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At least 397 records · Page 22
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Redox poise in R. rubrum phototrophic growth drives large-scale changes in macromolecular pathways

During photoheterotrophic growth on organic substrates, purple nonsulfur photosynthetic bacteria like Rhodospirillum rubrum can acquire electrons by multiple means, including oxidation of organic substrates, oxidation of inorganic electron donors (e.g., H 2 ), and by reverse electron flow from the photosynthetic electron transport chain. These electrons are stored as reduced electron-carrying cofactors (e.g., NAD(P)H and ferredoxin). The overall ratio of oxidized to reduced cofactors (e.g., NAD(P)+:NAD(P)H), or ’redox poise’, is difficult to understand or predict, as are the cellular processes for dissipating these reducing equivalents. Using physics-based models that capture mass action kinetics consistent with the thermodynamics of reactions and pathways, a range of redox conditions for heterophototrophic growth are evaluated, from conditions in which the NADP+/NADPH levels approach thermodynamic equilibrium to conditions in which the NADP+/NADPH ratio is far above the typical physiological values. Modeling predictions together with experimental measurements indicate that the redox poise of the cell results in large-scale changes in the activity of biosynthetic pathways and, thus, changes in cell macromolecule levels (DNA, RNA, proteins, and fatty acids). Furthermore, modeling predictions indicate that during phototrophic growth, reverse electron flow from the quinone pool is a minor contributor to the production of reduced cofactors (e.g., NAD(P)H) compared to other oxidative processes (H 2 and carbon substrate oxidation). Instead, the quinone pool primarily operates to aid ATP production. The high level of ATP, in turn, drives reduction processes even when NADPH levels are relatively low compared to NADP+ by coupling ATP hydrolysis to the reductive processes. The model, in agreement with experimental measurements of macromolecule ratios of cells growing on different carbon substrates, indicates that the dynamics of nucleotide versus lipid and protein production is likely a significant mechanism of balancing oxidation and reduction in the cell.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Developing Accelerated Test Protocols and Tuning Microstructures of the Common Materials to Improve Robustness, Reliability, and Endurance of SOFC Cells

This work is built on our prior work where we found that phase transformation in praseodymium nickelates, e.g. Pr 2 NiO 4 (PNO) and (Pr 1-x Nd x ) 2 NiO 4 (PNNO), can be electrochemically driven, and is substantially faster when compared to thermal annealing studies. The first task aims at an attempt to further accelerate the phase transformation in the oxygen electrode by alternating the current input in the cells, which lead to the development of accelerated test protocols (ATPs). ATPs showed up to 60x faster phase transformation and up to 10x faster performance degradation in (Pr 0.50 Nd 0.50 ) 2 NiO 4 electrodes, when compared to long-term operation under constant current density. Furthermore, the phase stable Nd 2 NiO 4 and (La 0.6 Sr 0.4 )(Co 0.8 Fe 0.2 )O 3 (LSCF6482) electrodes were tested in full cells under ATPs, and showed up to 10x faster performance degradation within 1,100 hours in a comparison with long-term thermal annealing studies and electrochemical operation under constant current density. The second task aims at the quantification of the contributions of cell components to the total impedance of a solid oxide fuel cell (SOFC) using electrochemical impedance spectroscopy (EIS) and distribution of relaxation times (DRT). Specifically, the role of gas composition at both anode and cathode was systematically studied, aiming at deconvoluting, identifying and quantifying the contributions of different electrode processes. This was achieved by first tuning the partial pressure of H 2 at the anode and subsequently varying the partial pressure of O 2 at the cathode. The results suggest that, while DRT offers a viable way of deconvoluting different times distributions, additional attention is needed before assigning a peak to a specific electrode process due to the significant overlap of the contributions from the cathode and the anode. Density function theory studies show that both Pr-vacancies and O-defects play a key role on the activity and stability for nickelates towards oxygen reduction reaction. The resident O-interstitials and oxygen ions in the PrO layer form peroxide (O 2 2- ) nearby Pr vacancies. The O 2 2- limits oxygen-ion transport due to the required additional energy to break its O-O bond. We further calculated the formation and segregation energies for different Ln ions (La, Pr, Nd, Pm, Sm, Gd, Tb, Dy, and Ho) in PNO and CeO 2 (111) surfaces. In addition to Nd, Pm and La are suggested as potential dopants in PNO to enhance it stability without decomposition due to their more negative formation energies, lower diffusion energies, and positive separation energies.

01 COAL, LIGNITE, AND PEAT↗
DOE OSTI2021

The CopA2-Type P 1B -Type ATPase CcoI Serves as Central Hub for cbb 3 -Type Cytochrome Oxidase Biogenesis

Copper (Cu)-transporting P 1B -type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P 1B -type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb 3 -type cytochrome oxidase (cbb 3 -Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb 3 -Cox. CcoI and cbb 3 -Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb 3 -Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb 3 -Cox assembly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Defects in the Expression of Chloroplast Proteins Leads to H2O2 Accumulation and Activation of Cyclic Electron Flow around Photosystem I

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H 2 O 2 . Surprisingly, hcef2 was mapped to a nonsense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash induced thylakoid electric field suggest that these defect lead to accumulation of H 2 O 2 in hcef2, which we have previously shown leads to activation of NDHrelated CEF. We observed similar increases in CEF and H 2 O 2 accumulation in other translation defective mutants, suggesting that loss of coordination in plastid protein levels lead to imbalances in the photosynthetic energy balance that leads to increased CEF. These results, together with a large body of previous observations, support a general model in which processes that imbalances in chloroplast energetics result in the production of H 2 O 2 , which activates CEF, either as a redox signal or by inducing deficits in ATP levels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Elucidation of Triacylglycerol Overproduction in the C4 Bioenergy Crop Sorghum bicolor by Constraint-Based Analysis

Upregulation of triacylglycerols (TAGs) in vegetative plant tissues such as leaves has the potential to drastically increase the energy density and biomass yield of bioenergy crops. In this context, constraint-based analysis has the promise to improve metabolic engineering strategies. Here we present a core metabolism model for the C 4 biomass crop Sorghum bicolor ( iTJC1414 ) along with a minimal model for photosynthetic CO 2 assimilation, sucrose and TAG biosynthesis in C 3 plants. Extending iTJC1414 to a four-cell diel model we simulate C 4 photosynthesis in mature leaves with the principal photo-assimilatory product being replaced by TAG produced at different levels. Independent of specific pathways and per unit carbon assimilated, energy content and biosynthetic demands in reducing equivalents are about 1.3 to 1.4 times higher for TAG than for sucrose. For plant generic pathways, ATP- and NADPH-demands per CO 2 assimilated are higher by 1.3- and 1.5-fold, respectively. If the photosynthetic supply in ATP and NADPH in iTJC1414 is adjusted to be balanced for sucrose as the sole photo-assimilatory product, overproduction of TAG is predicted to cause a substantial surplus in photosynthetic ATP. This means that if TAG synthesis was the sole photo-assimilatory process, there could be an energy imbalance that might impede the process. Adjusting iTJC1414 to a photo-assimilatory rate that approximates field conditions, we predict possible daily rates of TAG accumulation, dependent on varying ratios of carbon partitioning between exported assimilates and accumulated oil droplets (TAG, oleosin) and in dependence of activation of futile cycles of TAG synthesis and degradation. We find that, based on the capacity of leaves for photosynthetic synthesis of exported assimilates, mature leaves should be able to reach a 20% level of TAG per dry weight within one month if only 5% of the photosynthetic net assimilation can be allocated into oil droplets. From this we conclude that high TAG levels should be achievable if TAG synthesis is induced only during a final phase of the plant life cycle.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique

Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use of harsh chemicals and enzymes, which interfere with detection assays. In this work, a miniature acoustic nucleic acid extraction system is presented. Using a miniature bulk acoustic wave (BAW) transducer array based on 36° Y-cut lithium niobate, acoustic waves were coupled into disposable laminate-based microfluidic cartridges. To verify the lysing effectiveness, the amount of liberated ATP and the cell viability were measured and compared to untreated samples. The relationship between input power, energy dose, flow-rate, and lysing efficiency were determined. DNA was purified on-chip using three approaches implemented in the cartridges: a silica-based sol-gel silica-bead filled microchannel, nucleic acid binding magnetic beads, and Nafion-coated electrodes. Using E. coli, the lysing dose defined as ATP released per joule was 2.2× greater, releasing 6.1× more ATP for the miniature BAW array compared to a bench-top acoustic lysis system. An electric field-based nucleic acid purification approach using Nafion films yielded an extraction efficiency of 69.2% in 10 min for 50 µL samples.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Redox Poise during Rhodospirillum rubrum Phototrophic Growth Drives Large-scale Changes in Macromolecular Synthesis Pathways

During photoheterotrophic growth on organic substrates, purple nonsulfur photosynthetic bacteria like Rhodospirillum rubrum can acquire electrons by multiple means, including oxidation of organic substrates, oxidation of inorganic electron donors (e.g., H2), and by reverse electron flow from the photosynthetic electron transport chain. These electrons are stored as reduced electron-carrying cofactors (e.g., NAD(P)H and ferredoxin). The overall ratio of oxidized to reduced cofactors (e.g., NAD(P)+:NAD(P)H), or ’redox poise’, is difficult to understand or predict, as are the cellular processes for dissipating these reducing equivalents. Using physics-based models that capture mass action kinetics consistent with the thermodynamics of reactions and pathways, a range of redox conditions for heterophototrophic growth are evaluated, from conditions in which the NADP+/NADPH levels approach thermodynamic equilibrium to conditions in which the NADP+/NADPH ratio is far above the typical physiological values. Modeling predictions together with experimental measurements indicate that the redox poise of the cell results in large-scale changes in the activity of biosynthetic pathways and, thus, changes in cell macromolecule levels (DNA, RNA, proteins, and fatty acids). Furthermore, modeling predictions indicate that during phototrophic growth, reverse electron flow from the quinone pool is a minor contributor to the production of reduced cofactors (e.g., NAD(P)H) compared to other oxidative processes (H2 and carbon substrate oxidation). Instead, the quinone pool primarily operates to aid ATP production. The high level of ATP, in turn, drives reduction processes even when NADPH levels are relatively low compared to NADP+ by coupling ATP hydrolysis to the reductive processes. The model, in agreement with experimental measurements of macromolecule ratios of cells growing on different carbon substrates, indicates that the dynamics of nucleotide versus lipid and protein production is likely a significant mechanism of balancing oxidation and reduction in the cell.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

High-resolution structures of kinesin on microtubules provide a basis for nucleotide-gated force-generation

Microtubule-based transport by the kinesin motors, powered by ATP hydrolysis, is essential for a wide range of vital processes in eukaryotes. We obtained insight into this process by developing atomic models for no-nucleotide and ATP states of the monomeric kinesin motor domain on microtubules from cryo-EM reconstructions at 5–6 Å resolution. By comparing these models with existing X-ray structures of ADP-bound kinesin, we infer a mechanistic scheme in which microtubule attachment, mediated by a universally conserved ‘linchpin’ residue in kinesin (N255), triggers a clamshell opening of the nucleotide cleft and accompanying release of ADP. Binding of ATP re-closes the cleft in a manner that tightly couples to translocation of cargo, via kinesin's ‘neck linker’ element. These structural transitions are reminiscent of the analogous nucleotide-exchange steps in the myosin and F1-ATPase motors and inform how the two heads of a kinesin dimer ‘gate’ each other to promote coordinated stepping along microtubules.

Life Sciences & Biomedicine - Other Topics↗
DOE OSTI2015

Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication

To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant ‘Cdc6-E224Q’ promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Structural basis for subtype-specific inhibition of the P2X7 receptor

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1974

Improved methods for counting bacteria in physiological fluids

Bacterial population detection is based on detection of adenosine triphosphate (ATP), chemical present in all living matter. Amount of ATP in sample, after chemically removing all nonbacterial ATP, is directly related to bacterial population. Sensitivity is improved by concentration step; specificity is improved by lowering pH of solution.

Picciolo, G. L.↗
NASA NTRS1975

Bioluminescence for determining energy state of plants

Bioluminescence produced by the luciferin-luciferase system is a very sensitive assay for ATP content in extracts of plant materials. The ATP test for seed and pollen viability and vigor is presented, along with prediction of high growth potential and productivity in new crosses and selections of breeding materials. ATP as an indicator for environmental quality, stresses, and metabolic regulation is also considered.

Ching, T. M.↗
NASA NTRS1976

Method of detecting and counting bacteria

An improved method is provided for determining bacterial levels, especially in samples of aqueous physiological fluids. The method depends on the quantitative determination of bacterial adenosine triphosphate (ATP) in the presence of nonbacterial ATP. The bacterial ATP is released by cell rupture and is measured by an enzymatic bioluminescent assay. A concentration technique is included to make the method more sensitive. It is particularly useful where the fluid to be measured contains an unknown or low bacteria count.

Picciolo, G. L.↗
NASA NTRS1979

Determination of antimicrobial susceptibilities on infected urines without isolation

A method is described for the quick determination of the susceptibilities of various unidentified bacteria contained in an aqueous physiological fluid sample, particularly urine, to one or more antibiotics. A bacterial adenosine triphosphate (ATP) assay is carried out after the elimination of non-bacterial ATP to determine whether an infection exists. If an infection does exist, a portion of the sample is further processed, including subjecting parts of the portion to one or more antibiotics. Growth of the bacteria in the parts are determined, again by an ATP assay, to determine whether the unidentified bacteria in the sample are susceptible to the antibiotic or antibiotics under test.

Picciolo, G. L.↗
NASA NTRS1980

Effect of immobilization stress on the level of macroergic phosphates in the blood of rats

The effect of immobilization for 1, 2, and 24 hours, and of daily (for 2 hours for 7 days) immobilization on the blood nucleotide (ATP, ADP, AMP) level was studied on 79 male rats. A progressive reduction of the ATP content was most pronounced in immobilization for 24 hours. This was accompanied by an increase of lymphopenia and eosinopenia. A fall of the relative weight of the thymus and a weight gain of the adrenal glands was observed along with a reduction of ascorbic acid concentration in both of them. In case of daily immobilization for 2 hours, the ATP and ADP content on the 1st and 2nd day was below the normal level, and then showed a gradual increase, with complete normalization in 6-7 days, except for inorganic phosphorus the level of which remained lower than normal.

Pudov, V. I.↗
NASA NTRS1980

A quinone-assisted photoformation of energy-rich chemical bonds

In a study of biochemical means of solar energy conversion, ADP and inorganic phosphates were converted to ATP by white light in the nonaqueous solvent dimethylformamide in the presence of tetrachloro-p-quinone or ubiquinone. Conversion of ADP to ATP has been accomplished in aqueous suspension by the use of cell-like structures aggregated from poly(aspartic acid, glutamic acid, tyrosine). This is believed to occur through the formation of dopaquinone in the peptide structure during illumination. The way in which the quantitative yield of ATP has been influenced by pH and by added substances, such as FeCl2, was studied.

Fox, S. W.↗
NASA NTRS1980

The effects of cutting or of stretching skeletal muscle in vitro on the rates of protein synthesis and degradation

Skeletal muscle preparations using cut muscle fibers have often been used in studies of protein metabolism. The present paper reports an investigation of the effect of muscle cutting or stretching in vitro on the rates of protein synthesis and/or degradation. Protein synthesis and content, and ATP and phosphocreatine levels were monitored in soleus and extensor digitorum longus muscles from the rat with various extents of muscle fiber cuts and following stretching to about 120% the resting length. Rates of protein synthesis are found to be significantly lower and protein degradation higher in the cut muscles than in uncut controls, while ATP and phosphocreatine concentrations decreased. Stretched intact muscles, on the other hand, are observed to have higher concentrations of high-energy phosphates than unstretched muscles, while rates of protein degradation were not affected. Results thus demonstrate that the cutting of skeletal muscle fibers alters many aspects of muscle metabolism, and that moderate decreases in ATP concentration do not alter rates of protein concentration in intact muscles in vitro.

Seider, M. J.↗
NASA NTRS1986

Models of glycolysis: Glyceraldehyde as a source of energy and monomers for prebiotic condensation reactions

All organisms require energy in a chemical form for maintenance and growth. In contemporary life this chemical energy is obtained by the synthesis of the phosphoanhydride bonds of ATP. Among the biological processes that yield ATP, fermentation is generally considered primitive, because it operates under anaerobic conditions by substrate-level phosphorylation which does not require compartmentation by membranes. Fermentation by the glycolytic pathway, which is found in almost every living cell, is an especially attractive energy source for primitive life. Glycolysis not only produces useful chemical energy (ATP), but intermediates of this pathway are also involved in amino acid synthesis and photosynthetic carbon-fixation. It is believed that energy and substrates needed for the origin of life were provided by nonenzymatic chemical reactions that resemble the enzyme-mediated reactions of glycolysis. These nonenzymatic reactions would have provided a starting point for the evolutionary development of glycolysis.

Weber, A. L.↗