SEARCH · Search NASA

Results for “formate dehydrogenase”

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.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

11 records

All sources NASA NTRS DOE OSTI

NASA NTRS2002

Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity

A comprehensive assessment of lignin configuration in transgenic and mutant plants is long overdue. This review thus undertook the systematic analysis of trends manifested through genetic and mutational manipulations of the various steps associated with monolignol biosynthesis; this included consideration of the downstream effects on organized lignin assembly in the various cell types, on vascular function/integrity, and on plant growth and development. As previously noted for dirigent protein (homologs), distinct and sophisticated monolignol forming metabolic networks were operative in various cell types, tissues and organs, and form the cell-specific guaiacyl (G) and guaiacyl-syringyl (G-S) enriched lignin biopolymers, respectively. Regardless of cell type undergoing lignification, carbon allocation to the different monolignol pools is apparently determined by a combination of phenylalanine availability and cinnamate-4-hydroxylase/"p-coumarate-3-hydroxylase" (C4H/C3H) activities, as revealed by transcriptional and metabolic profiling. Downregulation of either phenylalanine ammonia lyase or cinnamate-4-hydroxylase thus predictably results in reduced lignin levels and impaired vascular integrity, as well as affecting related (phenylpropanoid-dependent) metabolism. Depletion of C3H activity also results in reduced lignin deposition, albeit with the latter being derived only from hydroxyphenyl (H) units, due to both the guaiacyl (G) and syringyl (S) pathways being blocked. Apparently the cells affected are unable to compensate for reduced G/S levels by increasing the amounts of H-components. The downstream metabolic networks for G-lignin enriched formation in both angiosperms and gymnosperms utilize specific cinnamoyl CoA O-methyltransferase (CCOMT), 4-coumarate:CoA ligase (4CL), cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) isoforms: however, these steps neither affect carbon allocation nor H/G designations, this being determined by C4H/C3H activities. Such enzymes thus fulfill subsidiary processing roles, with all (except CCOMT) apparently being bifunctional for both H and G substrates. Their severe downregulation does, however, predictably result in impaired monolignol biosynthesis, reduced lignin deposition/vascular integrity, (upstream) metabolite build-up and/or shunt pathway metabolism. There was no evidence for an alternative acid/ester O-methyltransferase (AEOMT) being involved in lignin biosynthesis.The G/S lignin pathway networks are operative in specific cell types in angiosperms and employ two additional biosynthetic steps to afford the corresponding S components, i.e. through introduction of an hydroxyl group at C-5 and its subsequent O-methylation. [These enzymes were originally classified as ferulate-5-hydroxylase (F5H) and caffeate O-methyltransferase (COMT), respectively.] As before, neither step has apparently any role in carbon allocation to the pathway; hence their individual downregulation/manipulation, respectively, gives either a G enriched lignin or formation of the well-known S-deficient bm3 "lignin" mutant, with cell walls of impaired vascular integrity. In the latter case, COMT downregulation/mutation apparently results in utilization of the isoelectronic 5-hydroxyconiferyl alcohol species albeit in an unsuccessful attempt to form G-S lignin proper. However, there is apparently no effect on overall G content, thereby indicating that deposition of both G and S moieties in the G/S lignin forming cells are kept spatially, and presumably temporally, fully separate. Downregulation/mutation of further downstream steps in the G/S network [i.e. utilizing 4CL, CCR and CAD isoforms] gives predictable effects in terms of their subsidiary processing roles: while severe downregulation of 4CL gave phenotypes with impaired vascular integrity due to reduced monolignol supply, there was no evidence in support of increased growth and/or enhanced cellulose biosynthesis. CCR and CAD downregulation/mutations also established that a depletion in monolignol supply reduced both lignin contents supply reduced both lignin contents and vascular integrity, with a concomitant shift towards (upstream) metabolite build-up and/or shunting.The extraordinary claims of involvement of surrogate monomers (2-methoxybenzaldehyde, feruloyl tyramine, vanillic acid, etc.) in lignification were fully disproven and put to rest, with the investigators themselves having largely retracted former claims. Furthermore analysis of the well-known bm1 mutation, a presumed CAD disrupted system, apparently revealed that both G and S lignin components were reduced. This seems to imply that there is no monolignol specific dehydrogenase, such as the recently described sinapyl alcohol dehydrogenase (SAD) for sinapyl alcohol formation. Nevertheless, different CAD isoforms of differing homology seem to be operative in different lignifying cell types, thereby giving the G-enriched and G/S-enriched lignin biopolymers, respectively. For the G-lignin forming network, however, the CAD isoform is apparently catalytically less efficient with all three monolignols than that additionally associated with the corresponding G/S lignin forming network(s), which can more efficiently use all three monolignols. However, since CAD does not determine either H, G, or S designation, it again serves in a subsidiary role-albeit using different isoforms for different cell wall developmental and cell wall type responses.The results from this analysis contrasts further with speculations of some early investigators, who had viewed lignin assembly as resulting from non-specific oxidative coupling of monolignols and subsequent random polymerization. At that time, though, the study of the complex biological (biochemical) process of lignin assembly had begun without any of the (bio)chemical tools to either address or answer the questions posed as to how its formation might actually occur. Today, by contrast, there is growing recognition of both sophisticated and differential control of monolignol biosynthetic networks in different cell types, which serve to underscore the fact that complexity of assembly need not be confused any further with random formation. Moreover, this analysis revealed another factor which continues to cloud interpretations of lignin downregulation/mutational analyses, namely the serious technical problems associated with all aspects of lignin characterization, whether for lignin quantification, isolation of lignin-enriched preparations and/or in determining monomeric compositions. For example, in the latter analyses, some 50-90% of the lignin components still cannot be detected using current methodologies, e.g. by thioacidolysis cleavage and nitrobenzene oxidative cleavage. This deficiency in lignin characterization thus represents one of the major hurdles remaining in delineating how lignin assembly (in distinct cell types) and their configuration actually occurs.

Review, Academic↗

NASA NTRS1990

Cosmos 2044

The effects of microgravity and hind limb suspension on the enzyme patterns are assessed within a slow twitch muscle (soleus) and a fast twitch muscle (tibialis anterior). Studies were made on 95 soleus fibers and about 300 tibialis anterior (TA) fibers. Over 2200 individual enzyme measurements were made. Six key metabolic enzymes (hexokinase, pyruvate kinease, citrate kinase, beta-hydroxyacyl CoA dehydrogenase, glucose-6-P dehydrogenase, and aspartate aminotransferase) plus glutaminase and glutamate decarboxylase, as well as glutamate, aspartate, and GABA, were measured in 11 regions of the hippocampal formation of synchronous, flight, and tail suspension rats. Major differences were observed in the normal distribution of each enzyme and amine acid, but no substantive effects of either microgravity or tail suspension on these patterns were clearly demonstrated.

Lowry, Oliver H.↗

NASA NTRS2002

Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism

Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two monolignols, p-coumaryl and coniferyl alcohols (lignin/lignan precursors). When the cells were transferred to a medium containing 8% sucrose and 20 mm potassium iodide, the monolignol/phenylpropanoid pathway was induced, and transcript levels for phenylalanine ammonia lyase, cinnamate 4-hydroxylase, p-coumarate 3-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase, and cinnamyl alcohol dehydrogenase were coordinately up-regulated. Provision of increasing levels of exogenously supplied Phe to saturating levels (40 mm) to the induction medium resulted in further up-regulation of their transcript levels in the P. taeda cell cultures; this in turn was accompanied by considerable increases in both p-coumaryl and coniferyl alcohol formation and excretion. By contrast, transcript levels for both cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase were only slightly up-regulated. These data, when considered together with metabolic profiling results and genetic manipulation of various plant species, reveal that carbon allocation to the pathway and its differential distribution into the two monolignols is controlled by Phe supply and differential modulation of cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase activities, respectively. The coordinated up-regulation of phenylalanine ammonia lyase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase in the presence of increasing concentrations of Phe also indicates that these steps are not truly rate-limiting, because they are modulated according to metabolic demand. Finally, the transcript profile of a putative acid/ester O-methyltransferase, proposed as an alternative catalyst for O-methylation leading to coniferyl alcohol, was not up-regulated under any of the conditions employed, suggesting that it is not, in fact, involved in monolignol biosynthesis.

NASA Discipline Plant Biology↗

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 NTRS1996

Effects of substrates and phosphate on INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli

The effects of substrates of primary aerobic dehydrogenases, and inorganic phosphate on aerobic INT and CTC reduction in Escherichia coli were examined. In general, INT produced less formazan than CTC, but INT (+) cell counts remained near values of CTC (+) cells. INT and CTC (+) cell numbers were higher than plate counts on R2A medium using succinate, formate, lactate, casamino acids, glucose, glycerol (INT only) and no substrate. Formate resulted in the greatest amount of INT and CTC formazan. Reduction of both INT and CTC was inhibited above 10 mmol l-1 phosphate, and this appeared to be related to decreased rates of O2 consumption. Formation of fluorescent CTC (+), but not INT (+) cells was also inhibited in a concentration dependent manner by phosphate above 10 mmol l-1. From light microscopic observations it appeared CTC formed increasing amounts of poorly or non-fluorescent formazan with increasing phosphate. Therefore, use of phosphate buffer in excess of 10 mmol l-1 may not be appropriate in CTC and INT reduction assays.

NASA Program Environmental Health↗

NASA NTRS1999

Gravity and Skeletal Growth

Two simultaneous experiments were performed using 5-week-old male Sprague Dawley rats; in one study, the rats were flown in low earth orbit; in the other study, the hindlimbs of the growing rats were elevated to prevent weight bearing. Following 9 d of unloading, weight bearing was restored for 4, 28, and 76 hrs. Afterwards, additional hindlimb unloading experiments were performed to evaluate the skeletal response to 0, 2, 4, 6, 8, 10, 12, 16, and 24 hrs of restored weight bearing following 7 d of unloading. Cancellous and cortical bone histomorphometry were evaluated in the left tibia at the proximal metaphysis and in the left femur at mid-diaphysis, respectively. Steady-state mRNA levels for bone matrix proteins and skeletal signaling peptides were determined in total cellular RNA extracted from trabeculae from the right proximal tibiametaphysis and periosteum from the right femur. Spaceflight and hindlimb unloading each resulted in cancellous osteopenia, as well as a tendency towards decreased periosteal bone formation. Both models for skeletal unloading resulted in site specific reductions in mRNA levels for transforming growth factor-beta (sub 1) (TGF-beta) osteocalcin (OC), and prepro-alpha (I) subunit of type 1 collagen (collagen) and little or no changes in mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAP) and insulin-like growth factor I (IGF-I). Restoration of normal weight bearing resulted in transient increases in mRNA levels for the bone matrix proteins and TGF-beta in the proximal metaphysis and periosteum and no changes in either GAP or IGF-I mRNA levels. The timecourse for the response differed between the two skeletal compartments; the tibial metaphysis responded much more quickly to reloading. These results suggest that the skeletal adaptation to acute physiological changes in mechanical usage are mediated, in part, by changes in mRNA levels for bone matrix proteins and TGF-beta.

Morey-Holton, Emily↗

NASA NTRS1974

Application of nitrogen metabolism in autotrophic bacteria to chemosynthetic bioregeneration in space missions, supplement

The chemolithotroph, Hydrogenomonas eutropha, was considered as a life support, bioregenerative system. This project focuses on several metabolic functions that are related to the proposed nitrogen cycle between man and this microbe. Specifically this organism has the capability to utilize as the sole nitrogen source such urine components as urea and fifteen individual amino acids, but not nine other amino acids. The effectiveness of utilization was high for many amino acids. Several specific growth inhibitions were also observed. The enzyme that catalyzes the incorporation of ammonia in the medium into amino acids was identified as a NADP-specific, L-glutamate dehydrogenase. This enzyme has a constitutive nature. This organism can synthesize all of its amino acids from carbon dioxide and ammonia. Therefore with the background literature of multiple pathways of individual amino acid biosyntheses, our evidence to date is consistent with the Hydrogeneomonas group having the same pathway of valine-isoleucine formation as the classical E. coli.

Wixom, R. L.↗

NASA NTRS2000

Induced phenylpropanoid metabolism during suberization and lignification: a comparative analysis

Induction of the biosynthesis of phenylpropanoids was monitored at the enzyme level through measurement of the temporal change in the activity of two marker enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase, (PAL, E.C. 4.1.3.5) and 4-coumaryl-CoA ligase (4-CL, E.C. 6.2.1.12) and two marker enzymes for hydroxycinnamyl alcohol biosynthesis, cinnamoyl-CoA:NADP+ oxidoreductase (CCR, E.C. 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) in both suberizing potato (Solanum tuberosum) tubers and lignifying loblolly pine (Pinus taeda) cell cultures. While measurable activities of PAL, 4-CL and CAD increased upon initiation of suberization in potato tubers, that of CCR did not. By contrast, all four enzymes were induced upon initiation of lignification in pine cell cultures. The lack of CCR induction in potato by wound treatment is consistent with the channelling of hydroxycinnamoyl-CoA derivatives away from monolignol formation and toward other hydroxycinnamoyl derivatives such as those that accumulate during suberization.

Non-NASA Center↗

NASA NTRS1998

Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

short duration↗

NASA NTRS2002

Study of Fluid Flow Control In Protein Crystallization Using Strong Magnetic Fields

Ramachandran, N.↗

NASA NTRS2002

Study of Fluid Flow Control in Protein Crystallization using Strong Magnetic Fields

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in "microgravity", researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. Once the basic understanding is obtained, the study will focus on testing the hypothesis on proteins of pyruvate dehydrogenase complex (PDC), proteins E1 and E3. Obtaining high crystal quality of these proteins is of great importance to structural biologists since their structures need to be determined. Specific goals for the investigation are: 1. To develop an understanding of convection control in diamagnetic fluids with concentration gradients through experimentation and numerical modeling. Specifically solutal buoyancy driven convection due to crystal growth will be considered. 2. To develop predictive measures for successful crystallization in a magnetic field using analyses and numerical modeling for use in future protein crystal growth experiments. This will establish criteria that can be used to estimate the efficacy of magnetic field flow damping on crystallization of candidate proteins. 3. To demonstrate the understanding of convection damping by high magnetic fields to a class of proteins that is of interest and whose structure is as yet not determined. 4. To compare quantitatively, the quality of the grown crystals with and without a magnetic field. X-ray diffraction techniques will be used for the comparative studies. In a preliminary set of experiments, we studied crystal dissolution effects in a 5 Tesla magnet available at NASA Marshall Space Flight Center (MSFC). Using a Schlieren setup, a 1mm crystal of Alum (Aluminum-Potassium Sulfate) was introduced in a 75% saturated solution and the resulting dissolution plume was observed. The experiment was conducted both in the presence and absence of a magnetic field gradient. The magnet produces a gradient field of approx. 1 Tesla2/cm. Image analysis of the recorded images indicated an enhanced plume velocity that was of the order of the measurement limit. For this experiment, both the gradient and gravity fields are in the same direction resulting in an enhanced effective gravity that tends to accelerate the observed plume velocity. While the results are not conclusive, pending further tests, it clearly points out the inadequacy of the MSFC magnet for conducting protein crystallization experiments and the need for a stronger magnet. In spacebased experiments, however, where the gravitational effects are small, only a weak magnetic field will be required to control or mitigate the effects of convective contamination.

Ramachandran, Narayanan↗