Search NASA⌕ Search

SEARCH · Search NASA

Results for “protein complex”

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.

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

At least 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
NASA NTRS2003

New Programs Utilizing Light Scattering and Flow Imaging Techniques for Macromolecular Crystal Growth and Fluid Dynamics Studies

Dr. Phil Segre, a physicist by training, is a recent addition to the Biotech group, SD46, having joined NASA in August of 2000. Over the past two years he has been developing a laboratory for the study of macromolecular and protein crystal growth. The main apparatus for this work is a Dynamic Light Scattering apparatus, DLS, which is capable of making highly precise measurements of size distributions of both protein solutions and protein crystals. With Drs. Chernov and Thomas (USRA), he has begun a collaboration studying the affects of protein impurities on protein crystal growth and subsequent crystal quality. One of the hypotheses behind the differences between Earth and space grown protein crystals is that the absorption of harmful impurities is reduced in space due to the absence of convective flows. Using DLS measurements we are examining crystal growth with varying amounts of impurities and testing whether there is a strong physical basis behind this hypothesis. With Dr. Joe Ng of UAH he has been collaborating on a project to examine the folding/unfolding dynamics of large RNA complexes. A detailed understanding of this process is necessary for the handling of RNA in biotech applications, and the DLS instrument gives details and results beyond that of other instruments. With Prof. Jim McClymer of the University of Maine (summer faculty visitor to NASA in 2001, 2002), we have been studying the crystallization process in model colloidal suspensions whose behavior in some cases can mimic that of much smaller protein solutions. An understanding of the self-assembly of colloids is the first step in the process of engineering novel materials for photonic and light switching applications. Finally, he has begun an investigation into the physics of particle sedimentation. In addition to the DLS instrument he also has an instrument (called PIV) that can measure flow fields of fluids. The applications are to the dynamics of protein crystal motions both on earth and in low-gravity.

Source record↗
NASA NTRS1995

Structural characterization and regulatory element analysis of the heart isoform of cytochrome c oxidase VIa

In order to investigate the mechanism(s) governing the striated muscle-specific expression of cytochrome c oxidase VIaH we have characterized the murine gene and analyzed its transcriptional regulatory elements in skeletal myogenic cell lines. The gene is single copy, spans 689 base pairs (bp), and is comprised of three exons. The 5'-ends of transcripts from the gene are heterogeneous, but the most abundant transcript includes a 5'-untranslated region of 30 nucleotides. When fused to the luciferase reporter gene, the 3.5-kilobase 5'-flanking region of the gene directed the expression of the heterologous protein selectively in differentiated Sol8 cells and transgenic mice, recapitulating the pattern of expression of the endogenous gene. Deletion analysis identified a 300-bp fragment sufficient to direct the myotube-specific expression of luciferase in Sol8 cells. The region lacks an apparent TATA element, and sequence motifs predicted to bind NRF-1, NRF-2, ox-box, or PPAR factors known to regulate other nuclear genes encoding mitochondrial proteins are not evident. Mutational analysis, however, identified two cis-elements necessary for the high level expression of the reporter protein: a MEF2 consensus element at -90 to -81 bp and an E-box element at -147 to -142 bp. Additional E-box motifs at closely located positions were mutated without loss of transcriptional activity. The dependence of transcriptional activation of cytochrome c oxidase VIaH on cis-elements similar to those found in contractile protein genes suggests that the striated muscle-specific expression is coregulated by mechanisms that control the lineage-specific expression of several contractile and cytosolic proteins.

Non-NASA Center↗
NASA NTRS2001

Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryo-preserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 T. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica. The microbial extremophiles recovered from permafrost, ice, cold pools and deep sea sediments may provide information relevant to the question of how and where we should search for evidence of extant or extinct microbial life elsewhere in the Cosmos.

Hoover, Richard B.↗
NASA NTRS2002

Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryopreserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 C. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep-sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica. The microbial extremophiles recovered from permafrost, ice, cold pools and deep-sea sediments may provide information relevant to the question of how and where we should search for evidence of extant or extinct microbial life elsewhere in the Cosmos.

Hoover, Richard B.↗
NASA NTRS1995

Integrin-extracellular matrix interactions in connective tissue remodeling and osteoblast differentiation

The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.

NASA Discipline Number 26-10↗
NASA NTRS2007

Solution Growth and Characterization of Single Crystals on Earth and in Microgravity

Crystal growth has been of interest to physicists and engineers for a long time because of their unique properties. Single crystals are utilized in such diverse applications as pharmaceuticals, computers, infrared detectors, frequency measurements, piezoelectric devices, a variety of high-technology devices, and sensors. Solution crystal growth is one of the important techniques to grow a variety of crystals when the material decomposes at the melting point and a suitable solvent is available to make a saturated solution at a desired temperature. In this Technical Memorandum (TM) an attempt is made to give the fundamentals of growing crystals from solution including improved designs of various crystallizers. Since the same solution crystal growth technique could not be used in microgravity, the authors proposed a new cooled-sting technique to grow crystals in space. The authors experience from conducting two Space Shuttle solution crystal growth experiments are also detailed in this TM and the complexity of solution growth experiments to grow crystals in space are also discussed. These happen to be some of the early experiments performed in space, and various lessons learned are described. A brief discussion of protein crystal growth that shares basic principles of the solution growth technique is given, along with some flight hardware information for growth in microgravity.

Aggarwal, M. D.↗
NASA NTRS2011

Growth and Characteristics of Bulk Single Crystals Grown from Solution on Earth and in Microgravity

The growth of crystals has been of interest to physicists and engineers for a long time because of their unique properties. Single crystals are utilized in such diverse applications as pharmaceuticals, computers, infrared detectors, frequency measurements, piezoelectric devices, a variety of high technology devices and sensors. Solution crystal growth is one of the important techniques to grow a variety of crystals when the material decomposes at the melting point and a suitable solvent is available to make a saturated solution at a desired temperature. In this chapter an attempt is made to give some fundamentals of growing crystals from solution including improved designs of various crystallizers. Since the same solution crystal growth technique could not be used in microgravity, authors had proposed a new cooled sting technique to grow crystals in space. Authors? experiences of conducting two space shuttle experiments relating to solution crystal growth are also detailed in this work. The complexity of these solution growth experiments to grow crystals in space are discussed. These happen to be some of the early experiments performed in space, and various lessons learned are described. A brief discussion of protein crystal growth that also shares basic principles of solution growth technique is given along with some flight hardware information for its growth in microgravity.

Aggarwal, M. D.↗
NASA NTRS2000

The chimeric eukaryote: origin of the nucleus from the karyomastigont in amitochondriate protists

We present a testable model for the origin of the nucleus, the membrane-bounded organelle that defines eukaryotes. A chimeric cell evolved via symbiogenesis by syntrophic merger between an archaebacterium and a eubacterium. The archaebacterium, a thermoacidophil resembling extant Thermoplasma, generated hydrogen sulfide to protect the eubacterium, a heterotrophic swimmer comparable to Spirochaeta or Hollandina that oxidized sulfide to sulfur. Selection pressure for speed swimming and oxygen avoidance led to an ancient analogue of the extant cosmopolitan bacterial consortium "Thiodendron latens." By eubacterial-archaebacterial genetic integration, the chimera, an amitochondriate heterotroph, evolved. This "earliest branching protist" that formed by permanent DNA recombination generated the nucleus as a component of the karyomastigont, an intracellular complex that assured genetic continuity of the former symbionts. The karyomastigont organellar system, common in extant amitochondriate protists as well as in presumed mitochondriate ancestors, minimally consists of a single nucleus, a single kinetosome and their protein connector. As predecessor of standard mitosis, the karyomastigont preceded free (unattached) nuclei. The nucleus evolved in karyomastigont ancestors by detachment at least five times (archamoebae, calonymphids, chlorophyte green algae, ciliates, foraminifera). This specific model of syntrophic chimeric fusion can be proved by sequence comparison of functional domains of motility proteins isolated from candidate taxa.

Review, Tutorial↗
NASA NTRS2001

Crosstalk between p38, Hsp25 and Akt in spinal motor neurons after sciatic nerve injury

The p38 stress-activated protein kinase pathway is involved in regulation of phosphorylation of Hsp25, which in turn regulates actin filament dynamic in non-neuronal cells. We report that p38, Hsp25 and Akt signaling pathways were specifically activated in spinal motor neurons after sciatic nerve axotomy. The activation of the p38 kinase was required for induction of Hsp25 expression. Furthermore, Hsp25 formed a complex with Akt, a member of PI-3 kinase pathway that prevents neuronal cell death. Together, our observations implicate Hsp25 as a central player in a complex system of signaling that may both promote regeneration of nerve fibers and prevent neuronal cell death in the injured spinal cord.

NASA Discipline Developmental Biology↗
NASA NTRS2022

CryoEM Structures of the Nitrogenase Complex During Catalytic Turnover

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multi-electron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here we report the cryogenic electron microscopic interrogation of the nitrogenase complex under enzymatic turnover conditions, which has enabled the structural characterization of the nitrogenase reaction intermediates at high resolution for the first time. Our structures show that asymmetry governs all aspects of nitrogenase mechanism including ATP hydrolysis, protein-protein interactions, and catalysis. Furthermore, they reveal several previously unobserved, mechanistically relevant conformational changes near the catalytic iron-molybdenum cofactor that are correlated with the nucleotide-hydrolysis state of the enzyme.

Hannah L. Rutledge↗
NASA NTRS1999

Correlation of Fos expression and circling asymmetry during gerbil vestibular compensation

Vestibular compensation is a central nervous system process resulting in recovery of functional movement and control following a unilateral vestibular lesion. Small pressure injections of phosphorothioate 20mer oligonucleotides were used to probe the role of the Fos transcription protein during vestibular compensation in the gerbil brainstem. During isoflurane gas anesthesia, antisense probes against the c-fos mRNA sequence were injected into the medial vestibular and prepositus nuclei unilaterally prior to a unilateral surgical labyrinthectomy. Anionic dyes, which did not interact with the oligonucleotides, were used to mark the injection site and help determine the extent of diffusion. The antiFos oligonucleotide injections reduced Fos expression at the injection site in neurons which normally express Fos after the lesion, and also affected circling behavior induced by hemilabyrinthectomy. With both ipsilateral and contralateral medial vestibular and prepositus nuclei injections, less ipsilateral and more contralateral circling was noted in animals injected with antiFos injections as compared to non-injected controls. The degree of change in these behaviors was dependent upon the side of the injection. Histologically, antiFos injections reduced the number of Fos immunolabeled neurons around the injection site, and increased Fos expression contralaterally. The correlation of the number of neurons with Fos expression to turning behavior was stronger for contralateral versus ipsilateral turns, and for neurons in the caudal and ipsilateral sub-regions of the medial vestibular and prepositus nuclei. The results are discussed in terms of neuronal firing activity versus translational activity based on the asymmetrical expression of the Fos inducible transcription factor in the medial vestibular and prepositus nuclei. Although ubiquitous in the brain, transcription factors like Fos can serve localized and specific roles in sensory-specific adaptive stimuli. Antisense injections can be an effective procedure for localized intervention into complex physiological functions, e.g. vestibular compensation. Copyright 1999 Elsevier Science B.V.

NASA Discipline Neuroscience↗
NASA NTRS1987

Structural elements and organization of the ancestral translational machinery

The molecular mechanisms of the primitive translational apparatus are discussed in the framework of present-day protein biosynthesis. The structural necessities of an early adaptor and the multipoint recognition properties of such an adaptor are investigated on the basis of structure/function relationships found in a contemporary system and a molecular model of the contemporary transpeptidation complex. A model of the tRNA(Tyr)-tyrosyl tRNA synthetase complex including the positioning of the disordered region is proposed; the model is used to illustrate the required recognition properties of the ancestor aminoacyl synthetase.

Rein, R.↗
NASA NTRS2004

Variation in sulfide tolerance of photosystem II in phylogenetically diverse cyanobacteria from sulfidic habitats

Physiological and molecular phylogenetic approaches were used to investigate variation among 12 cyanobacterial strains in their tolerance of sulfide, an inhibitor of oxygenic photosynthesis. Cyanobacteria from sulfidic habitats were found to be phylogenetically diverse and exhibited an approximately 50-fold variation in photosystem II performance in the presence of sulfide. Whereas the degree of tolerance was positively correlated with sulfide levels in the environment, a strain's phenotype could not be predicted from the tolerance of its closest relatives. These observations suggest that sulfide tolerance is a dynamic trait primarily shaped by environmental variation. Despite differences in absolute tolerance, similarities among strains in the effects of sulfide on chlorophyll fluorescence induction indicated a common mode of toxicity. Based on similarities with treatments known to disrupt the oxygen-evolving complex, it was concluded that sulfide toxicity resulted from inhibition of the donor side of photosystem II.

Phylogeny↗
NASA NTRS2011

Differential Processing of Low and High LET Radiation Induced DNA Damage: Investigation of Switch from ATM to ATR Signaling

The members of the phosphatidylinositol kinase-like kinase family of proteins namely ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) are directly responsible for the maintenance of genomic integrity by mounting DDR through signaling and facilitating the recruitment of repair factors at the sites of DNA damage along with coordinating the deployment of cell cycle checkpoints to permit repair by phosphorylating Checkpoint kinase Chk1, Chk2 and p53. High LET radiation from GCR (Galactic Cosmic Rays) consisting mainly of protons and high energy and charged (HZE) particles from SPE (Solar Particle Event) pose a major health risk for astronauts on their space flight missions. The determination of these risks and the design of potential safeguards require sound knowledge of the biological consequences of lesion induction and the capability of the cells to counter them. We here strive to determine the coordination of ATM and ATR kinases at the break sites directly affecting checkpoint signaling and DNA repair and whether differential processing of breaks induced by low and high LET radiation leads to possible augmentation of swap of these damage sensors at the sites of DNA damage. Exposure of cells to IR triggers rapid autophosphorylation of serine-1981 that causes dimer dissociation and initiates monomer formation of ATM. ATM kinase activity depends on the disruption of the dimer, which allows access and phosphorylation of downstream ATM substrates like Chk2. Evidence suggests that ATM is activated by the alterations in higher-order chromatin structure although direct binding of ATM to DSB ends may be a crucial step in its activation. On the other hand, in case of ATR, RPA (replication protein A)-coated ssDNA (single-stranded DNA) generated as a result of stalled DNA replication or during processing of chromosomal lesions is crucial for the localization of ATR to sites of DNA damage in association with ATR-interacting protein (ATRIP). Although the majority of RPA-coated ssDNA is generally present only during DNA replication, ATR activation in G1 and G2-phase might still require formation of RPA-coated ssDNA, probably initiated by the MRN-CtIP complex and then extended by the Exo1- or BLM-dependent mechanisms at the sites of DSBs. Evidence accumulates that activation of ATM and ATR are oppositely regulated by the length of single stranded overhangs generated at the break sites by processes mentioned above and these stretches of single stranded overhangs hold the clue for ATM to ATR switch at broken DNA ends. We irradiated 82-6hTERT human fibroblast cells with low LET gamma-rays and high LET Fe and Si particles. Preliminary results with cells exposed to 1Gy gamma-rays show that the kinetics of pChk2-pT68 foci formation is comparable to that of gamma-H2AX although they appear to recede quicker. The number and intensity of observed foci reaches a maximum at 30 min and 60 min post IR for Chk2-pT68 and gamma-H2AX foci respectively and all Chk2-pT68 foci colocalize with gamma-H2AX foci. The kinetics of Chk1-pS345 and ATRIP are being determined. Results of Chk2-pT68 foci kinetics was also corroborated by western blot experiments, although phosphorylation was detected as early as 10 min and started receding 30 min post IR with 2Gy of gamma-rays. On the other hand, level of ATR-pS428 reached its maximum between 60 and 120 min and was maintained until the last measured time point of 4 hours post IR as determined by western blotting. Experiments performed with high LET Fe and Si particles will be reported.

Saha, Janapriya↗
NASA NTRS2001

Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle

The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T(3), 3,5,3'-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T(3)/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the beta-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T(3) or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T(3) and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of heterogeneity among the individual fibers would ensure a large functional diversity in performing complex movement patterns. Future studies must now focus on 1) the signaling pathways and the underlying mechanisms governing the transcriptional/translational machinery that control this marked degree of plasticity and 2) the morphological organization and functional implications of the muscle fiber's capacity to express such a diversity of motor proteins.

NASA Discipline Musculoskeletal↗
NASA NTRS2007

Involvement of DNA-PK(sub cs) in DSB Repair Following Fe-56 Ion Irradiation

When cells are exposed to radiation, cellular lesions are induced in the DNA including double strand breaks (DSBs), single strand breaks and clustered DNA damage, which if not repaired with high fidelity may lead to detrimental biological consequences. Complex DSBs are induced by ionizing radiation and characterized by the presence of base lesions close to the break termini. They are believed to be one of the major causes of the biological effects of IR. The complexity of DSBs increases with the ionization density of the radiation and these complex DSBs are distinct from the damage induced by sparsely ionizing gamma-radiation. It has been hypothesized that complex DSBs produced by heavy ions in space pose problems to the DNA repair machinery. We have used imm uno-cyto-chemical staining of phosphorylated histone H2AX (gamma-H2AX) foci, as a marker of DSBs. We have investigated the formation and loss of gamma-H2AX foci and RAD51 foci (a protein involved in the homologous recombination pathway) in mammalian cells induced by low fluences of low-LET gamma-radiation and high-LET Fe-56 ions (1GeV/n, 151 keV/micron LET). M059J and M059K cells, which are deficient and proficient in DNA-PK(sub cs) activity respectively, were used to examine the role of DNA-PK(sub cs), a key protein in the non-homologous end joining (NHEJ) pathway of DSB repair, along with HF19 human fibroblasts. Followi ng irradiation with Fe-56 ions the rate of repair was slower in M059J cells compared with that in M059K, indicating a role for DNA-PK(sub cs) in the repair of DSB induced by Fe-56 ions. However a small percentage of DSBs induced are rejoined within 5 h although many DSBs still persist up to 24 h. When RAD51 was examined in M059J/K cells, RAD51 foci are visible 24 hours after irradiation in approximately 40% of M059J cells compared with <5% of M059K cells indicating that persistent DSBs or those formed at stalled replication forks recruit RAD51 in DNA-PK(sub cs) deficient cells. Following 1 Gy gamma-radiation the induction of gamma-H2AX foci is similar in M059J and M059K cells. However, the repair rate of DSBs is slower in M059J cells than in M059K as shown previously but faster than seen with DSB induced by 56Fe ions. Vanillin, an inhibitor of DNA-PK(sub cs), reduces significantly the rate of DSB repair in HF19 cells following 1 Gy gamma-radiation but at 0.25 Gy gamma-irradiation the rate of DSB repair is similar in the presence or absence vanillin, thus suggesting the repair of a sub-set of DSBs induced by low dose, low-LET radiation does not require DNA-PK(sub cs). This sub-set of DSBs is formed in lower yield with high LET radiation. T he complexity of DNA DSBs induced by HZE radiation will be discussed in terms of reduced repair efficiency and provide scope to model different sub-classes of DSBs as precursors that may lead to the detrimental health effects of HZE radiation.

O'Neill, Peter↗
NASA NTRS1994

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

Non-NASA Center↗
NASA NTRS1980

Impregnated metal-polymeric functional beads

Amine containing polymeric microspheres such as polyvinyl pyridine are complexed with metal salts or acids containing metals such as gold, platinum or iron. After reduction with sodium borohydride, the salt is reduced to finely divided free metal or metal oxides, useful as catalysts. Microspheres containing covalent bonding sites can be used for labeling or separating proteins.

Rembaum, Alan↗