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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 NTRS1976

Thorium-uranium fission radiography

Results are described for studies designed to develop routine methods for in-situ measurement of the abundance of Th and U on a microscale in heterogeneous samples, especially rocks, using the secondary high-energy neutron flux developed when the 650 MeV proton beam of an accelerator is stopped in a 42 x 42 cm diam Cu cylinder. Irradiations were performed at three different locations in a rabbit tube in the beam stop area, and thick metal foils of Bi, Th, and natural U as well as polished silicate glasses of known U and Th contents were used as targets and were placed in contact with mica which served as a fission track detector. In many cases both bare and Cd-covered detectors were exposed. The exposed mica samples were etched in 48% HF and the fission tracks counted by conventional transmitted light microscopy. Relative fission cross sections are examined, along with absolute Th track production rates, interaction tracks, and a comparison of measured and calculated fission rates. The practicality of fast neutron radiography revealed by experiments to data is discussed primarily for Th/U measurements, and mixtures of other fissionable nuclei are briefly considered.

Haines, E. L.↗
DOE OSTI2011

COMSOL-based Nuclear Reactor Kinetics Studies at the HFIR

The computational ability to accurately predict the dynamic behavior of a nuclear reactor core in response to reactivity-induced perturbations is an important subject in reactor physics. Space-time and point kinetics methodologies were developed for the purpose of studying the transient-induced behavior of the High Flux Isotope Reactor s (HFIR) compact core. The space-time simulations employed the three-energy-group neutron diffusion equations, and transients initiated by control cylinder and hydraulic tube rabbit ejections were studied. The work presented here is the first step towards creating a comprehensive multiphysics methodology for studying the dynamic behavior of the HFIR core during reactivity perturbations. The results of these studies show that point kinetics is adequate for small perturbations in which the power distribution is assumed to be time-independent, but space-time methods must be utilized to determine localized effects.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗
DOE OSTI2017

Assembly and Delivery of Rabbit Capsules for Irradiation of Silicon Carbide Cladding Tube Specimens in the High Flux Isotope Reactor

Neutron irradiation of silicon carbide (SiC)-based fuel cladding under a high radial heat flux presents a critical challenge for SiC cladding concepts in light water reactors (LWRs). Fission heating in the fuel provides a high heat flux through the cladding, which, combined with the degraded thermal conductivity of SiC under irradiation, results in a large temperature gradient through the thickness of the cladding. The strong temperature dependence of swelling in SiC creates a complex stress profile in SiCbased cladding tubes as a result of differential swelling. The Nuclear Science User Facilities (NSUF) Program within the US Department of Energy Office of Nuclear Energy is supporting research efforts to improve the scientific understanding of the effects of irradiation on SiC cladding tubes. Ultimately, the results of this project will provide experimental validation of multi-physics models for SiC-based fuel cladding during LWR operation. The first objective of this project is to irradiate tube specimens using a previously developed design that allows for irradiation testing of miniature SiC tube specimens subjected to a high radial heat flux. The previous “rabbit” capsule design uses the gamma heating in the core of the High Flux Isotope Reactor (HFIR) to drive a high heat flux through the cladding tube specimens. A compressible aluminum foil allows for a constant thermal contact conductance between the cladding tubes and the rabbit housing despite swelling of the SiC tubes. To allow separation of the effects of irradiation from those due to differential swelling under a high heat flux, a new design was developed under the NSUF program. This design allows for irradiation of similar SiC cladding tube specimens without a high radial heat flux. This report briefly describes the irradiation experiment design concepts, summarizes the irradiation test matrix, and reports on the successful delivery of six rabbit capsules to the HFIR. Rabbits of both low and high heat flux configurations have been assembled, welded, evaluated, and delivered to the HFIR along with a complete quality assurance fabrication package. These rabbits contain a wide variety of specimens including monolith tubes, SiC fiber SiC matrix (SiC/SiC) composites, duplex specimens (inner composite, outer monolith), and specimens with a variety of metallic or ceramic coatings on the outer surface. The rabbits are targeted for insertion during HFIR cycle 475, which is scheduled for September 2017.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗
DOE OSTI2014

Dose equations for shift-variant CT acquisition modes using variable pitch, tube current, and aperture, and the meaning of their associated CTDI{sub vol}

Purpose: With the increasing clinical use of shift-variant CT protocols involving tube current modulation (TCM), variable pitch or pitch modulation (PM), and variable aperture a(t), the interpretation of the scanner-reported CTDI{sub vol} is called into question. This was addressed for TCM in their previous paper published by Dixon and Boone [Med. Phys. 40, 111920 (14pp.) (2013)] and is extended to PM and concurrent TCM/PM as well as variable aperture in this work. Methods: Rigorous convolution equations are derived to describe the accumulated dose distributions for TCM, PM, and concurrent TCM/PM. A comparison with scanner-reported CTDI{sub vol} formulae clearly identifies the source of their differences with the traditional CTDI{sub vol}. Dose distribution simulations using the convolution are provided for a variety of TCM and PM scenarios including a helical shuttle used for perfusion studies (as well as constant mA)—all having the same scanner-reported CTDI{sub vol}. These new convolution simulations for TCM are validated by comparison with their previous discrete summations. Results: These equations show that PM is equivalent to TCM if the pitch variation p(z) is proportional to 1/i(z), where i(z) is the local tube current. The simulations show that the local dose at z depends only weakly on the local tube current i(z) or local pitch p(z) due to scatter from all other locations along z, and that the “local CTDI{sub vol}(z)” or “CTDI{sub vol} per slice” do not represent a local dose but rather only a relative i(z) or p(z). The CTDI-paradigm does not apply to shift-variant techniques and the scanner-reported CTDI{sub vol} for the same lacks physical significance and relevance. Conclusions: While the traditional CTDI{sub vol} at constant tube current and pitch conveys useful information (the peak dose at the center of the scan length), CTDI{sub vol} for shift-variant techniques (TCM or PM) conveys no useful information about the associated dose distribution it purportedly represents. On the other hand, the total energy absorbed E (“integral dose”) as well as its surrogate DLP remain robust (invariant) with respect to shift-variance, depending only on the total mAs = 〈i〉t{sub 0} accumulated during the total beam-on time t{sub 0} and aperture a, where 〈i〉 is the average current.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2011

Influence of Carbon Sources and Electron Shuttles on Ferric Iron Reduction by Cellulomonas sp. Strain ES6

Microbially reduced iron minerals can reductively transform a variety of contaminants including heavy metals, radionuclides, chlorinated aliphatics, and nitroaromatics. A number of Cellulomonas spp. strains, including strain ES6, isolated from aquifer samples obtained at the U.S. Department of Energy's Hanford site in Washington, have been shown to be capable of reducing Cr(VI), TNT, natural organic matter, and soluble ferric iron [Fe(III)]. This research investigated the ability of Cellulomonas sp. strain ES6 to reduce solid phase and dissolved Fe(III) utilizing different carbon sources and various electron shuttling compounds. Results suggest that Fe(III) reduction by and growth of strain ES6 was dependent upon the type of electron donor, the form of iron present, and the presence of synthetic or natural organic matter, such as anthraquinone-2,6-disulfonate (AQDS) or humic substances. This research suggests that Cellulomonas sp. strain ES6 could play a significant role in metal reduction in the Hanford subsurface and that the choice of carbon source and organic matter addition can allow for independent control of growth and iron reduction activity.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2017

Proton Induced Modulation of ICT and PET Processes in an Imidazo-phenanthroline Based BODIPY Fluorophores

BODIPY fluorophores linked with an imidazo-phenanthroline donor at α and β positions have been synthesized. Intriguing intramolecular charge transfer phenomenon is observed in both the dyes which has been extensively investigated using UV–vis absorption, steady-state and time-resolved fluorescence measurements. H-bonding and intrinsic polarity of the solvents has modulated the absorption and emission bands of these fluorophores strongly causing significant increase in the Stokes shifts. In spite of having difference only in terms of the position of donor subunit, the photophysics of these dyes are not only significantly different from each other, but contradictory too. Interestingly, acidochromic studies revealed the shuttling mechanism between ICT and PET processes for BDP 2. Quantum chemical calculations have been employed further to support experimental findings. DFT and TD-DFT method of analysis have been used to optimize ground and excited state geometries of the synthesized dyes.

74 ATOMIC AND MOLECULAR PHYSICS↗
DOE OSTI2011

Sensing actin dynamics: Structural basis for G-actin-sensitive nuclear import of MAL

Highlights: {yields} MAL has a bipartite NLS that binds to Imp{alpha} in an extended conformation. {yields} Mutational analyses verified the functional significance of MAL-Imp{alpha} interactions. {yields} Induced folding and NLS-masking by G-actins inhibit nuclear import of MAL. -- Abstract: The coordination of cytoskeletal actin dynamics with gene expression reprogramming is emerging as a crucial mechanism to control diverse cellular processes, including cell migration, differentiation and neuronal circuit assembly. The actin-binding transcriptional coactivator MAL (also known as MRTF-A/MKL1/BSAC) senses G-actin concentration and transduces Rho GTPase signals to serum response factor (SRF). MAL rapidly shuttles between the cytoplasm and the nucleus in unstimulated cells but Rho-induced depletion of G-actin leads to MAL nuclear accumulation and activation of transcription of SRF:MAL-target genes. Although the molecular and structural basis of actin-regulated nucleocytoplasmic shuttling of MAL is not understood fully, it is proposed that nuclear import of MAL is mediated by importin {alpha}/{beta} heterodimer, and that G-actin competes with importin {alpha}/{beta} for the binding to MAL. Here we present structural, biochemical and cell biological evidence that MAL has a classical bipartite nuclear localization signal (NLS) in the N-terminal 'RPEL' domain containing Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs. The NLS residues of MAL adopt an extended conformation and bind along the surface groove of importin-{alpha}, interacting with the major- and minor-NLS binding sites. We also present a crystal structure of wild-type MAL RPEL domain in complex with five G-actins. Comparison of the importin-{alpha}- and actin-complexes revealed that the binding of G-actins to MAL is associated with folding of NLS residues into a helical conformation that is inappropriate for importin-{alpha} recognition.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

Triptolide inhibits COX-2 expression by regulating mRNA stability in TNF-{alpha}-treated A549 cells

Highlights: Black-Right-Pointing-Pointer Triptolide inhibited COX-2 expression and the half-life of COX-2 mRNA is decreased. Black-Right-Pointing-Pointer The HuR protein shuttling from nucleus to cytoplasm is inhibited by triptolide. Black-Right-Pointing-Pointer Triptolide inhibited 3 Prime -UTR fluorescence reporter gene activity. Black-Right-Pointing-Pointer COX-2 mRNA binding to HuR is decreased by triptolide in pull-down experiments. -- Abstract: Cyclooxygenase-2 (COX-2) over-expression is frequently associated with human non-small-cell lung cancer (NSCLC) and involved in tumor proliferation, invasion, angiogenesis and resistance to apoptosis. In the present study, the effects of triptolide on COX-2 expression in A549 cells were investigated and triptolide was found to inhibit TNF-{alpha}-induced COX-2 expression. In our further studies, it was found that triptolide decreased the half-life of COX-2 mRNA dramatically and that it inhibited 3 Prime -untranslated region (3 Prime -UTR) fluorescence reporter gene activity. Meanwhile, triptolide inhibited the HuR shuttling from nucleus to cytoplasm. After triptolide treatment, decreased COX-2 mRNA in pull-down experiments with anti-HuR antibodies was observed, indicating that the decreased cytoplasmic HuR is responsible for the decreased COX-2 mRNA. Taken together, our results provided evidence for the first time that triptolide inhibited COX-2 expression by COX-2 mRNA stability modulation and post-transcriptional regulation. These results provide a novel mechanism of action for triptolide which may be important in the treatment of lung cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Engineering of a novel Ca{sup 2+}-regulated kinesin molecular motor using a calmodulin dimer linker

Highlights: Black-Right-Pointing-Pointer Engineered kinesin-M13 and calmodulin involving single cysteine were prepared. Black-Right-Pointing-Pointer CaM mutant was cross-linked to dimer by bifunctional thiol reactive reagent. Black-Right-Pointing-Pointer Kinesin-M13 was dimerized via CaM dimer in the presence of calcium. Black-Right-Pointing-Pointer Function of the engineered kinesin was regulated by a Ca{sup 2+}-calmodulin dimer linker. -- Abstract: The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have 'on-off' control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesin monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca{sup 2+}-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca{sup 2+}-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Structural determination of importin alpha in complex with beak and feather disease virus capsid nuclear localization signal

Highlights: •Circovirus capsid proteins contain large nuclear localization signals (NLS). •A method of nuclear import has not been elucidated. •Beak and feather disease virus (BFDV) capsid NLS was crystallized with importin α. •The structure showed BFDV NLS binding to the major site of importin α. •Result shows implications for mechanism of nuclear transport for all circoviruses. -- Abstract: Circoviruses represent a rapidly increasing genus of viruses that infect a variety of vertebrates. Replication requires shuttling viral molecules into the host cell nucleus, a process facilitated by capsid-associated protein (Cap). Whilst a nuclear localization signal (NLS) has been shown to mediate nuclear translocation, the mode of nuclear transport remains to be elucidated. To better understand this process, beak and feather disease virus (BFDV) Cap NLS was crystallized with nuclear import receptor importin-α (Impα). Diffraction yielded structural data to 2.9 Å resolution, and the binding site on both Impα and BFDV Cap NLS were well resolved. The binding mechanism for the major site is likely conserved across circoviruses as supported by the similarity of NLSs in circovirus Caps. This finding illuminates a crucial step for infection of host cells by this viral family, and provides a platform for rational drug design against the binding interface.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Crystal structure of the karyopherin Kap121p bound to the extreme C-terminus of the protein phosphatase Cdc14p

In Saccharomyces cerevisiae, the protein phosphatase Cdc14p is an antagonist of mitotic cyclin-dependent kinases and is a key regulator of late mitotic events such as chromosome segregation, spindle disassembly and cytokinesis. The activity of Cdc14p is controlled by cell-cycle dependent changes in its association with its competitive inhibitor Net1p (also known as Cfi1p) in the nucleolus. For most of the cell cycle up to metaphase, Cdc14p is sequestered in the nucleolus in an inactive state. During anaphase, Cdc14p is released from Net1p, spreads into the nucleus and cytoplasm, and dephosphorylates key mitotic targets. Although regulated nucleocytoplasmic shuttling of Cdc14p has been suggested to be important for exit from mitosis, the mechanism underlying Cdc14p nuclear trafficking remains poorly understood. Here we show that the C-terminal region (residues 517–551) of Cdc14p can function as a nuclear localization signal (NLS) in vivo and also binds to Kap121p (also known as Pse1p), an essential nuclear import carrier in yeast, in a Gsp1p-GTP-dependent manner in vitro. Moreover we report a crystal structure, at 2.4 Å resolution, of Kap121p bound to the C-terminal region of Cdc14p. The structure and structure-based mutational analyses suggest that either the last five residues at the extreme C-terminus of Cdc14p (residues 547–551; Gly-Ser-Ile-Lys-Lys) or adjacent residues with similar sequence (residues 540–544; Gly-Gly-Ile-Arg-Lys) can bind to the NLS-binding site of Kap121p, with two residues (Ile in the middle and Lys at the end of the five residues) of Cdc14p making key contributions to the binding specificity. Based on comparison with other structures of Kap121p-ligand complexes, we propose “IK-NLS” as an appropriate term to refer to the Kap121p-specific NLS. - Highlights: • The C-terminus of Cdc14p binds to Kap121p in a Gsp1p-GTP-dependent manner. • The crystal structure of Kap121p-Cdc14p complex is determined. • The structure reveals how Cdc14p binds to the NLS-binding site of Kap121p. • “IK-NLS” is proposed as an appropriate name to refer to the Kap121p-specific NLS.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Versatile function of the circadian protein CIPC as a regulator of Erk activation

The CLOCK-interacting protein, Circadian (CIPC), has been identified as an additional negative-feedback regulator of the circadian clock. However, recent study on CIPC knockout mice has shown that CIPC is not critically required for basic circadian clock function, suggesting other unknown biological roles for CIPC. In this study, we focused on the cell cycle dependent nuclear-cytoplasmic shuttling function of CIPC and on identifying its binding proteins. Lys186 and 187 were identified as the essential amino acid residues within the nuclear localization signal (NLS) of CIPC. We identified CIPC-binding proteins such as the multifunctional enzyme CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), which is a key enzyme for de novo pyrimidine synthesis. Compared to control cells, HEK293 cells overexpressing wild-type CIPC showed suppressed cell proliferation and retardation of cell cycle. We also found that PMA-induced Erk activation was inhibited with expression of wild-type CIPC. In contrast, the NLS mutant of CIPC, which reduced the ability of CIPC to translocate into the nucleus, did not exhibit these biological effects. Since CAD and Erk have significant roles in cell proliferation and cell cycle, CIPC may work as a cell cycle regulator by interacting with these binding proteins. - Highlights: • CIPC is a cell cycle dependent nuclear-cytoplasmic shuttling protein. • K186 and 187are the essential amino acid residues within the NLS of CIPC. • CAD was identified as a novel CIPC-binding protein. • CIPC might regulate the activity and translocation of CAD in the cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Development of exosome surface display technology in living human cells

Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell–cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

PAK1 translocates into nucleus in response to prolactin but not to estrogen

Tyrosyl phosphorylation of the p21-activated serine–threonine kinase 1 (PAK1) has an essential role in regulating PAK1 functions in breast cancer cells. We previously demonstrated that PAK1 serves as a common node for estrogen (E2)- and prolactin (PRL)-dependent pathways. We hypothesize herein that intracellular localization of PAK1 is affected by PRL and E2 treatments differently. We demonstrate by immunocytochemical analysis that PAK1 nuclear translocation is ligand-dependent: only PRL but not E2 stimulated PAK1 nuclear translocation. Tyrosyl phosphorylation of PAK1 is essential for this nuclear translocation because phospho-tyrosyl-deficient PAK1 Y3F mutant is retained in the cytoplasm in response to PRL. We confirmed these data by Western blot analysis of subcellular fractions. In 30 min of PRL treatment, only 48% of pTyr-PAK1 is retained in the cytoplasm of PAK1 WT clone while 52% re-distributes into the nucleus and pTyr-PAK1 shuttles back to the cytoplasm by 60 min of PRL treatment. In contrast, PAK1 Y3F is retained in the cytoplasm. E2 treatment causes nuclear translocation of neither PAK1 WT nor PAK1 Y3F. Finally, we show by an in vitro kinase assay that PRL but not E2 stimulates PAK1 kinase activity in the nuclear fraction. Thus, PAK1 nuclear translocation is ligand-dependent: PRL activates PAK1 and induces translocation of activated pTyr-PAK1 into nucleus while E2 activates pTyr-PAK1 only in the cytoplasm. - Highlights: • Prolactin but not estrogen causes translocation of PAK1 into nucleus. • Tyrosyl phosphorylation of PAK1 is required for nuclear localization. • Prolactin but not estrogen stimulates PAK1 kinase activity in nucleus.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Zinc fingers 1, 2, 5 and 6 of transcriptional regulator, PRDM4, are required for its nuclear localisation

PRDM4 is a member of the PRDM family of transcriptional regulators which control various aspects of cellular differentiation and proliferation. PRDM proteins exert their biological functions both in the cytosol and the nucleus of cells. All PRDM proteins are characterised by the presence of two distinct structural motifs, the PR/SET domain and the zinc finger (ZF) motifs. We previously observed that deletion of all six zinc fingers found in PRDM4 leads to its accumulation in the cytosol, whereas overexpressed full length PRDM4 is found predominantly in the nucleus. Here, we investigated the requirements for single zinc fingers in the nuclear localisation of PRDM4. We demonstrate that ZF's 1, 2, 5 and 6 contribute to the accumulation of PRDM4 in the nucleus. Their effect is additive as deleting either ZF1-2 or ZF 5–6 redistributes PRDM4 protein from being almost exclusively nuclear to cytosolic and nuclear. We investigated the potential mechanism of nuclear shuttling of PRDM4 via the importin α/β-mediated pathway and find that PRDM4 nuclear targeting is independent of α/β-mediated nuclear import. -- Highlights: •Zinc fingers 1, 2, 5, and 6 are necessary for efficient nuclear localisation of PRDM4. •Zinc fingers 3 and 4 are dispensable for nuclear localisation of PRDM4. •Zinc knuckle is dispensable for nuclear localisation of PRDM4. •PRDM4 nuclear transport is independent of importin α/β-mediated pathway of nuclear import.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling

Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reduced following the activation of adenosine 5′ monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

A novel role for the nuclear localization signal in regulating hnRNP K protein stability in vivo

hnRNP K is a highly conserved nucleocytoplasmic shuttling protein, which associates with RNAs through synergistic binding via its three KH domains. hnRNP K is required for proper nuclear export and translational control of its mRNA targets, and these processes are controlled by hnRNP K's movement between subcellular compartments. Whereas the nuclear export and localization of hnRNP K that is associated with mRNP complexes has been well studied, the trafficking of hnRNP K that is unbound to mRNA has yet to be elucidated. To that end, we expressed an EGFP-tagged RNA binding-defective form of hnRNP K in intact Xenopus embryos, and found it was rapidly degraded in vivo. Deleting hnRNP K's nuclear localization signal (NLS), which contains two prospective ubiquitination sites, rescued the protein from degradation. These data demonstrate a novel activity for the NLS of hnRNP K in regulating the protein's stability in vivo when it is unbound to nucleic acids. - Highlights: • An RNA-binding mutant of Xenopus hnRNP K is degraded in vivo. • hnRNP K's nuclear localization signal contains two predicted ubiquitination sites. • Deletion of the nuclear localization signal rescues RNA-binding mutant degradation.

60 APPLIED LIFE SCIENCES↗