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At least 631 records · Page 35
All sourcesNASA NTRSDOE OSTI
NASA NTRS1992

Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov

Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.

Non-NASA Center↗
NASA NTRS1992

Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories

In the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies. The main focus of this study is the classification of individual EF-hand domains. Five subfamilies--calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin--and three uniques--call, squidulin, and calcium-dependent protein kinase--are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC. The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.

Non-NASA Center↗
NASA NTRS1997

Mitochondrial gene arrangement of the horseshoe crab Limulus polyphemus L.: conservation of major features among arthropod classes

Numerous complete mitochondrial DNA sequences have been determined for species within two arthropod groups, insects and crustaceans, but there are none for a third, the chelicerates. Most mitochondrial gene arrangements reported for crustaceans and insect species are identical or nearly identical to that of Drosophila yakuba. Sequences across 36 of the gene boundaries in the mitochondrial DNA (mtDNA) of a representative chelicerate. Limulus polyphemus L., also reveal an arrangement like that of Drosophila yakuba. Only the position of the tRNA(LEU)(UUR) gene differs; in Limulus it is between the genes for tRNA(LEU)(CUN) and ND1. This positioning is also found in onychophorans, mollusks, and annelids, but not in insects and crustaceans, and indicates that tRNA(LEU)(CUN)-tRNA(LEU)(UUR)-ND1 was the ancestral gene arrangement for these groups, as suggested earlier. There are no differences in the relative arrangements of protein-coding and ribosomal RNA genes between Limulus and Drosophila, and none have been observed within arthropods. The high degree of similarity of mitochondrial gene arrangements within arthropods is striking, since some taxa last shared a common ancestor before the Cambrian, and contrasts with the extensive mtDNA rearrangements occasionally observed within some other metazoan phyla (e.g., mollusks and nematodes).

Non-NASA Center↗
NASA NTRS1992

Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases

Two protein inhibitors of metalloproteinases (TIMP) were isolated from medium conditioned by the clonal rat osteosarcoma line UMR 106-01. Initial purification of both a 30-kDa inhibitor and a 20-kDa inhibitor was accomplished using heparin-Sepharose chromatography with dextran sulfate elution followed by DEAE-Sepharose and CM-Sepharose chromatography. Purification of the 20-kDa inhibitor to homogeneity was completed with reverse-phase high-performance liquid chromatography. The 20-kDa inhibitor was identified as rat TIMP-2. The 30-kDa inhibitor, although not purified to homogeneity, was identified as rat TIMP-1. Amino terminal amino acid sequence analysis of the 30-kDa inhibitor demonstrated 86% identity to human TIMP-1 for the first 22 amino acids while the sequence of the 20-kDa inhibitor was identical to that of human TIMP-2 for the first 22 residues. Treatment with peptide:N-glycosidase F indicated that the 30-kDa rat inhibitor is glycosylated while the 20-kDa inhibitor is apparently unglycosylated. Inhibition of both rat and human interstitial collagenase by rat TIMP-2 was stoichiometric, with a 1:1 molar ratio required for complete inhibition. Exposure of UMR 106-01 cells to 10(-7) M parathyroid hormone resulted in approximately a 40% increase in total inhibitor production over basal levels.

NASA Discipline Musculoskeletal↗
NASA NTRS1980

Studies on chemical evolution using carbon suboxide

It was learned that when carbon suboxide polymers react with hydroxylamine in modified sea water the amino acids glycine and lysine are produced and that they react with urea to produce nucleic acid-like substances. These results lead to the supposition that it is possible that carbon suboxide polymers may have accumulated in the primeval sea some three or more billion years ago and were transformed into the procursors of living molecules.

Yanagawa, H.↗
NASA NTRS1991

Two-dimensional life?

A model [Wachtershauser, G. (1988) Microbiol. Rev. 52, 452-484], according to which life started in the form of a monomolecular layer of interacting anionic metabolites electrostatically bound to a positively charged surface, is examined critically. The model raises a number of thermodynamic and kinetic difficulties.

NASA Discipline Number 52-20↗
NASA NTRS1993

Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique

Amino acid sequence from tryptic peptides of three different bovine corneal keratan sulfate proteoglycan (KSPG) core proteins (designated 37A, 37B, and 25) showed similarities to the sequence of a chicken KSPG core protein lumican. Bovine lumican cDNA was isolated from a bovine corneal expression library by screening with chicken lumican cDNA. The bovine cDNA codes for a 342-amino acid protein, M(r) 38,712, containing amino acid sequences identified in the 37B KSPG core protein. The bovine lumican is 68% identical to chicken lumican, with an 83% identity excluding the N-terminal 40 amino acids. Location of 6 cysteine and 4 consensus N-glycosylation sites in the bovine sequence were identical to those in chicken lumican. Bovine lumican had about 50% identity to bovine fibromodulin and 20% identity to bovine decorin and biglycan. About two-thirds of the lumican protein consists of a series of 10 amino acid leucine-rich repeats that occur in regions of calculated high beta-hydrophobic moment, suggesting that the leucine-rich repeats contribute to beta-sheet formation in these proteins. Sequences obtained from 37A and 25 core proteins were absent in bovine lumican, thus predicting a unique primary structure and separate mRNA for each of the three bovine KSPG core proteins.

NASA Discipline Cell Biology↗
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 NTRS2004

cWINNOWER algorithm for finding fuzzy dna motifs

The cWINNOWER algorithm detects fuzzy motifs in DNA sequences rich in protein-binding signals. A signal is defined as any short nucleotide pattern having up to d mutations differing from a motif of length l. The algorithm finds such motifs if a clique consisting of a sufficiently large number of mutated copies of the motif (i.e., the signals) is present in the DNA sequence. The cWINNOWER algorithm substantially improves the sensitivity of the winnower method of Pevzner and Sze by imposing a consensus constraint, enabling it to detect much weaker signals. We studied the minimum detectable clique size qc as a function of sequence length N for random sequences. We found that qc increases linearly with N for a fast version of the algorithm based on counting three-member sub-cliques. Imposing consensus constraints reduces qc by a factor of three in this case, which makes the algorithm dramatically more sensitive. Our most sensitive algorithm, which counts four-member sub-cliques, needs a minimum of only 13 signals to detect motifs in a sequence of length N = 12,000 for (l, d) = (15, 4). Copyright Imperial College Press.

Evaluation Studies↗
DOE OSTI2024

Effects of Charge Sequence Pattern and Lysine-to-Arginine Substitution on the Structural Stability of Bioinspired Polyampholytes

A comprehensive study focusing on the combined influence of the charge sequence pattern and the type of positively charged amino acids on the formation of secondary structures in sequence-specific polyampholytes is presented. The sequences of interest consisting exclusively of ionizable amino acids (lysine, K; arginine, R; and glutamic acid, E) are (EKEK) 5 , (EKKE) 5 , (ERER) 5 , (ERRE) 5 , and (EKER) 5 . The stability of the secondary structure was examined at three pH values in the presence of urea and NaCl. The results presented here underscore the combined prominent effects of the charge sequence pattern and the type of positively charged monomers on secondary structure formation. Additionally, (ERRE) 5 readily aggregated across a wide range of pH. In contrast, sequences with the same charge pattern, (EKKE) 5 , as well as the sequences with the equivalent amino acid content, (ERER) 5 , exhibited no aggregate formation under equivalent pH and concentration conditions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1997

The organization of the actin cytoskeleton in vertical and graviresponding primary roots of maize

To determine whether actin microfilament (MF) organization is correlated with differential elongation, primary roots of Zea mays cv Merit maintained vertically or reoriented horizontally for 15 to 120 min were stained with rhodamine phalloidin and examined with a confocal microscope. Root curvature was measured with a computer-controlled video digitizer. In vertical roots bundles of MFs in the elongation and maturation zone were oriented parallel to the longitudinal axis of cells. MFs in the vascular parenchyma cells were more abundant than in the cortex and epidermis. Epidermal and proendodermal cells in the meristematic region contained transverse cortical MFs. The organization of MFs of graviresponding roots was similar to that of vertical roots. Application of cytochalasin B or cytochalasin D resulted in extensive disruption of MFs in the cortex and epidermis, but only partially affected MFs in the stele. Despite the cytochalasin B-induced depolymerization of MFs, gravicurvature exceeded that of controls. In contrast, the auxin transport inhibitor N-1 naphthylphthalamic acid suppressed root curvature but had no observable effect on the integrity of the MFs. The data indicate that MFs may not be involved in the graviresponse of maize roots.

NASA Discipline Plant Biology↗
DOE OSTI2021

Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering

Abstract Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

Haloanaerobium kushneri sp. nov., an obligately halophilic, anaerobic bacterium from an oil brine

Three strains, designated VS-751T, VS-511 and VS-732, of a strictly anaerobic, moderately halophilic, Gram-negative, rod-shaped bacterium were isolated from a highly saline (15-20%) brine from an oil reservoir in central Oklahoma, USA. The optimal concentration of NaCl for growth of these three strains was 2 M (12%), and the strains also grew in the presence of an additional 1 M MgCl2. The strains were mesophilic and grew at a pH range of 6-8. Carbohydrates used by all three strains included glucose, fructose, arabinose, galactose, maltose, mannose, cellobiose, sucrose and inulin. Glucose fermentation products included ethanol, acetate, H2 and CO2, with formate produced by two of the three strains. Differences were noted among strains in the optimal temperature and pH for growth, the maximum and minimum NaCl concentration that supported growth, substrate utilization and cellular fatty acid composition. Despite the phenotypic differences among the three strains, analysis of the 16S rRNA gene sequences and DNA-DNA hybridizations showed that these three strains were members of the same genospecies which belonged to the genus Haloanaerobium. The phenotypic and genotypic characteristics of strains VS-751T, VS-511 and VS-732 are different from those of previously described species of Haloanaerobium. It is proposed that strain VS-751T (ATCC 700103T) be established as the type strain of a new species, Haloanaerobium kushneri.

NASA Discipline Exobiology↗
DOE OSTI2022

Frontal Polymerization of Dihydrofuran Comonomer Facilitates Thermoset Deconstruction

Frontal ring-opening metathesis polymerization (FROMP) is a rapid, low-energy manufacturing reaction that is useful for curing thermosetting materials. FROMP of dicyclopentadiene (DCPD) results in poly(dicyclopentadiene) (p(DCPD)), a tough thermoset with excellent mechanical performance and chemical stability. Like most thermosets, p(DCPD) cannot be reprocessed and is therefore difficult to recycle. Previous work demonstrated that the incorporation of a small quantity of cleavable units in the strand segments of p(DCPD) networks enables their deconstruction. Here, we report that a commercially available multifunctional comonomer, 2,3-dihydrofuran (DHF), both acts as a potent Grubbs catalyst inhibitor during FROMP and introduces acid cleavable units. The resulting materials retain high performance characteristics, including glass-transition temperatures ranging from 115 to 165 °C and ultimate strength ranging from 35 to 40 MPa. The addition of DHF above critical loading levels enables deconstructable thermosets. Here, we further demonstrate freeform three-dimensional (3D) printing of deconstructable thermosets via frontal polymerization.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Dynamic Bottlebrush Polymer Networks: Self-Healing in Super-Soft Materials

Here, we introduce a design strategy to expand the range of accessible mechanical properties in covalent adaptable networks (CANs) using bottlebrush polymer building blocks. Well-defined bottlebrush polymers with rubbery poly(4-methylcaprolactone) side-chains were crosslinked in formulations that include a bislactone and strong Lewis acid (tin ethylhexanoate). The resulting materials exhibit tunable stress relaxation rates at elevated temperatures (160–180 °C) due to dynamic ester crosslinks that undergo transesterification with residual hydroxy groups. Varying the crosslinker loading or bottlebrush backbone degree of polymerization yields predictable low-frequency shear moduli ca. 10–100 kPa, well below values typical of linear polymer CANs (1 MPa). These extensible networks can be stretched to strains as large as 350% before failure and undergo efficient self-healing to recover >85% of their original toughness upon repeated fracture and melt processing. In summary, molecular architecture creates new opportunities to tailor the mechanical properties of CANs in ways that are otherwise difficult to achieve.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1999

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

Non-NASA Center↗
DOE OSTI2026

Integrated Strategies for Overcoming Resolution Limits in Electron Beam Lithography of Chemically Amplified Resists

Electron beam lithography (EBL) of chemically amplified resists (CARs) faces fundamental challenges, including stochastic electron scattering and acid diffusion, that limit resolution and reproducibility. Using SU-8 as a model CAR, this study systematically investigated complementary strategies to address these challenges, combining multipass exposure, proximity effect correction (PEC) with midrange correction factors, base quencher incorporation, and post-exposure bake (PEB) suppression. Monte Carlo simulations and calibrated PEC modeling revealed that extending the point spread function to include a midrange scattering component significantly improved critical dimension (CD) control across varying pattern densities, correcting deviations that conventional two-term PEC failed to capture. Multipass exposure, particularly 4-pass writing with a 25% offset, redistributed the dose to average stochastic beam and scattering fluctuations, reducing line-width roughness by more than 50% and yielding more uniform nanoscale features. Photoacid confinement was investigated by adding urea as a base quencher, which successfully reduced acid diffusion but introduced substantial sensitivity penalties without improving ultimate resolution or Z-factor performance, underscoring the trade-offs of chemical versus physical confinement. Suppressing PEB most directly minimized acid diffusion, resulting in improved Z-factors and reproducible 30 nm half-pitch dense line/space patterns. Overall, these results demonstrated that PEC with midrange correction, multipass strategies, quencher additives, and PEB-free processing addresses different aspects of the EBL process window and that their integration provides a comprehensive framework for managing stochastic scattering, diffusion, and chemical amplification effects. This framework advances dense nanoscale patterning in CARs and establishes guiding principles for optimizing resist design and process strategies in high-resolution EBL and potentially other advanced lithographies, such as extreme ultraviolet (EUV) lithography.

36 MATERIALS SCIENCE↗