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

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At least 847 records · Page 47
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Roles for Countercharge in the Voltage Sensor Domain of Ion Channels

Voltage-gated ion channels share a common structure typified by peripheral, voltage sensor domains. Their S4 segments respond to alteration in membrane potential with translocation coupled to ion permeation through a central pore domain. The mechanisms of gating in these channels have been intensely studied using pioneering methods such as measurement of charge displacement across a membrane, sequencing of genes coding for voltage-gated ion channels, and the development of all-atom molecular dynamics simulations using structural information from prokaryotic and eukaryotic channel proteins. One aspect of this work has been the description of the role of conserved negative countercharges in S1, S2, and S3 transmembrane segments to promote sequential salt-bridge formation with positively charged residues in S4 segments. These interactions facilitate S4 translocation through the lipid bilayer. In this review, we describe functional and computational work investigating the role of these countercharges in S4 translocation, voltage sensor domain hydration, and in diseases resulting from countercharge mutations.

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

Rapid tachyphylaxis to hemodynamic effects of PACAP-27 after inhibition of nitric oxide synthesis

The vasodilator effects of pituitary adenylate cyclase-activating polypeptide (PACAP)-27 are subject to tachyphylaxis in rats treated with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). We examined whether this tachyphylaxis could be prevented by administration of the putative endothelium-derived nitrosyl factor S-nitroso-L-cysteine (L-SNC) and whether L-SNC may exert its effects via increases in cGMP levels in vascular smooth muscle. Five doses of PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats. These responses were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME-treated (50 micromol/kg iv) rats produced vasodilator responses similar to those in saline-treated rats, whereas subsequent injections produced progressively smaller responses. The injection of L-SNC (1,200 nmol/kg iv) before each injection of PACAP-27 prevented tachyphylaxis to the Gs protein-coupled receptor agonist in L-NAME-treated rats, whereas equihypotensive doses of the NO donor sodium nitroprusside (100 micrograms/kg iv) did not. The injection of the membrane-permeant cGMP analog 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-CPT-cGMP; 30 micromol/kg iv) to L-NAME-treated rats restored resting hemodynamic values to pre-L-NAME levels but did not prevent the development of tachyphylaxis to PACAP-27. These results suggest that nitrosyl factors prevent the development of tachyphylaxis to the hemodynamic actions of PACAP-27. These nitrosyl factors may act independently of their ability to generate cGMP in vascular smooth muscle.

NASA Discipline Regulatory Physiology↗
DOE OSTI2025

From cytoplasm to lumen—mapping the free pools of protein subunits of three photosynthetic complexes using quantitative mass spectrometry

The phycobilisome (PBS) captures light energy and transfers it to photosystem I (PSI) and photosystem II (PSII). Which and how many copies of protein subunits in PBSs, PSI, and PSII remain unbound in thylakoids are unknown. Here, quantitative mass spectrometry (QMS) was used to quantify substantial pools of free extrinsic subunits of PSII and PSI. Interestingly, the membrane intrinsic PsaL is 3-fold higher than PsaA/B. This scenario complements the static structures of these complexes as revealed by X-ray crystallography and cryo-EM. Furthermore, the ratios of ApcG and photoprotective OCP over PBS indicate a pool of extra ApcG. The 2.5 ratio of CpcG-PBS over CpcL-PBS improves our understanding of these light-harvesting complexes involved in energy capture and photoprotection in cyanobacteria.

cyanobacteria↗
DOE OSTI2021

PLEKHA7 signaling is necessary for the growth of mutant KRAS driven colorectal cancer

Plekha7 (Pleckstrin homology [PH] domain containing, family A member 7) regulates the assembly of proteins of the cytoplasmic apical zonula adherens junction (AJ), thus ensuring cell-cell adhesion and tight-junction barrier integrity. Little is known of Plekha7 function in cancer. In colorectal cancer (CRC) Plekha7 expression is elevated compared to adjacent normal tissue levels, increasing with clinical stage. Plekha7 was present at plasma membrane AJ with wild-type KRas (wt-KRas) but was dispersed in cells expressing mutant KRas (mut-KRas). Fluorescence lifetime imaging microscopy (FLIM) indicated a direct Plekha7 interaction with wt-KRas but scantily with mut-KRas. Inhibiting Plekha7 specifically decreased mut-KRas cell signaling, proliferation, attachment, migration, and retarded mut-KRAS CRC tumor growth. Binding of diC8-phosphoinositides (PI) to the PH domain of Plekha7 was relatively low affinity. This may be because a D175 amino acid residue plays a “sentry” role preventing PI(3,4)P{sub 2} and PI(3,4,5)P{sub 3} binding. Molecular or pharmacological inhibition of the Plekha7 PH domain prevented the growth of mut-KRas but not wt-KRas cells. Taken together the studies suggest that Plekha7, in addition to maintaining AJ structure plays a role in mut-KRas signaling and phenotype through interaction of its PH domain with membrane mut-KRas, but not wt-KRas, to increase the efficiency of mut-KRas downstream signaling.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2012

Quality Control Method for a Micro-Nano-Channel Microfabricated Device

A variety of silicon-fabricated devices is used in medical applications such as drug and cell delivery, and DNA and protein separation and analysis. When a fluidic device inlet is connected to a compressed gas reservoir, and the outlet is at a lower pressure, a gas flow occurs through the membrane toward the outside. The method relies on the measurement of the gas pressure over the elapsed time inside the upstream and downstream environments. By knowing the volume of the upstream reservoir, the gas flow rate through the membrane over the pressure drop can be calculated. This quality control method consists of measuring the gas flow through a device and comparing the results with a standard curve, which can be obtained by testing standard devices. Standard devices can be selected through a variety of techniques, both destructive and nondestructive, such as SEM, AFM, and standard particle filtration.

Grattoni, Alessandro↗
NASA NTRS1993

Monoclonal Antibody Testing for Cancer Metastasis

Malignant cells are characterized by the ability to invade surrounding normal tissues. Tumor invasion is abetted by proteolytic enzymes that have been correlated with recurrent disease and metastasis. These enzymes are involved in a cascade of proteolytic interactions with other enzymes and inhibitors which allow cancer cells to dissolve surrounding extracellular matrix, thereby enabling the cells to rapidly invade adjacent tissues and migrate to metastatic sites distant from the primary tumor. Among these proteases are the plasminogen activators (PA), collagenase IV, faminase, and in some cases cathepsin D, which together mediate key steps in the invasion process of metastasis. Cells which have the selective advantage for invasion and metastasis are those capable of regulating their proteolytic activity and proliferation. Cells in the process of invasion would be probably down-regulated for proliferation, but subsequent to attachment and adhesion at a distant site, would then be in a proliferative mode, up-regulating DNA replication. Urokinase (uPA) can be present in the tissues in several molecular forms. The inactive proenzyme is a single chain protein (scuPA) that is cleaved at Lys. 158 to form the double chain, high molecular weight active form (HMW-uPA) of 54 kD. A low molecular weight form (LMW-uPA) can also be produced by cleavage of the HMW-U PA at Lys. 135 - Lys. 136 giving a 35 kD active enzyme. Recently, it has been shown that the HMW active form of urokinase, bound to the tumor cell membrane, is responsible for the local lysis of the extracellular matrix, hence the tissue invasion mechanism for metastasis (Andreasen et al, 19861. Receptor- (membrane) bound uPA is twice as efficient (catalytically) as free fluid-phase uPA. Tho unbound uPA and the LMW form is not responsible for most of the local dissolution of extracellular matrix in the immediate vicinity of the metastatic tumor cell. High levels of urokinase (greater than 3.49 ng/mg of total protein) extracted from breast tumor tissues have recently been shown, together with plasminogen activator inhibitor 1 (PAI-1), to be a good prognostic indicator for high risk of recurrence and shorter patient survival times. In this project, we have attempted to develop immunocytochemical methodologies for the clinical assessment of the expression of urokinase plasminogen activator, which has been implicated to be important for initial steps in tumor invasion, and to relate it to cell proliferation and DNA replication at the single-cell level.

Source record↗
NASA NTRS1989

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.

NASA Discipline Number 61-10↗
NASA NTRS2003

Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

Non-NASA Center↗
DOE OSTI2022

Effect of Foot-and-Mouth Disease Virus 2B Viroporin on Expression and Extraction of Mammalian Cell Culture Produced Foot-and-Mouth Disease Virus-like Particles

To improve the production of foot-and-mouth disease (FMD) molecular vaccines, we sought to understand the effects of the FMD virus (FMDV) 2B viroporin in an experimental, plasmid-based, virus-like particle (VLP) vaccine. Inclusion of the FMDV viroporin 2B into the human Adenovirus 5 vectored FMD vaccine enhanced transgene expression despite independent 2B expression negatively affecting cell viability. Evaluating both wildtype 2B and mutants with disrupted viroporin activity, we confirmed that viroporin activity is detrimental to overall transgene expression when expressed independently. However, the incorporation of 2B into an FMD molecular vaccine construct containing a wildtype FMDV 3C protease, a viral encoded protease responsible for processing structural proteins, resulted in enhancement of transgene expression, validating previous observations. This benefit to transgene expression was negated when using the FMDV 3CL127P mutant, which has reduced processing of host cellular proteins, a reversion resulting from 2B viroporin activity. Inclusion of 2B into VLP production constructs also adversely impacted antigen extraction, a possible side effect of 2B-dependent rearrangement of cellular membranes. These results demonstrate that inclusion of 2B enhanced transgene expression when a wildtype 3C protease is present but was detrimental to transgene expression with the 3CL127P mutant. This has implications for future molecular FMD vaccine constructs, which may utilize mutant FMDV 3C proteases.

2B↗
NASA NTRS1998

Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat)

Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space-flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post-flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time-dependent increase of the apoptosis-associated Fas/ APO-1 protein in static flown, but not the in-flight 1 g centrifuged or ground controls, confirmed microgravity-associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space-flown lymphocytes are concomitant with increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.

STS-76 Shuttle Project↗
NASA NTRS1991

Palmitate stimulates glucose transport in rat adipocytes by a mechanism involving translocation of the insulin sensitive glucose transporter (GLUT4)

In rat adipocytes, palmitate: a) increases basal 2-deoxyglucose transport 129 +/- 27% (p less than 0.02), b) decreases the insulin sensitive glucose transporter (GLUT4) in low density microsomes and increases GLUT4 in plasma membranes and c) increases the activity of the insulin receptor tyrosine kinase. Palmitate-stimulated glucose transport is not additive with the effect of insulin and is not inhibited by the protein kinase C inhibitors staurosporine and sphingosine. In rat muscle, palmitate: a) does not affect basal glucose transport in either the soleus or epitrochlearis and b) inhibits insulin-stimulated glucose transport by 28% (p less than 0.005) in soleus but not in epitrochlearis muscle. These studies demonstrate a potentially important differential role for fatty acids in the regulation of glucose transport in different insulin target tissues.

NASA Discipline Cell Biology↗
DOE OSTI2025

Abbr. Final report: Self-assembled molecular containers as artificial water channels: towards biomimetic desalination membranes

Global water scarcity demands advances in desalination technologies that can deliver more fresh water with less energy. Current reverse osmosis membranes are fundamentally limited by a trade-off between how much water they can pass and how well they block salts. To address this challenge, we developed a bottom-up strategy to design and test artificial water channels that mimic the efficiency of biological proteins but are built from robust synthetic molecules. Over two years, we synthesized and evaluated more than twenty molecular channel candidates, including supramolecular macrocycles and nanographene pores with atomically precise structures. We showed that small chemical modifications allow direct control over pore size and chemistry, which in turn govern water permeability and salt rejection. In collaboration with university partners, we reported the first experimental demonstration of water transport through a nanographene pore, bridging a long-standing gap between simulation and experiment. Several of the artificial channels we developed achieved water–salt selectivity beyond conventional polymer membranes, highlighting their potential for next-generation desalination and precision separations.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Tissue distribution and subcellular localization of the family of Kidney Ankyrin Repeat Domain (KANK) proteins

Kidney Ankyrin Repeat-containing Proteins (KANKs) comprise a family of four evolutionary conserved proteins (KANK1 to 4) that localize to the belt of mature focal adhesions (FAs) where they regulate integrin-mediated adhesion, actomyosin contractility, and link FAs to the cortical microtubule stabilization complex (CMSC). The human KANK proteins were first identified in kidney and have been associated with kidney cancer and nephrotic syndrome. Here, we report the distributions and subcellular localizations of the four Kank mRNAs and proteins in mouse tissues. We found that the KANK family members display distinct and rarely overlapping expression patterns. Whereas KANK1 is expressed at the basal side of epithelial cells of all tissues tested, KANK2 expression is mainly observed at the plasma membrane and/or cytoplasm of mesenchymal cells and KANK3 exclusively in vascular and lymphatic endothelial cells. KANK4 shows the least widespread expression pattern and when present, overlaps with KANK2 in contractile cells, such as smooth muscle cells and pericytes. Our findings show that KANKs are widely expressed in a cell type-specific manner, which suggests that they have cell- and tissue-specific functions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Covalent labeling of the Arabidopsis plasma membrane H + ‐ ATPase reveals 3D conformational changes involving the C‐terminal regulatory domain

The plasma membrane proton pump is the primary energy transducing, electrogenic ion pump of the plasma membrane in plants and fungi. Compared to its fungal counterpart, the plant plasma membrane proton pump's regulatory C‐terminal domain (CTD) contains an additional regulatory segment that links multiple sensory pathways regulating plant cell length through phosphorylation and recruitment of regulatory 14‐3‐3 proteins. However, a complete structural model of a plant proton pump is lacking. Here, we performed covalent labeling with mass spectrometric analysis (CL‐MS) on the Arabidopsis pump AHA2 to identify potential interactions between the CTD and the catalytic domains. Our results suggest that autoinhibition in the plant enzyme is much more structurally complex than in the fungal enzyme.

Blackburn, Matthew R. [Department of Biochemistry ↗
DOE OSTI2021

Chloride facilitates Mn(III) formation during photoassembly of the Photosystem II oxygen-evolving complex

The Mn 4 Ca oxygen-evolving complex (OEC) in Photosystem II (PSII) is assembled in situ from free Mn 2+ , Ca 2+ , and water. In an early light-driven step, Mn 2+ in a protein high-affinity site is oxidized to Mn 3+ . Using dual-mode electron paramagnetic resonance spectroscopy, we observed that Mn 3+ accumulation increases as chloride concentration increases in spinach PSII membranes depleted of all extrinsic subunits. At physiologically relevant pH values, this effect requires the presence of calcium. Here, when combined with pH studies, we conclude that the first Mn 2+ oxidation event in OEC assembly requires a deprotonation that is facilitated by chloride.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1995

Apparatus for diffusion controlled dialysis under microgravity conditions

Apparatus for implementing crystal growth by allowing mixing of solutions under microgravity conditions includes a housing within which a number of pairs of chambers are formed. The chambers of each pair are aligned and a rotary valve is positioned between the chambers of each pair. When the valve is in a first position one chamber of each pair may communicate with the other chamber. A separate valve is provided for each pair of chambers so that each pair of chambers may be activated independently of the others and sequentially at selected intervals. Protein solution may be located within a small cavity in a cap which closes one of the chambers of a pair, and the cavity in the cap is closed by a dialysis membrane. The length of certain pairs of chambers may differ from the length of other pairs of chambers to optimize conditions for various dialysis productions, and wicking material may be incorporated into selected chambers for controlling the critical approach to supersaturation.

Carter, Daniel C.↗