyl esters will be products in the studies of peptide formation on the ribose of 5'-AMP. Other studies were on the rate of synthesis and the identity of the product when producing 3'Ac-Phe-2'tBOC-Phe-AMP diester. HPLC purification and identification of this product were accomplished.

Lacey, James C., Jr.↗
NASA NTRS1994

Negative regulators of cell proliferation

Cell proliferation is governed by the influence of both mitogens and inhibitors. Although cell contact has long been thought to play a fundamental role in cell cycling regulation, and negative regulators have long been suspected to exist, their isolation and purification has been complicated by a variety of technical difficulties. Nevertheless, over recent years an ever-expanding list of putative negative regulators have emerged. In many cases, their biological inhibitory activities are consistent with density-dependent growth inhibition. Most likely their interactions with mitogenic agents, at an intracellular level, are responsible for either mitotic arrest or continued cell cycling. A review of naturally occurring cell growth inhibitors is presented with an emphasis on those factors shown to be residents of the cell surface membrane. Particular attention is focused on a cell surface sialoglycopeptide, isolated from intact bovine cerebral cortex cells, which has been shown to inhibit the proliferation of an unusually wide range of target cells. The glycopeptide arrest cells obtained from diverse species, both fibroblasts and epithelial cells, and a broad variety of transformed cells. Signal transduction events and a limited spectrum of cells that are refractory to the sialoglycopeptide have provided insight into the molecular events mediated by this cell surface inhibitor.

Review↗
DOE OSTI2021

Structural Characterization of the Human Cytosolic Malate Dehydrogenase I

The first crystal structure of the human cytosolic malate dehydrogenase I (MDH1) is described. Structure determination at a high resolution (1.65 Å) followed production, isolation, and purification of human MDH1 using a bacterial expression system. The structure is a binary complex of MDH1 with only a bound malonate molecule in the substrate binding site. Comparisons of this structure with malate dehydrogenase enzymes from other species confirm that the human enzyme adopts similar secondary, tertiary, and quaternary structures and that the enzyme retains a similar conformation even when nicotinamide adenine dinucleotide (NAD + ) is not bound. A comparison to the highly homologous porcine (sus scrofa) MDH1 ternary structures leads to the conclusion that only small conformational differences are needed to accommodate binding by NAD + or other NAD + mimetics. Conformational differences observed in the second subunit show that the NAD + binding elements are nevertheless quite flexible. Comparison of hMDH1 to the human mitochondrial malate dehydrogenase (hMDH2) reveals some key differences in the α7–α8 loop, which lies directly beneath the substrate binding pocket. These differences might be exploited in the structure-assisted design of selective small molecule inhibitors of hMDH1, an emerging target for the development of anticancer therapeutics.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1995

Isolation, characterization, and primary structure of rubredoxin from the photosynthetic bacterium, Heliobacillus mobilis

Rubredoxin is a small nonheme iron protein that serves as an electron carrier in bacterial systems. Rubredoxin has now been isolated and characterized from the strictly anaerobic phototroph, Heliobacillus mobilis. THe molecular mass (5671.3 Da from the amino acid sequence) was confirmed and partial formylation of the N-terminal methionyl residue was established by matrix-assisted laser desorption mass spectroscopy. The complete 52-amino-acid sequence was determined by a combination of N-terminal sequencing by Edman degradation and C-terminal sequencing by a novel method using carboxypeptidase treatment in conjunction with amino acid analysis and laser desorption time of flight mass spectrometry. The molar absorption coefficient of Hc. mobilis rubredoxin at 490 nm is 6.9 mM-1 cm-1 and the midpoint redox potential at pH 8.0 is -46 mV. The EPR spectrum of the oxidized form shows resonances at g = 9.66 and 4.30 due to a high-spin ferric iron. The amino acid sequence is homologous to those of rubredoxins from other species, in particular, the gram-positive bacteria, and the phototrophic green sulfur bacteria, and the evolutionary implications of this are discussed.

NASA Discipline Number 52-30↗
NASA NTRS1993

Z-DNA binding protein from chicken blood nuclei

A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.

NASA Discipline Exobiology↗
NASA NTRS1995

The molecular response of bone to growth hormone during skeletal unloading: regional differences

Hind limb elevation of the growing rat provides a good model for the skeletal changes that occur during space flight. In this model the bones of the forelimbs (normally loaded) are used as an internal control for the changes that occur in the unloaded bones of the hind limbs. Previous studies have shown that skeletal unloading of the hind limbs results in a transient reduction of bone formation in the tibia and femur, with no change in the humerus. This fall in bone formation is accompanied by a fall in serum osteocalcin (bone Gla protein, BGP) and bone BGP messenger RNA (mRNA) levels, but a rise in bone insulin-like growth factor-I (IGF-I) protein and mRNA levels and resistance to the skeletal growth-promoting actions of IGF-I. To determine whether skeletal unloading also induced resistance to GH, we evaluated the response of the femur and humerus of sham and hypophysectomized rats, control and hind limb elevated, to GH (two doses), measuring mRNA levels of IGF-I, BGP, rat bone alkaline phosphatase (RAP), and alpha 1(1)-procollagen (coll). Hypophysectomy (HPX) decreased the mRNA levels of IGF-I, BGP, and coll in the femur, but was either less effective or had the opposite effect in the humerus. GH at the higher dose (500 micrograms/day) restored these mRNA levels to or above the sham control values in the femur, but generally had little or no effect on the humerus. RAP mRNA levels were increased by HPX, especially in the femur. The lower dose of GH (50 micrograms/day) inhibited this rise in RAP, whereas the higher dose raised the mRNA levels and resulted in the appearance of additional transcripts not seen in controls. As for the other mRNAs, RAP mRNA in the humerus was less affected by HPX or GH than that in the femur. Hind limb elevation led to an increase in IGF-I, coll, and RAP mRNAs and a reduction in BGP mRNA in the femur and either had no effect or potentiated the response of these mRNAs to GH. We conclude that GH stimulates a number of markers of bone formation by raising their mRNA levels, and that skeletal unloading does not block this response, but the response varies substantially from bone to bone.

Non-NASA Center↗