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At least 1,189 records · Page 66

All sources NASA NTRS DOE OSTI

NASA NTRS1997

Isolation of novel ribozymes that ligate AMP-activated RNA substrates

BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

NASA Discipline Exobiology↗

NASA NTRS1992

The contribution of the extracellular matrix to gravisensing in characean cells

The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.

NASA Discipline Plant Biology↗

NASA NTRS1991

Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans

A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.

Halobacteriaceae/enzymology↗

NASA NTRS2004

Optical Sensors for Biomolecules Using Nanoporous Sol-Gel Materials

An important consideration for space missions to Mars is the ability to detect biosignatures. Solid-state sensing elements for optical detection of biological entities are possible using sol-gel based biologically active materials. We have used these materials as optical sensing elements in a variety of bioassays, including immunoassays and enzyme assays. By immobilizing an appropriate biomolecule in the sol-gel sensing element, we have successfully detected analytes such as amino acids and hormones. In the case of the amino acid glutamate, the enzyme glutamate dehydrogenase was the immobilized molecule, whereas in the case of the hormone cortisol, an anti-cortisol antibody was immobilized in the sensing element. In this previous work with immobilized enzymes and antibodies, excellent sensitivity and specificity were demonstrated in a variety of formats including bulk materials, thin films and fibers. We believe that the sol-gel approach is an attractive platform for bioastronautics sensing applications because of the ability to detect a wide range of entities such as amino acids, fatty acids, hopanes, porphyrins, etc. The sol-gel approach produces an optically transparent 3D silica matrix that forms around the biomolecule of interest, thus stabilizing its structure and functionality while allowing for optical detection. This encapsulation process protects the biomolecule and leads to a more "rugged" sensor. The nanoporous structure of the sol-gel matrix allows diffusion of small target molecules but keeps larger, biomolecules immobilized in the pores. We are currently developing these biologically active sol-gel materials into small portable devices for on-orbit cortisol detection

Fang, Jonathan↗

NASA NTRS2007

Antibodies Against Three Forms of Urokinase

Antibodies that bind to preselected regions of the urokinase molecule have been developed. These antibodies can be used to measure small quantities of each of three molecular forms of urokinase that could be contained in microsamples or conditioned media harvested from cultures of mammalian cells. Previously available antibodies and assay techniques do not yield both clear distinctions among, and measurements of, all three forms. Urokinase is a zymogen that is synthesized in a single-chain form, called ScuPA, which is composed of 411 amino acid residues (see figure). ScuPA has very little enzyme activity, but it can be activated in two ways: (1) by cleavage of the peptide bond lysine 158/isoleucine 159 and the loss of lysine 158 to obtain the high molecular-weight (HMW) form of the enzyme or (2) by cleavage of the bond lysine 135/lysine 136 to obtain the low-molecular-weight (LMW) form of the enzyme. The antibodies in question were produced in mice and rabbits by use of peptides as immunogens. The peptides were selected to obtain antibodies that bind to regions of ScuPA that include the lysine 158/isoleucine 159 and the lysine 135/lysine 136 bonds. The antibodies include monoclonal and polyclonal ones that yield indications as to whether either of these bonds is intact. The polyclonal antibodies include ones that preferentially bind to the HMW or LMW forms of the urokinase molecule. The monoclonal antibodies include ones that discriminate between the ScuPA and the HMW form. A combination of these molecular-specific antibodies will enable simultaneous assays of the ScuPA, HMW, and LMW forms in the same specimen of culture medium.

Morrison, Dennis R.↗

NASA NTRS2010

Electrochemical Detection of Multiple Bioprocess Analytes

An apparatus that includes highly miniaturized thin-film electrochemical sensor array has been demonstrated as a prototype of instruments for simultaneous detection of multiple substances of interest (analytes) and measurement of acidity or alkalinity in bioprocess streams. Measurements of pH and of concentrations of nutrients and wastes in cell-culture media, made by use of these instruments, are to be used as feedback for optimizing the growth of cells or the production of desired substances by the cultured cells. The apparatus is designed to utilize samples of minimal volume so as to minimize any perturbation of monitored processes. The apparatus can function in a potentiometric mode (for measuring pH), an amperometric mode (detecting analytes via oxidation/reduction reactions), or both. The sensor array is planar and includes multiple thin-film microelectrodes covered with hydrous iridium oxide. The oxide layer on each electrode serves as both a protective and electrochemical transducing layer. In its transducing role, the oxide provides electrical conductivity for amperometric measurement or pH response for potentiometric measurement. The oxide on an electrode can also serve as a matrix for one or more enzymes that render the electrode sensitive to a specific analyte. In addition to transducing electrodes, the array includes electrodes for potential control. The array can be fabricated by techniques familiar to the microelectronics industry. The sensor array is housed in a thin-film liquid-flow cell that has a total volume of about 100 mL. The flow cell is connected to a computer-controlled subsystem that periodically draws samples from the bioprocess stream to be monitored. Before entering the cell, each 100-mL sample is subjected to tangential-flow filtration to remove particles. In the present version of the apparatus, the electrodes are operated under control by a potentiostat and are used to simultaneously measure the pH and the concentration of glucose. It is anticipated that development of procedures for trapping more enzymes into hydrous iridium oxide (and possibly into other electroactive metal oxides) and of means for imparting long-term stability to the transducer layers should make it possible to monitor concentrations of products of many enzyme reactions for example, such key bioprocess analytes as amino acids, vitamins, lactose, and acetate.

Rauh, R. David↗

NASA NTRS2003

Growth Conditions To Reduce Oxalic Acid Content of Spinach

A controlled-environment agricultural (CEA) technique to increase the nutritive value of spinach has been developed. This technique makes it possible to reduce the concentration of oxalic acid in spinach leaves. It is desirable to reduce the oxalic acid content because oxalic acid acts as an anti-nutritive calcium-binding component. More than 30 years ago, an enzyme (an oxidase) that breaks down oxalic acid into CO2 and H2O2 was discovered and found to be naturally present in spinach leaves. However, nitrate, which can also be present because of the use of common nitratebased fertilizers, inactivates the enzyme. In the CEA technique, one cuts off the supply of nitrate and keeps the spinach plants cool while providing sufficient oxygen. This technique provides the precise environment that enables the enzyme to naturally break down oxalate. The result of application of this technique is that the oxalate content is reduced by 2/3 in one week.

Johnson-Rutzke, Corinne↗

NASA NTRS2012

Saliva Preservative for Diagnostic Purposes

Saliva is an important body fluid for diagnostic purposes. Glycoproteins, glucose, steroids, DNA, and other molecules of diagnostic value are found in saliva. It is easier to collect as compared to blood or urine. Unfortunately, saliva also contains large numbers of bacteria that can release enzymes, which can degrade proteins and nucleic acids. These degradative enzymes destroy or reduce saliva s diagnostic value. This innovation describes the formulation of a chemical preservative that prevents microbial growth and inactivates the degradative enzymes. This extends the time that saliva can be stored or transported without losing its diagnostic value. Multiple samples of saliva can be collected if needed without causing discomfort to the subject and it does not require any special facilities to handle after it is collected.

Pierson, Duane L.↗

NASA NTRS2014

Maximizing Science Return from Future Rodent Experiments on the International Space Station (ISS): Tissue Preservation

To better understand how mammals adapt to long duration habitation in space, a system for performing rodent experiments on the ISS is under development; Rodent Research-1 is the first flight and will include validation of both on-orbit animal support and tissue preservation. To evaluate plans for on-orbit sample dissection and preservation, we simulated conditions for euthanasia, tissue dissection, and prolonged sample storage on the ISS, and we also developed methods for post-flight dissection and recovery of high quality RNA from multiple tissues following prolonged storage in situ for future science. Mouse livers and spleens were harvested under conditions that simulated nominal, on-orbit euthanasia and dissection operations including storage at -80 C for 4 months. The RNA recovered was of high quality (RNA Integrity Number, RIN(is) greater than 8) and quantity, and the liver enzyme contents and activities (catalase, glutathione reductase, GAPDH) were similar to positive controls, which were collected under standard laboratory conditions. We also assessed the impact of possible delayed on-orbit dissection scenarios (off-nominal) by dissecting and preserving the spleen (RNAlater) and liver (fast-freezing) at various time points post-euthanasia (from 5 min up to 105 min). The RNA recovered was of high quality (spleen, RIN (is) greater than 8; liver, RIN (is) greater than 6) and liver enzyme activities were similar to positive controls at all time points, although an apparent decline in select enzyme activities was evident at the latest time (105 min). Additionally, various tissues were harvested from either intact or partially dissected, frozen carcasses after storage for approximately 2 months; most of the tissues (brain, heart, kidney, eye, adrenal glands and muscle) were of acceptable RNA quality for science return, whereas some tissues (small intestine, bone marrow and bones) were not. These data demonstrate: 1) The protocols developed for future flight experiments will support science return despite delayed preservation post-euthanasia or prolonged storage, and 2) Many additional tissues for gene expression analysis can be obtained by dissection following prolonged storage of the tissue in situ at -80 C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to all future spaceflight experiments that entail on-orbit sample recovery by the ISS crew.

tissues↗

NASA NTRS2014

Maximizing Science Return from Future Rodent Experiments on the International Space Station (ISS): Tissue Preservation

To better understand how mammals adapt to long duration habitation in space, a system for performing rodent experiments on the ISS is under development. Rodent Research-1 is the first flight and will include validation of both on-orbit animal support and tissue preservation. To evaluate plans for on-orbit sample dissection and preservation, we simulated conditions for euthanasia, tissue dissection, and prolonged sample storage on the ISS, and we also developed methods for post-flight dissection and recovery of high quality RNA from multiple tissues following prolonged storage in situ for future science return. Livers and spleens from mice were harvested under conditions that simulated nominal, on-orbit euthanasia and dissection procedures including storage at minus 80 degrees Centigrade for 4 months. The RNA recovered was of high quality (RNA Integrity Number, RNA Integrity Number (RIN) greater than 8) and quantity, and the liver enzyme contents and activities (catalase, glutathione reductase, GAPDH) were similar to positive controls, which were collected under standard laboratory conditions. We also assessed the impact of possible delayed on-orbit dissection scenarios (off-nominal) by dissecting and preserving the spleen (RNA, later) and liver (fast-freezing) at various time points post-euthanasia (from 5 minutes up to 105 minutes). The RNA recovered was of high quality (spleen, RIN greater than 8; liver, RIN greater than 6) and liver enzyme activities were similar to positive controls at all time points, although an apparent decline in select enzyme activities was evident at 105 minutes. Additionally, various tissues were harvested from either intact or partially dissected, frozen carcasses after storage for approximately 2 months; most of the tissues (brain, heart, kidney, eye, adrenal glands and muscle) were of acceptable RNA quality for science return, whereas some tissues (small intestine, bone marrow and bones) were not. These data demonstrate: 1) The protocols developed for future flight experiments will support science return despite delayed preservation post-euthanasia or prolonged storage, and 2) High-quality RNA samples from many different tissues can be recovered by dissection following prolonged storage of the tissue in situ at minus 80 degrees Centigrade. These findings have relevance both to high-value, ground-based experiments when sample collection capability is severely constrained, and to future spaceflight experiments that entail on-orbit sample recovery by the ISS crew.

tissues↗

NASA NTRS2023

Design of a Minimal di-Nickel Hydrogenase Peptide

The most ancient processes for energy production in the evolution of life involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13 amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe]-hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase (ACS), two ancient, extant proteins central to metabolism. These experimental results clearly demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth. One Sentence Summary: Small metal-binding peptides were the likely precursors of modern enzymes.

Joshua A. Mancini↗

DOE OSTI2020

Cell-based composite materials with programmed structures and functions

The present invention is directed to the use of silicic acid to transform biological materials, including cellular architecture into inorganic materials to provide biocomposites (nanomaterials) with stabilized structure and function. In the present invention, there has been discovered a means to stabilize the structure and function of biological materials, including cells, biomolecules, peptides, proteins (especially including enzymes), lipids, lipid vesicles, polysaccharides, cytoskeletal filaments, tissue and organs with silicic acid such that these materials may be used as biocomposites. In many instances, these materials retain their original biological activity and may be used in harsh conditions which would otherwise destroy the integrity of the biological material. In certain instances, these biomaterials may be storage stable for long periods of time and reconstituted after storage to return the biological material back to its original form. In addition, by exposing an entire cell to form CSCs, the CSCs may function to provide a unique system to study enzymes or a cascade of enzymes which are otherwise unavailable.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

Engineering an increase in ethanol production by altering cofactor specificity

The present invention provides for the manipulation of cofactor usage in a recombinant host cell to increase the formation of desirable products. In some embodiments, the invention provides for a recombinant microorganism comprising a mutation in one or more native enzymes such that their cofactor specificity is altered in such a way that overall cofactor usage in the cell is balanced for a specified pathway and there is an increase in a specific product formation within the cell. In some embodiments, endogenous enzymes are replaced by enzymes with an alternate cofactor specificity from a different species.

09 BIOMASS FUELS↗

DOE OSTI2020

In vitro methods for processing lignin and other aromatic compounds

Enzymes for depolymerizing lignin. The enzymes include dehydrogenases, β-etherases, and glutathione lyases. The dehydrogenases can comprise one or more or LigD, LigO, LigN, and LigL. The β-etherases can comprise one or more of LigE, LigF, LigP, and BaeA. The glutathione lyases can comprise any one or more of LigG and a number of non-stereospecific, optionally recombinant glutathione lyases derived from Sphingobium sp. SYK-6, Novosphingobium aromaticivorans, Escherichia coli, Streptococcus sanguinis, Phanerochaete chrysosporium, and other microorganisms. The enzymes can be combined in compositions and/or used in methods of processing lignin or other aromatic compounds in vitro.

Donohue, Timothy James↗

DOE OSTI2021

Feruloyl-CoA:monolignol transferases

The invention relates to feruloyl-CoA:monolignol transferase enzymes and nucleic acids encoding the feruloyl-CoA:monolignol transferase enzymes. The enzymes and/or the nucleic acids enable incorporation of monolignol ferulates into the lignin of plants. The monolignol ferulates include, for example, p-coumaryl ferulate, coniferyl ferulate, and/or sinapyl ferulate.

Ralph, John↗

DOE OSTI2021

Electrochemical flow-cell for hydrogen production and nicotinamide dependent target reduction, and related methods and systems

Methods and systems for hydrogen production or production of a reduced target molecule are described, wherein a nicotinamide co-factor dependent membrane hydrogenase or a nicotinamide co-factor dependent membrane enzyme presented on a nanolipoprotein adsorbed onto an electrically conductive supporting structure, which can preferably be chemically inert, is contacted with protons or a target molecule to be reduced and nicotinamide cofactors in presence of an electric current and one or more electrically driven redox mediators. Methods and systems for production of hydrogen or a reduced target molecule are also described wherein a membrane-bound hydrogenase enzyme or enzyme capable or reducing a target molecule is contacted with protons or the target molecule, a nicotinamide co-factor and a nicotinamide co-factor dependent membrane hydrogenase presented on a nanolipoprotein particle for a time and under condition to allow hydrogen production or production of a reduced target molecule in presence of an electrical current and of an electrically driven redox mediator.

Hoeprich, Jr., Paul D.↗

DOE OSTI2021

Radiolabeled and fluorescent PARP inhibitors for imaging and radiotherapy

The present disclosure relates to compounds of Formula I and II, wherein R1-R20 and FL are defined herein. Also provided are methods of targeting alpha-radiation to poly(ADP-ribose)polymerase 1 (PARP-1) enzyme expression, reducing proliferation of cancer cells, reducing proliferation of cancer cells, detecting intact and enzymatically active poly(ADP-ribose)polymerase 1 (PARP-1) enzyme expression, detecting PARP-1 enzyme expression in a subjects tissue sample, monitoring cancer treatment in a subject, or detecting a PARP-1 receptive cancer in a subject.

Mach, Robert H.↗

DOE OSTI2022

Picoliter droplet sample processing and deposition for mass spectrometry

Disclosed herein include systems, devices, and methods for droplet deposition for mass spectrometry. In some embodiments, a microfluidic device comprising wells is reversibly sealed to a mass spectrometry surface and used to deposit contents of droplets (e.g., enzymes and substrates), or products thereof, onto the mass spectrometry surface. The contents of droplets can be analyzed by laser desorption/ionization to, for example, identify a substrate of an enzyme or an enzyme capable of catalyzing a substrate to a product.

Ha, Noel↗