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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
NASA NTRS2003

The Potato virus X TGBp3 protein associates with the ER network for virus cell-to-cell movement

Potato virus X (PVX) TGBp3 is required for virus cell-to-cell movement. Cell-to-cell movement of TGBp3 was studied using biolistic bombardment of plasmids expressing GFP:TGBp3. TGBp3 moves between cells in Nicotiana benthamiana, but requires TGBp1 to move in N. tabacum leaves. In tobacco leaves GFP:TGBp3 accumulated in a pattern resembling the endoplasmic reticulum (ER). To determine if the ER network is important for GFP:TGBp3 and for PVX cell-to-cell movement, a single mutation inhibiting membrane binding of TGBp3 was introduced into GFP:TGBp3 and into PVX. This mutation disrupted movement of GFP:TGBp3 and PVX. Brefeldin A, which disrupts the ER network, also inhibited GFP:TGBp3 movement in both Nicotiana species. Two deletion mutations, that do not affect membrane binding, hindered GFP:TGBp3 and PVX cell-to-cell movement. Plasmids expressing GFP:TGBp2 and GFP:TGBp3 were bombarded to several other PVX hosts and neither protein moved between adjacent cells. In most hosts, TGBp2 or TGBp3 cannot move cell-to-cell.

NASA Discipline Plant Biology↗
NASA NTRS2003

Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers

The present work utilizes electrospinning to fabricate synthetic polymer/DNA composite scaffolds for therapeutic application in gene delivery for tissue engineering. The scaffolds are non-woven, nano-fibered, membranous structures composed predominantly of poly(lactide-co-glycolide) (PLGA) random copolymer and a poly(D,L-lactide)-poly(ethylene glycol) (PLA-PEG) block copolymer. Release of plasmid DNA from the scaffolds was sustained over a 20-day study period, with maximum release occurring at approximately 2 h. Cumulative release profiles indicated amounts released were approximately 68-80% of the initially loaded DNA. Variations in the PLGA to PLA-PEG block copolymer ratio vastly affected the overall structural morphology, as well as both the rate and efficiency of DNA release. Results indicated that DNA released directly from these electrospun scaffolds was indeed intact, capable of cellular transfection, and successfully encoded the protein beta-galactosidase. When tested under tensile loads, the electrospun polymer/DNA composite scaffolds exhibited tensile moduli of approximately 35 MPa, with approximately 45% strain initially. These values approximate those of skin and cartilage. Taken together, this work represents the first successful demonstration of plasmid DNA incorporation into a polymer scaffold using electrospinning.

Non-NASA Center↗
NASA NTRS1997

CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast nuclear proteins. These results identify C/EBPdelta as a hormonally activated inducer of IGF-I gene transcription in osteoblasts and show that the HS3D element within IGF-I promoter 1 is a high affinity binding site for this protein.

NASA Discipline Musculoskeletal↗
NASA NTRS1995

Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element

Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of IGF-I exon 1 as being essential for this hormonal regulation.

Non-NASA Center↗
NASA NTRS1992

Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae

A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

NASA Discipline Exobiology↗
NASA NTRS2011

Introduction of structural affinity handles as a tool in selective nucleic acid separations

The method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications.

Willson, III, Richard Coale↗
NASA NTRS2011

Stress-Driven Selection of Novel Phenotypes

A process has been developed that can confer novel properties, such as metal resistance, to a host bacterium. This same process can also be used to produce RNAs and peptides that have novel properties, such as the ability to bind particular compounds. It is inherent in the method that the peptide or RNA will behave as expected in the target organism. Plasmid-born mini-gene libraries coding for either a population of combinatorial peptides or stable, artificial RNAs carrying random inserts are produced. These libraries, which have no bias towards any biological function, are used to transform the organism of interest and to serve as an initial source of genetic variation for stress-driven evolution. The transformed bacteria are propagated under selective pressure in order to obtain variants with the desired properties. The process is highly distinct from in vitro methods because the variants are selected in the context of the cell while it is experiencing stress. Hence, the selected peptide or RNA will, by definition, work as expected in the target cell as the cell adapts to its presence during the selection process. Once the novel gene, which produces the sought phenotype, is obtained, it can be transferred to the main genome to increase the genetic stability in the organism. Alternatively, the cell line can be used to produce novel RNAs or peptides with selectable properties in large quantity for separate purposes. The system allows for easy, large-scale purification of the RNAs or peptide products. The process has been reduced to practice by imposing sub-inhibitory concentrations of NiCl2 on cells of the bacterium Escherichia coli that were transformed separately with the peptide library and RNA library. The evolved resistant clones were isolated, and sequences of the selected mini-gene variants were established. Clones resistant to NiCl2 were found to carry identical plasmid variants with a functional mini-gene that specifically conferred significant nickel tolerance on the host cells. Sequencing of the selected mini-gene revealed a propensity of the encoded peptide to bind transient metal ions. Expression of the mini-gene markedly improved growth parameters of the evolved clones at sub-inhibitory concentrations of NiCl2 while being slightly detrimental in the absence of stress. Similar results have been obtained with the RNA libraries. Overall, the results demonstrate a very natural outcome of the selection experiments in which the mini-genes were expected to be either successfully integrated into bacterial genetic networks, or rejected depending upon their effect on host fitness. This described approach can be useful as a laboratory model to study the dynamics of bacterial adaptive evolution on the molecular level. It can also provide a strategy for screening expressed DNA libraries in search of novel genes with desirable properties.

Fox, George E.↗
NASA NTRS2019

Nasa GeneLab Computomics Reveal Horizontal Gene Transfer on International Space Station Environmental Metagenomes

Prokaryotic lifeforms can be observed to demonstrate many keen adaptive advantages, perhaps facilitated by a nature simplistic relative to divergent domains of life. In particular, decompartmentalized gene expression facilitates adaptation by allowing free exchange of genetic material, albeit at the cost of increased susceptibility to genetic damage. Thus, these lifeforms must compensate by embracing diverse investment strategies in an attempt to “brute force” the evolvability equation through precipitous genesis, lean metabolic efficiency, and sheer population. This prokaryotic archetype also enables symbiotic relationships with secondary mobile genetic elements known as plasmids, which have been shown to drive evolution on rapid temporal scales through processes such as conjugation and transformation. This study attempts to decipher whether these mechanisms of horizontal gene transfer (HGT) are major factors in determining prokaryote fitness within a unique isolated environment, the International Space Station (ISS). The ISS Microbial Tracking (MT) project has generated a wealth of data concerning the successive reigns of microbial genera that appear to thrive amidst harsh conditions for life. Despite relatively higher doses of ionizing radiation as compared to Earth, complications associated with microgravity, and the anti-microbial mélange deployed, microbial life still persists in this environment. The NASA GeneLab serves as a data repository and analysis platform to enable researchers to access space flight factor related data. With the use of GeneLab’s modern computational suites (computomics), phylogenetic and functional genomic investigations of HGT events were conducted on the data generated from the MT-1 project. The putative data concerning the plasmid population (plasmidome) of the ISS was algorithmically derived and compared to those of habitats with similar environmental dynamics- such as living quarters and hospitals- to investigate whether these HGT elements may play crucial role(s) in shaping the microbiome of this closed habitat that serves as the only inhabited structure in space.

Bense, Nicholas↗
NASA NTRS1999

Myogenic expression of an injectable protease-resistant growth hormone-releasing hormone augments long-term growth in pigs

Ectopic expression of a new serum protease-resistant porcine growth hormone-releasing hormone, directed by an injectable muscle-specific synthetic promoter plasmid vector (pSP-HV-GHRH), elicits growth in pigs. A single 10 mg intramuscular injection of pSP-HV-GHRH DNA followed by electroporation in three-week-old piglets elevated serum GHRH levels by twofold to fourfold, enhanced growth hormone secretion, and increased serum insulin-like growth factor-I by threefold to sixfold over control pigs. After 65 days the average body weight of the pigs injected with pSP-HV-GHRH was approximately 37% greater than the placebo-injected controls and resulted in a significant reduction in serum urea concentration, indicating a decrease in amino acid catabolism. Evaluation of body composition indicated a uniform increase in mass, with no organomegaly or associated pathology.

Non-NASA Center↗
NASA NTRS1998

Evaluation of pGL1-TNF-alpha therapy in combination with radiation

Long-term control of high-grade brain tumors is rarely achieved with current therapeutic regimens. In this study a new plasmid-based human tumor necrosis factor-alpha (TNF-alpha) expression vector was synthesized (pGL1-TNF-alpha) and evaluated together with radiation in the aggressive, rapidly growing C6 rat glioma model. pGL1-TNF-alpha was successfully transfected into C6 cells in vitro using a cationic polyamine method. Expression was detected up to 7 days and averaged 0.4 ng of TNF-alpha in the culture medium from 1x10(5) cells. The expressed protein was biologically functional, as evidenced by growth inhibition of L929, a TNF-alpha-susceptible cell line. Using fluorescence-labeled monoclonal antibodies and laser scanning cytometry, we confirmed that both the P55 and P75 receptors for TNF-alpha were present on the C6 cell membrane. However, the receptors were present at low density and P55 was expressed more than the P75 receptor. These findings were in contrast to results obtained with TNF-alpha-susceptible L929 cells. Tests in athymic mice showed that pGL1-TNF-alpha administered intratumorally 16-18 h before radiation (each modality given three times) significantly inhibited C6 tumor progression (P<0.05). This effect was more than additive, because pGL1-TNF-alpha alone did not slow tumor growth and radiation alone had little effect on tumor growth. These results indicate that pGL1-TNF-alpha has potential to augment the antitumor effects of radiation against a tumor type that is virtually incurable.

Non-NASA Center↗
NASA NTRS1984

Algal culture studies related to a Closed Ecological Life Support System (CELSS)

In many respects, algae would be the ideal plant component for a biologically based controlled life support system, since they are eminently suited to the closely coupled functions of atmosphere regeneration and food production. Scenedesmus obliquus and Spirulina platensis were grown in three continuous culture apparatuses. Culture vessels their operation and relative merits are described. Both light and nitrogen utilization efficiency are examined. Long term culture issues are detailed and a discussion of a plasmid search in Spirulina is included.

Radmer, R.↗
NASA NTRS1991

Exploration of RNA structure spaces

In order to understand the structure of real structure spaces, we are studying the 5S rRNA structure space experimentally. A plasmid containing a synthetic 5S rRNA gene, two rRNA promoters, and transcription terminators has been assembled. Assays are conducted to determine if the foreign 5S rRNA is expressed, and to see whether or not it is incorporated into ribosomes. Evolutionary competition is used to determine the relative fitness of strains containing the foreign 5S rRNA and a control 5S rRNA. By using site directed mutagenesis, a number of mutants can be made in order to study the boundaries of the structure space and how sharply defined they are. By making similar studies in the vicinity of structure space, it will be possible to determine how homogeneous the 5S rRNA structure space is. Useable experimental protocols have been developed, and a number of mutants have already been studied. Initial results suggest an explanation of why single stranded regions of the RNA are less subject to mutation than double stranded regions.

Fox, G. E.↗
NASA NTRS1994

Investigation of evolution-related aspects of bacterial rhodopsins

We have investigated evolution-related aspects of bacterial rhodopsins, the unique retinal-based energy transducing systems of halophilic archae. The approach was to describe both structural and functional aspects: the structure by sequencing genes to explore which regions are conserved, and the function by comparing proton and chloride transport in the closely related systems, bacteriorhodopsin and halorhodopsin, respectively. In the latter, we have made a good start toward the ultimate goal of separating the attributes of the general principles of retinal-based ionic pumps from those of the specific ion specificities, by determining the thermodynamics of the internal steps of the protein-mediated active transport process, as well as some of the intraprotein ion-transfer steps. Our present emphasis is on continuing to acquire the tools for studying what distinguishes proton transport from chloride transport. We consider it important, therefore, that we have been able to provide firm mathematical grounds for the kinetics analyses which underlies these studies. Our molecular biological studies have received a great boost from the expression vector for the bop gene based on a halobacterial plasmid, that we recently developed.

Source record↗
NASA NTRS1994

Fusion proteins as alternate crystallization paths to difficult structure problems

The three-dimensional structure of a peptide fusion product with glutathione transferase from Schistosoma japonicum (SjGST) has been solved by crystallographic methods to 2.5 A resolution. Peptides or proteins can be fused to SjGST and expressed in a plasmid for rapid synthesis in Escherichia coli. Fusion proteins created by this commercial method can be purified rapidly by chromatography on immobilized glutathione. The potential utility of using SjGST fusion proteins as alternate paths to the crystallization and structure determination of proteins is demonstrated.

Carter, Daniel C.↗
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 NTRS1999

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.↗
NASA NTRS2002

The Hematopoietic Stem Cell Therapy for Exploration of Space

Astronauts experience severe/invasive disorders caused by space environments. These include hematological/cardiac abnormalities, bone and muscle losses, immunodeficiency, neurological disorders and cancer. While the cause of these symptoms are not yet fully delineated, one possible explanation could be the inhibition of hematopoietic stem cell (HSC) growth and hematopoiesis in space. HSCs differentiate into all types of blood cells, and growing evidence indicates that the HSCs also have the ability to transdifferentiate to various tissues, including muscle, skin, liver, neuronal cells and possibly bone. Therefore, a hypothesis was advanced in this laboratory that the hematopoietic stem cell-based therapy, herein called the hematopoietic stem cell therapy (HSCT), could mitigate some of the disorders described above. Due to the magnitude of this project our laboratory has subdivided it into 3 sections: a) HSCT for space anemia; b) HSCT for muscle and bone losses; and c) HSCT for immunodeficiency. Toward developing the HSCT protocol for space anemia, the HSC transplantation procedure was established using a mouse model of beta thalassemia. In addition, the NASA Rotating Wall Vessel (RWV) culture system was used to grow HSCs in space condition. To investigate the HSCT for muscle loss and bone loss, donor HSCs were genetically marked either by transfecting the beta-galactosidase-containing plasmid, pCMV.SPORT-beta-gal or by preparing from b-galactosidase transgenic mice. The transdifferentiation of HSCs to muscle is traced by the reporter gene expression in the hindlimb suspended mice with some positive outcome, as studied by the X-gal staining procedure. The possible structural contribution of HSCs against muscle loss is being investigated histochemically.

Roach, Allana Nicole↗