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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 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1986

An improved approach to the analysis of drug-protein binding by distance geometry

The calculation of side chain centers of coordinates and the subsequent generation of side chain-side chain and side chain-backbone distance matrices is suggested as an improved method for viewing interactions inside proteins and for the comparison of protein structures. The use of side chain distance matrices is demonstrated with free PTI, and the use of difference distance matrices for side chains is shown for free and trypsin-bound PTI as well as for the X-ray structures of trypsin complexes with PTI and with benzamidine. It is found that conformational variations are reflected in the side chain distance matrices much more than in the standard C-C distance representations.

NASA Discipline Exobiology↗
NASA NTRS1982

Synthesis of adriamycin-coupled polyglutaraldehyde microspheres and evaluation of their cytostatic activity

Adriamycin was coupled to polyglutaraldehyde microspheres having an average diameter of 4500 A. The coupled microspheres remained stable during incubation with cells. Full cytostatic activity was observed when the coupled adriamycin was tested with murine or human leukemia and murine sarcoma cell lines. A 10-fold increase in sensitivity was obtained with drug-resistant human leukemia cell lines. Repeated use of the coupled microspheres in the cytostatic assays did not decrease their activity, indicating that these complexes can be recycled. The results suggest that coupled adriamycin sufficiently perturbs the plasma membrane to lead to cytostatic activity. It is proposed that this mode of drug delivery provides multiple and repetitious sites for drug-cell interactions. In addition, the drug-polymer complexes may overcome those forms of resistance that are the result of decreased drug binding at the cell surface.

Tokes, Z. A.↗
NASA NTRS1989

Human serum albumin crystals and method of preparation

Human serum albumin (HSA) crystals are provided in the form of tetragonal plates having the space groups P42(sub 1)2, the crystals being grown to sizes in excess of 0.5 mm in two dimensions and a thickness of 0.1 mm. Growth of the crystals is carried out by a hanging drop method wherein a precipitant solution containing polyethylene glycol (PEG) and a phosphate buffer is mixed with an HSA solution, and a droplet of mixed solution is suspended over a well of precipitant solution. Crystals grow to the desired size in 3 to 7 days. Concentration of reagents, pH and other parameters are controlled within prescribed limits. The resulting crystals exhibit a size and quality such as to allow performance of x ray diffraction studies and enable the conduct of drug binding studies as well as genetic engineering studies.

Carter, Daniel C.↗
NASA NTRS1996

Crystals of Serum Albumin for Use in Genetic Engineering and Rational Drug Design

Serum albumin crystal forms have been produced which exhibit superior x-ray diffraction quality. The crystals are produced from both recombinant and wild-type human serum albumin, canine, and baboon serum albumin and allow the performance of drug-binding studies as well as genetic engineering studies. The crystals are grown from solutions of polyethylene glycol or ammonium sulphate within prescribed limits during growth times from one to several weeks and include the following space groups: P2(sub 1), C2, P1.

Carter, Daniel C.↗
NASA NTRS1992

Commercial researcher perspective

Protein crystallography--a research tool used to study the structure of the complex building blocks of living systems--has a lot to gain from space-based research. In order to know how a protein works in the human body, researchers must understand its molecular structure. Researchers have identified 150,000 different proteins in the body, but they now know the structure of less than a third of them. The only viable technique for analyzing the structure of these proteins is x-ray diffraction of the proteins in their crystal form. The better the quality of a protein crystal, the more useful it is to researchers who are trying to delineate its structure. The microgravity environment of space allows protein crystals to grow nearly undisturbed by convection and other gravity-driven forces that cause flaws to form in them on the ground. In space, lack of convection enables protein crystals to grow more slowly than they do on Earth, and the slower a protein crystal grows, the fewer flaws it will have. Protein crystal growth experiments have already flown on 14 Space Shuttle missions. This year's USML-1 Spacelab mission included protein crystal growth experiments conducted for commercial researchers. The results of protein crystal experiments flown thus far have been larger crystals with more uniform morphologies. The Center for Macromolecular Crystallography (A NASA-cosponsored CCDS) currently builds flight hardware to meet researchers' needs and handles sample loading and retrieval for flight experiments. Protein crystallography enables 'rational drug design': the development of drugs that bind only with the target protein and, hence, do not cause side effects. For example, pharmaceutical companies presently are interested in developing drugs that can inhibit purine nucleoside phosphorylase (PNP), a protein that plays a role in auto-immune diseases. To continue these kinds of investigations, researchers need a constant supply of protein crystals that are as free of flaws as possible. Space Station Freedom will provide the kind of research environment that will enable the production of such supplies. In addition, Freedom will provide the kind of long-duration facility required by protein crystal researchers: 40 percent of proteins require more than two weeks to crystallize.

Delucas, Larry↗
NASA NTRS2002

A site-directed mutagenesis analysis of tNOX functional domains

Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.

Non-NASA Center↗
NASA NTRS1986

Interactions of the interferon system with cellular metabolism

The results of studies concerning the interaction of the interferon (Inf) system with the activities of carcinogens, tumor promoters, and cytochrome P-450 are presented. The results show that the addition of a tumor promoter (TPA or 4-O-methyl-TPA) to a tissue culture enhances virus-induced Inf-gamma production, suggesting a potential value of tumor promoters in the biosynthesis of commercial Inf. On the other hand, the carcinogens were reported to inhibit the induction of Inf-alpha/beta in cultured cells and in intact animals (with no effect on the administered or preformed Inf). The demonstration of a correlation between the carcinogenic potential of a compound and its inhibitive effect on Inf production suggests a possible use of the Inf production assay in the evaluation of the carcinogenicity of chemicals. In addition, it was shown that the induction of Inf-alpha/beta as well as the administration of this Inf depresses the levels of rat liver cytochrome P-450 which is responsible for binding lipophilic drugs, steroids, and carcinogens, thus increasing the toxicity of the respective chemical.

Sonnenfeld, Gerald↗
NASA NTRS1994

Crystals of Human Serum Albumin for Use in Genetic Engineering and Rational Drug Design

This invention pertains to crystals of serum albumin and processes for growing them. The purpose of the invention is to provide crystals of serum albumin which can be studied to determine binding sites for drugs. Form 2 crystals grow in the monoclinic space P2(sub 1), and possesses the following unit cell constraints: a = 58.9 +/- 7, b = 88.3 +/- 7, c = 60.7 +/- 7, Beta = 101.0 +/- 2 degrees. One advantage of the invention is that it will allow rational drug design

Carter, Daniel C.↗
NASA NTRS1982

Quantitation of chlorpromazine-bound calmodulin during chlorpromazine inhibition of gravitropism

The regulatory protein, calmodulin (CaM), controls the activity of a plasma membrane localized ATPase in plants which serves to pump calcium out of cells. Recent data are consistent with the hypothesis that activation of this pump is one of the early steps necessary for gravitropism. Chlorpromazine (CPZ), a CaM antagonist, reversibly inhibits gravitropism in oat coleoptiles at concentrations which permit normal growth rates. C-14-labeled CPZ was used to photo-affinity label endogenous CaM in vivo to learn whether the drug is actually binding to some portion of endogenous CaM when it inhibits gravitropism. Under conditions in which CPZ inhibits gravitropism for over an hour, at least 11% of the CaM in gravitropically stimulated coleoptiles is bound to CPZ. In a given CPZ experiment the degree of inhibition of gravitropism correlates well with the amount of CaM bound to CPZ.

Roux, S. J.↗
NASA NTRS1996

Protein crystallization studies

The Structural Biology laboratory at NASA Marshall Spaceflight Center uses x-ray crystallographic techniques to conduct research into the three-dimensional structure of a wide variety of proteins. A major effort in the laboratory involves an ongoing study of human serum albumin (the principal protein in human plasma) and its interaction with various endogenous substances and pharmaceutical agents. Another focus is on antigenic and functional proteins from several pathogenic organisms including the human immunodeficiency virus (HIV) and the widespread parasitic genus, Schistosoma. My efforts this summer have been twofold: first, to identify clinically significant drug interactions involving albumin binding displacement and to initiate studies of the three-dimensional structure of albumin complexed with these agents, and secondly, to establish collaborative efforts to extend the lab's work on human pathogens.

Lyne, James Evans↗
NASA NTRS1991

Three-dimensional structure of human serum albumin

The binding locations to human serum albumin (HSA) of several drug molecules were determined at low resolution using crystallographic methods. The principal binding sites are located within subdomains IIA and IIIA. Preliminary studies suggest that an approach to increasing the in vivo efficacy of drugs which are rendered less effective or ineffective by virtue of their interaction with HSA, would be the use of competitive displacement in drug therapies and/or the development of a general inhibitor to the site within subdomain IIIA. These findings also suggest that the facilitated transfer of various ligands across organ/circulatory interfaces such as liver, kidney, and brain may be associated with binding to the IIIA subdomain.

Carter, Daniel C.↗
NASA NTRS2014

Effects of Radiation and Dietary Iron on Expression of Genes and Proteins Involved in Drug Metabolism

Liver function, especially the rate of metabolic enzyme activities, determines the concentration of circulating drugs and the duration of their efficacy. Most pharmaceuticals are metabolized by the liver, and clinically-used medication doses are given with normal liver function in mind. A drug overdose can result in the case of a liver that is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism, we want to understand any effects of spaceflight on the enzymes of the liver. Dietary factors and exposure to radiation are aspects of spaceflight that are potential oxidative stressors and both can be modeled in ground experiments. In this experiment, we examined the effects of high dietary iron and low dose gamma radiation (individually and combined) on the gene expression of enzymes involved in drug metabolism, redox homeostasis, and DNA repair. METHODS All procedures were approved by the JSC Animal Care and Use Committee. Male Sprague-Dawley rats were divided into 4 groups (n=8); control, high Fe diet (650 mg iron/kg), radiation (fractionated 3 Gy exposure from a Cs- 137 source) and combined high Fe diet + radiation exposure. Animals were euthanized 24h after the last treatment of radiation; livers were removed immediately and flash -frozen in liquid nitrogen. Expression of genes thought to be involved in redox homeostasis, drug metabolism and DNA damage repair was measured by RT-qPCR. Where possible, protein expression of the same genes was measured by western blotting. All data are expressed as % change in expression normalized to reference gene expression; comparisons were then made of each treatment group to the sham exposed/ normal diet control group. Data was considered significant at p< 0.5. RESULTS Among the redox homeostasis genes examined, metallothionein showed a significant down regulation in the radiation treated group (-3.85 fold) and a trend toward down regulation in the high Fe + rad group. Metallothionein is involved in the regulation of physiological metals and also has antioxidant activities. Among the drug metabolism genes examined, ATP binding cassette subfamily B (Abcb1b) gene expression increased more than 10-fold in both groups that received radiation treatments. This increased expression was also seen at the protein level. This ABC transporter carries many different compounds across cell membranes, including administered medications. The cytochrome P450 2E1 enzyme, a mixed-function oxidase that deactivates some medications and activates others, showed about a 2-fold increase in gene expression in both radiation-treated groups, with a trend toward increased expression at the protein level. Expression of epoxide hydrolase, which detoxifies polycyclic aromatic hydrocarbons, showed similar 2-fold increases. Among the DNA repair genes examined, expression of RAD51 was significantly down regulated (1.5 fold) in the radiation treated group. RAD51 is involved in repair of double-stranded DNA breaks. CONCLUSION This experiment used 2 different sources of physiological oxidative stress, administered separately and together, and examined their impacts on liver gene and protein expression. It is clear that significant changes occurred in expression of several genes and proteins in the radiation-treated animals. If the results from this ground analog of portions of the spaceflight environment hold true for the spaceflight environment itself, the physiological roles of the affected enzymes (drug transport and metabolism, redox homeostasis) could mean consequences in redox homeostasis or the pharmacokinetics of administered medications

Faust, K. M.↗
NASA NTRS2012

Protein Innovations Advance Drug Treatments, Skin Care

Dan Carter carefully layered the sheets of tracing paper on the light box. On each sheet were renderings of the atomic components of an essential human protein, one whose structure had long been a mystery. With each layer Carter laid down, a never-before-seen image became clearer. Carter joined NASA s Marshall Space Flight Center in 1985 and began exploring processes of protein crystal growth in space. By bouncing intense X-rays off the crystals, researchers can determine the electron densities around the thousands of atoms forming the protein molecules, unveiling their atomic structures. Cultivating crystals of sufficient quality on Earth was problematic; the microgravity conditions of space were far more accommodating. At the time, only a few hundred protein structures had been mapped, and the methods were time consuming and tedious. Carter hoped his work would help reveal the structure of human serum albumin, a major protein in the human circulatory system responsible for ferrying numerous small molecules in the blood. More was at stake than scientific curiosity. Albumin has a high affinity for most of the world s pharmaceuticals, Carter explains, and its interaction with drugs can change their safety and efficacy. When a medication enters the bloodstream a cancer chemotherapy drug, for example a majority of it can bind with albumin, leaving only a small percentage active for treatment. How a drug interacts with albumin can influence considerations like the necessary effective dosage, playing a significant role in the design and application of therapeutic measures. In spite of numerous difficulties, including having no access to microgravity following the 1986 Space Shuttle Challenger disaster, the image Carter had hoped to see was finally clarifying. In 1988, his lab had acquired specialized X-ray and detection equipment a tipping point. Carter and his colleagues began to piece together albumin s portrait, the formation of its electron densities coalescing on the sheets of tracing paper he arranged on the light box. While space-grown crystals were ultimately not involved in the achievement, a year later, Carter says, we were on the cover of Science magazine, having determined the atomic structure of albumin.

Source record↗
NASA NTRS1998

In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

Non-NASA Center↗
NASA NTRS2002

Subcutaneous administration of insulin-like growth factor (IGF)-II/IGF binding protein-2 complex stimulates bone formation and prevents loss of bone mineral density in a rat model of disuse osteoporosis

Elevated serum levels of insulin-like growth factor binding protein-2 (IGFBP-2) and a precursor form of IGF-II are associated with marked increases in bone formation and skeletal mass in patients with hepatitis C-associated osteosclerosis. In vitro studies indicate that IGF-II in complex with IGFBP-2 has high affinity for bone matrix and is able to stimulate osteoblast proliferation. The purpose of this study was to determine the ability of the IGF-II/IGFBP-2 complex to increase bone mass in vivo. Osteopenia of the femur was induced by unilateral sciatic neurectomy in rats. At the time of surgery, 14-day osmotic minipumps containing vehicle or 2 microg IGF-II+9 microg IGFBP-2/100g body weight/day were implanted subcutaneously in the neck. Bone mineral density (BMD) measurements were taken the day of surgery and 14 days later using a PIXImus small animal densitometer. Neurectomy of the right hindlimb resulted in a 9% decrease in right femur BMD (P<0.05 vs. baseline). This loss in BMD was completely prevented by treatment with IGF-II/IGFBP-2. On the control limb, there was no loss of BMD over the 14 days and IGF-II/IGFBP-2 treatment resulted in a 9% increase in left femur BMD (P<0.05). Bone histomorphometry indicated increases in endocortical and cancellous bone formation rates and in trabecular thickness. These results demonstrate that short-term administration of the IGF-II/IGFBP-2 complex can prevent loss of BMD associated with disuse osteoporosis and stimulate bone formation in adult rats. Furthermore, they provide proof of concept for a novel anabolic approach to increasing bone mass in humans with osteoporosis.

NASA Discipline Regulatory Physiology↗
NASA NTRS2022

Free Energy Landscapes for Elucidating the Structural Consequences of Exon-20 mutations on the ErbB Family of Protein Kinases

The ErbB family of protein kinases plays an important role in major cellular functions and consequently mutations in the functional regions of these proteins are implicated in several types of cancer growths. To envision rational design of small molecule drugs that target the diseased proteins it is important to quantify the structural effects of the mutations, as some of these mutants render the protein resistant to tyrosine kinase inhibitors (TKIs). Herein we use advanced sampling techniques and long-timescale molecular dynamics simulations to predict the effect of major exon 20 mutations on the ErbB family, specifically EGFR and HER2 proteins. Exon 20 mutations have been clinically known to induce TKI resistance, though the mechanisms of such an effect is poorly understood. By mapping out the free energy landscape of the mutants and comparing them against the wild-type, we elucidate the structural differences in the binding pocket region that alter the nature of drug-protein interactions. We believe that these insights will play a pivotal role in developing small molecule drugs that overcome the TKI resistance.

Ashwin Ravichandran↗
NASA NTRS2003

Caspase inhibitors promote vestibular hair cell survival and function after aminoglycoside treatment in vivo

The sensory hair cells of the inner ear undergo apoptosis after acoustic trauma or aminoglycoside antibiotic treatment, causing permanent auditory and vestibular deficits in humans. Previous studies have demonstrated a role for caspase activation in hair cell death and ototoxic injury that can be reduced by concurrent treatment with caspase inhibitors in vitro. In this study, we examined the protective effects of caspase inhibition on hair cell death in vivo after systemic injections of aminoglycosides. In one series of experiments, chickens were implanted with osmotic pumps that administrated the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD) into inner ear fluids. One day after the surgery, the animals received a 5 d course of treatment with streptomycin, a vestibulotoxic aminoglycoside. Direct infusion of zVAD into the vestibule significantly increased hair cell survival after streptomycin treatment. A second series of experiments determined whether rescued hair cells could function as sensory receptors. Animals treated with streptomycin displayed vestibular system impairment as measured by a greatly reduced vestibulo-ocular response (VOR). In contrast, animals that received concurrent systemic administration of zVAD with streptomycin had both significantly greater hair cell survival and significantly increased VOR responses, as compared with animals treated with streptomycin alone. These findings suggest that inhibiting the activation of caspases promotes the survival of hair cells and protects against vestibular function deficits after aminoglycoside treatment.

NASA Discipline Neuroscience↗
NASA NTRS2003

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

NASA Discipline Cell Biology↗