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Sample records for functional nuclear localization

  1. Multiple nuclear localization signals function in the nuclear import of the transcription factor Nrf2.

    PubMed

    Theodore, Melanie; Kawai, Yumiko; Yang, Jianqi; Kleshchenko, Yuliya; Reddy, Sekhar P; Villalta, Fernando; Arinze, Ifeanyi J

    2008-04-04

    Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494-511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins alpha5 and beta1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin alpha-beta heterodimer nuclear import receptor system plays a critical role in the import process.

  2. Intracellular localization of human cytidine deaminase. Identification of a functional nuclear localization signal.

    PubMed

    Somasekaram, A; Jarmuz, A; How, A; Scott, J; Navaratnam, N

    1999-10-01

    The cytidine deaminases belong to the family of multisubunit enzymes that catalyze the hydrolytic deamination of their substrate to a corresponding uracil product. They play a major role in pyrimidine nucleoside and nucleotide salvage. The intracellular distribution of cytidine deaminase and related enzymes has previously been considered to be cytosolic. Here we show that human cytidine deaminase (HCDA) is present in the nucleus. A highly specific, affinity purified polyclonal antibody against HCDA was used to analyze the intracellular localization of native HCDA in a variety of mammalian cells by in situ immunochemistry. Native HCDA was found to be present in the nucleus as well as the cytoplasm in several cell types. Indirect immunofluorescence microscopy indicated a predominantly nuclear localization of FLAG-tagged HCDA overexpressed in these cells. We have identified an amino-terminal bipartite nuclear localization signal that is both necessary and sufficient to direct HCDA and a non-nuclear reporter protein to the nucleus. We also show HCDA binding to the nuclear import receptor, importin alpha. Similar putative bipartite nuclear localization sequences are found in other cytidine/deoxycytidylate deaminases. The results presented here suggest that the pyrimidine nucleotide salvage pathway may operate in the nucleus. This localization may have implications in the regulation of nucleoside and nucleotide metabolism and nucleic acid biosynthesis.

  3. Identification and functional characterization of a novel bipartite nuclear localization sequence in ARID1A.

    PubMed

    Bateman, Nicholas W; Shoji, Yutaka; Conrads, Kelly A; Stroop, Kevin D; Hamilton, Chad A; Darcy, Kathleen M; Maxwell, George L; Risinger, John I; Conrads, Thomas P

    2016-01-01

    AT-rich interactive domain-containing protein 1A (ARID1A) is a recently identified nuclear tumor suppressor frequently altered in solid tumor malignancies. We have identified a bipartite-like nuclear localization sequence (NLS) that contributes to nuclear import of ARID1A not previously described. We functionally confirm activity using GFP constructs fused with wild-type or mutant NLS sequences. We further show that cyto-nuclear localized, bipartite NLS mutant ARID1A exhibits greater stability than nuclear-localized, wild-type ARID1A. Identification of this undescribed functional NLS within ARID1A contributes vital insights to rationalize the impact of ARID1A missense mutations observed in patient tumors.

  4. Transverse isospin response function of asymmetric nuclear matter from a local isospin density functional

    NASA Astrophysics Data System (ADS)

    Lipparini, Enrico; Pederiva, Francesco

    2016-08-01

    The time dependent local isospin density approximation (TDLIDA) has been extended to the study of the transverse isospin response function in nuclear matter with an arbitrary neutron-proton asymmetry parameter ξ . The energy density functional has been chosen in order to fit existing accurate quantum Monte Carlo calculations with a density dependent potential. The evolution of the response with ξ in the Δ Tz=±1 channels is quite different. While the strength of the Δ Tz=+1 channel disappears rather quickly by increasing the asymmetry, the Δ Tz=-1 channel develops a stronger and stronger collective mode that in the regime typical of neutron star matter at β equilibrium almost completely exhausts the excitation spectrum of the system. The neutrino mean free paths obtained from the TDLIDA responses are strongly dependent on ξ and on the presence of collective modes, leading to a sizable difference with respect to the prediction of the Fermi gas model.

  5. Identification of a functional nuclear localization signal within the human USP22 protein

    SciTech Connect

    Xiong, Jianjun; Wang, Yaqin; Gong, Zhen; Liu, Jianyun; Li, Weidong

    2014-06-20

    Highlights: • USP22 was accumulated in nucleus. • We identified of a functional USP22 NLS. • The KRRK amino acid residues are indispensable in NLS. • The KRRK motif is conserved in USP22 homologues. - Abstract: Ubiquitin-specific processing enzyme 22 (USP22), a member of the deubiquitinase family, is over-expressed in most human cancers and has been implicated in tumorigenesis. Because it is an enzymatic subunit of the human SAGA transcriptional cofactor, USP22 deubiquitylates histone H2A and H2B in the nucleus, thus participating in gene regulation and cell-cycle progression. However, the mechanisms regulating its nuclear translocation have not yet been elucidated. It was here demonstrated that USP22 is imported into the nucleus through a mechanism mediated by nuclear localization signal (NLS). The bipartite NLS sequence KRELELLKHNPKRRKIT (aa152–168), was identified as the functional NLS for its nuclear localization. Furthermore, a short cluster of basic amino acid residues KRRK within this bipartite NLS plays the primary role in nuclear localization and is evolutionarily conserved in USP22 homologues. In the present study, a functional NLS and the minimal sequences required for the active targeting of USP22 to the nucleus were identified. These findings may provide a molecular basis for the mechanism underlying USP22 nuclear trafficking and function.

  6. Functional and structural basis of the nuclear localization signal in the ZIC3 zinc finger domain

    PubMed Central

    Hatayama, Minoru; Tomizawa, Tadashi; Sakai-Kato, Kumiko; Bouvagnet, Patrice; Kose, Shingo; Imamoto, Naoko; Yokoyama, Shigeyuki; Utsunomiya-Tate, Naoko; Mikoshiba, Katsuhiko; Kigawa, Takanori; Aruga, Jun

    2008-01-01

    Disruptions in ZIC3 cause heterotaxy, a congenital anomaly of the left–right axis. ZIC3 encodes a nuclear protein with a zinc finger (ZF) domain that contains five tandem C2H2 ZF motifs. Missense mutations in the first ZF motif (ZF1) result in defective nuclear localization, which may underlie the pathogenesis of heterotaxy. Here we revealed the structural and functional basis of the nuclear localization signal (NLS) of ZIC3 and investigated its relationship to the defect caused by ZF1 mutation. The ZIC3 NLS was located in the ZF2 and ZF3 regions, rather than ZF1. Several basic residues interspersed throughout these regions were responsible for the nuclear localization, but R320, K337 and R350 were particularly important. NMR structure analysis revealed that ZF1–4 had a similar structure to GLI ZF, and the basic side chains of the NLS clustered together in two regions on the protein surface, similar to classical bipartite NLSs. Among the residues for the ZF1 mutations, C253 and H286 were positioned for the metal chelation, whereas W255 was positioned in the hydrophobic core formed by ZF1 and ZF2. Tryptophan 255 was a highly conserved inter-finger connector and formed part of a structural motif (tandem CXW-C-H-H) that is shared with GLI, Glis and some fungal ZF proteins. Furthermore, we found that knockdown of Karyopherin α1/α6 impaired ZIC3 nuclear localization, and physical interactions between the NLS and the nuclear import adapter proteins were disturbed by mutations in the NLS but not by W255G. These results indicate that ZIC3 is imported into the cell nucleus by the Karyopherin (Importin) system and that the impaired nuclear localization by the ZF1 mutation is not due to a direct influence on the NLS. PMID:18716025

  7. Functional analysis of the Cucumber mosaic virus 2b protein: pathogenicity and nuclear localization.

    PubMed

    Wang, Yongzeng; Tzfira, Tzvi; Gaba, Victor; Citovsky, Vitaly; Palukaitis, Peter; Gal-On, Amit

    2004-10-01

    The 2b protein encoded by Cucumber mosaic virus (CMV) has been shown to be a silencing suppressor and pathogenicity determinant in solanaceous hosts, but a movement determinant in cucumber. In addition, synergistic interactions between CMV and Zucchini yellow mosaic virus (ZYMV) have been described in several cucurbit species. Here, it was shown that deletion of the 2b gene from CMV prevented extensive systemic movement of the virus in zucchini squash, which could not be complemented by co-infection with ZYMV. Thus, ZYMV expressing a silencing suppressor with a different target could not complement the CMV 2b-specific movement function. Expression of the 2b protein from an attenuated ZYMV vector resulted in a synergistic response, largely restoring infection symptoms of wild-type ZYMV in several cucurbit species. Deletion or alteration of either of two nuclear localization signals (NLSs) did not affect nuclear localization in two assays, but did affect pathogenicity in several cucurbit species, whilst deletion of both NLSs led to loss of nuclear localization. The 2b protein interacted with an Arabidopsis thaliana karyopherin alpha protein (AtKAPalpha) in the yeast two-hybrid system, as did each of the two single NLS-deletion mutants. However, 2b protein containing a deletion of both NLSs was unable to interact with AtKAPalpha. These data suggest that the 2b protein localizes to the nucleus by using the karyopherin alpha-mediated system, but demonstrate that nuclear localization was insufficient for enhancement of the 2b-mediated pathogenic response in cucurbit hosts. Thus, the sequences corresponding to the two NLSs must have another role leading to pathogenicity enhancement.

  8. A functional nuclear localization sequence in the C. elegans TRPV channel OCR-2.

    PubMed

    Ezak, Meredith J; Ferkey, Denise M

    2011-01-01

    The ability to modulate gene expression in response to sensory experience is critical to the normal development and function of the nervous system. Calcium is a key activator of the signal transduction cascades that mediate the process of translating a cellular stimulus into transcriptional changes. With the recent discovery that the mammalian Ca(v)1.2 calcium channel can be cleaved, enter the nucleus and act as a transcription factor to control neuronal gene expression, a more direct role for the calcium channels themselves in regulating transcription has begun to be appreciated. Here we report the identification of a nuclear localization sequence (NLS) in the C. elegans transient receptor potential vanilloid (TRPV) cation channel OCR-2. TRPV channels have previously been implicated in transcriptional regulation of neuronal genes in the nematode, although the precise mechanism remains unclear. We show that the NLS in OCR-2 is functional, being able to direct nuclear accumulation of a synthetic cargo protein as well as the carboxy-terminal cytosolic tail of OCR-2 where it is endogenously found. Furthermore, we discovered that a carboxy-terminal portion of the full-length channel can localize to the nucleus of neuronal cells. These results suggest that the OCR-2 TRPV cation channel may have a direct nuclear function in neuronal cells that was not previously appreciated.

  9. A Functional Nuclear Localization Sequence in the C. elegans TRPV Channel OCR-2

    PubMed Central

    Ezak, Meredith J.; Ferkey, Denise M.

    2011-01-01

    The ability to modulate gene expression in response to sensory experience is critical to the normal development and function of the nervous system. Calcium is a key activator of the signal transduction cascades that mediate the process of translating a cellular stimulus into transcriptional changes. With the recent discovery that the mammalian Cav1.2 calcium channel can be cleaved, enter the nucleus and act as a transcription factor to control neuronal gene expression, a more direct role for the calcium channels themselves in regulating transcription has begun to be appreciated. Here we report the identification of a nuclear localization sequence (NLS) in the C. elegans transient receptor potential vanilloid (TRPV) cation channel OCR-2. TRPV channels have previously been implicated in transcriptional regulation of neuronal genes in the nematode, although the precise mechanism remains unclear. We show that the NLS in OCR-2 is functional, being able to direct nuclear accumulation of a synthetic cargo protein as well as the carboxy-terminal cytosolic tail of OCR-2 where it is endogenously found. Furthermore, we discovered that a carboxy-terminal portion of the full-length channel can localize to the nucleus of neuronal cells. These results suggest that the OCR-2 TRPV cation channel may have a direct nuclear function in neuronal cells that was not previously appreciated. PMID:21957475

  10. Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages.

    PubMed

    Redrejo-Rodríguez, Modesto; Muñoz-Espín, Daniel; Holguera, Isabel; Mencía, Mario; Salas, Margarita

    2012-11-06

    A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of Φ29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of Φ29 TP led us to identify a bona fide NLS within residues 1-37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of Φ29 TP attached to the 5' DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes.

  11. A functional nuclear localization sequence in the VP1 capsid protein of coxsackievirus B3

    SciTech Connect

    Wang, Tianying; Yu, Bohai; Lin, Lexun; Zhai, Xia; Han, Yelu; Qin, Ying; Guo, Zhiwei; Wu, Shuo; Zhong, Xiaoyan; Wang, Yan; Tong, Lei; Zhang, Fengmin; Si, Xiaoning; Zhao, Wenran; Zhong, Zhaohua

    2012-11-25

    The capsid proteins of some RNA viruses can translocate to the nucleus and interfere with cellular phenotypes. In this study we found that the VP1 capsid protein of coxsackievirus B3 (CVB3) was dominantly localized in the nucleus of the cells transfected with VP1-expressing plasmid. The VP1 nuclear localization also occurred in the cells infected with CVB3. Truncation analysis indicated that the VP1 nuclear localization sequence located near the C-terminal. The substitution of His220 with threonine completely abolished its translocation. The VP1 proteins of other CVB types might have the nuclear localization potential because this region was highly conserved. Moreover, the VP1 nuclear localization induced cell cycle deregulation, including a prolonged S phase and shortened G2-M phase. Besides these findings, we also found a domain between Ala72 and Phe106 that caused the VP1 truncates dotted distributed in the cytoplasm. Our results suggest a new pathogenic mechanism of CVB. - Highlights: Black-Right-Pointing-Pointer The VP1 protein of coxsackievirus B3 can specifically localize in the nucleus. Black-Right-Pointing-Pointer The nuclear localization signal of coxsackievirus B3 VP1 protein locates near its C-terminal. Black-Right-Pointing-Pointer The VP1 nuclear localization of coxsackievirus B3 can deregulate cell cycle. Black-Right-Pointing-Pointer There is a domain in the VP1 that determines it dotted distributed in the cytoplasm.

  12. Nuclear localization and functional characteristics of voltage-gated potassium channel Kv1.3.

    PubMed

    Jang, Soo Hwa; Byun, Jun Kyu; Jeon, Won-Il; Choi, Seon Young; Park, Jin; Lee, Bo Hyung; Yang, Ji Eun; Park, Jin Bong; O'Grady, Scott M; Kim, Dae-Yong; Ryu, Pan Dong; Joo, Sang-Woo; Lee, So Yeong

    2015-05-15

    It is widely known that ion channels are expressed in the plasma membrane. However, a few studies have suggested that several ion channels including voltage-gated K(+) (Kv) channels also exist in intracellular organelles where they are involved in the biochemical events associated with cell signaling. In the present study, Western blot analysis using fractionated protein clearly indicates that Kv1.3 channels are expressed in the nuclei of MCF7, A549, and SNU-484 cancer cells and human brain tissues. In addition, Kv1.3 is located in the plasma membrane and the nucleus of Jurkat T cells. Nuclear membrane hyperpolarization after treatment with margatoxin (MgTX), a specific blocker of Kv1.3 channels, provides evidence for functional channels at the nuclear membrane of A549 cells. MgTX-induced hyperpolarization is abolished in the nuclei of Kv1.3 silenced cells, and the effects of MgTX are dependent on the magnitude of the K(+) gradient across the nuclear membrane. Selective Kv1.3 blockers induce the phosphorylation of cAMP response element-binding protein (CREB) and c-Fos activation. Moreover, Kv1.3 is shown to form a complex with the upstream binding factor 1 in the nucleus. Chromatin immunoprecipitation assay reveals that Sp1 transcription factor is directly bound to the promoter region of the Kv1.3 gene, and the Sp1 regulates Kv1.3 expression in the nucleus of A549 cells. These results demonstrate that Kv1.3 channels are primarily localized in the nucleus of several types of cancer cells and human brain tissues where they are capable of regulating nuclear membrane potential and activation of transcription factors, such as phosphorylated CREB and c-Fos.

  13. Nuclear Localization and Functional Characteristics of Voltage-gated Potassium Channel Kv1.3*

    PubMed Central

    Jang, Soo Hwa; Byun, Jun Kyu; Jeon, Won-Il; Choi, Seon Young; Park, Jin; Lee, Bo Hyung; Yang, Ji Eun; Park, Jin Bong; O'Grady, Scott M.; Kim, Dae-Yong; Ryu, Pan Dong; Joo, Sang-Woo; Lee, So Yeong

    2015-01-01

    It is widely known that ion channels are expressed in the plasma membrane. However, a few studies have suggested that several ion channels including voltage-gated K+ (Kv) channels also exist in intracellular organelles where they are involved in the biochemical events associated with cell signaling. In the present study, Western blot analysis using fractionated protein clearly indicates that Kv1.3 channels are expressed in the nuclei of MCF7, A549, and SNU-484 cancer cells and human brain tissues. In addition, Kv1.3 is located in the plasma membrane and the nucleus of Jurkat T cells. Nuclear membrane hyperpolarization after treatment with margatoxin (MgTX), a specific blocker of Kv1.3 channels, provides evidence for functional channels at the nuclear membrane of A549 cells. MgTX-induced hyperpolarization is abolished in the nuclei of Kv1.3 silenced cells, and the effects of MgTX are dependent on the magnitude of the K+ gradient across the nuclear membrane. Selective Kv1.3 blockers induce the phosphorylation of cAMP response element-binding protein (CREB) and c-Fos activation. Moreover, Kv1.3 is shown to form a complex with the upstream binding factor 1 in the nucleus. Chromatin immunoprecipitation assay reveals that Sp1 transcription factor is directly bound to the promoter region of the Kv1.3 gene, and the Sp1 regulates Kv1.3 expression in the nucleus of A549 cells. These results demonstrate that Kv1.3 channels are primarily localized in the nucleus of several types of cancer cells and human brain tissues where they are capable of regulating nuclear membrane potential and activation of transcription factors, such as phosphorylated CREB and c-Fos. PMID:25829491

  14. The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function

    PubMed Central

    Cattaruzzi, Giacomo; Altamura, Sandro; Tessari, Michela A.; Rustighi, Alessandra; Giancotti, Vincenzo; Pucillo, Carlo; Manfioletti, Guidalberto

    2007-01-01

    High Mobility Group A (HMGA) is a family of architectural nuclear factors which play an important role in neoplastic transformation. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes including transcription. HMGA localization is exclusively nuclear but, to date, the mechanism of nuclear import for these proteins remains unknown. Here, we report the identification and characterization of a nuclear localization signal (NLS) for HMGA2, a member of the HMGA family. The NLS overlaps with the second of the three AT-hooks, the DNA-binding domains characteristic for this group of proteins. The functionality of this NLS was demonstrated by its ability to target a heterologous β-galactosidase/green fluorescent protein fusion protein to the nucleus. Mutations to alanine of basic residues within the second AT-hook resulted in inhibition of HMGA2 nuclear localization and impairment of its function in activating the cyclin A promoter. In addition, HMGA2 was shown to directly interact with the nuclear import receptor importin-α2 via the second AT-hook. HMGA proteins are overexpressed and rearranged in a variety of tumors; our findings can thus help elucidating their role in neoplastic transformation. PMID:17324944

  15. Breast Cancer–Associated Abraxas Mutation Disrupts Nuclear Localization and DNA Damage Response Functions

    PubMed Central

    Solyom, Szilvia; Aressy, Bernadette; Pylkäs, Katri; Patterson-Fortin, Jeffrey; Hartikainen, Jaana M.; Kallioniemi, Anne; Kauppila, Saila; Nikkilä, Jenni; Kosma, Veli-Matti; Mannermaa, Arto; Greenberg, Roger A.; Winqvist, Robert

    2013-01-01

    Breast cancer is the most common cancer in women in developed countries and has a well-established genetic component. Germline mutations in a network of genes encoding BRCA1, BRCA2, and their interacting partners confer hereditary susceptibility to breast cancer. Abraxas directly interacts with the BRCA1 BRCT (BRCA1 carboxyl-terminal) repeats and contributes to BRCA1-dependent DNA damage responses, making Abraxas a candidate for yet unexplained disease susceptibility. Here, we have screened 125 Northern Finnish breast cancer families for coding region and splice-site Abraxas mutations and genotyped three tagging single-nucleotide polymorphisms within the gene from 991 unselected breast cancer cases and 868 female controls for common cancer-associated variants. A novel heterozygous alteration, c.1082G>A (Arg361Gln), that results in abrogated nuclear localization and DNA response activities was identified in three breast cancer families and in one additional familial case from an unselected breast cancer cohort, but not in healthy controls (P = 0.002). On the basis of its exclusive occurrence in familial cancers, disease cosegregation, evolutionary conservation, and disruption of critical BRCA1 functions, the recurrent Abraxas c.1082G>A mutation connects to cancer predisposition. These findings contribute to the concept of a BRCA-centered tumor suppressor network and provide the identity of Abraxas as a new breast cancer susceptibility gene. PMID:22357538

  16. The nuclear localization signal of mitotic kinesin-like protein Mklp-1: effect on Mklp-1 function during cytokinesis.

    PubMed

    Liu, Xiaoqi; Erikson, Raymond L

    2007-02-23

    The mitotic kinesin-like protein (Mklp-1) localizes in the nucleus during interphase due to the presence of nuclear localization signal(s) [NLS(s)] within its sequence. Here, we mapped two NLSs to be 899SRKRRSST906 and 949KRKKP953 in the tail domain of Mklp-1, and showed that ectopic expression of a mutant Mklp-1 without the NLSs leads to cell cycle arrest at cytokinesis, indicating that the NLSs are necessary for Mklp-1 to execute its normal function during cell division. Furthermore, mutation of two serine residues in the first NLS to aspartic acid, which mimics phosphorylation, attenuated its nuclear localization function, suggesting that the function of this NLS might be regulated by phosphorylation.

  17. Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

    SciTech Connect

    Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2014-11-15

    The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

  18. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

    SciTech Connect

    Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin; Sun, Ya-Ni; Gao, Ji-Ming; Xie, Zhi-Jing; Wang, Yu; Zhu, Yan-Li; Jiang, Shi-Jin

    2013-02-05

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

  19. The N-terminal nuclear localization sequences of liver X receptors alpha and beta bind to importin alpha and are essential for both nuclear import and transactivating functions.

    PubMed

    Miller, Anna; Crumbley, Christine; Prüfer, Kirsten

    2009-04-01

    Liver X receptors (LXRs) alpha and beta are nuclear receptors, which form obligate heterodimers with the retinoid X receptor (RXR). The LXRs regulate both redundantly and non-redundantly the transcription of genes controlling cholesterol metabolism and transport as well as lipogenesis. Previously, we showed that mutations in putative N-terminal nuclear localization sequences (NLSs) within both LXRs inhibit nuclear import. Through in vitro studies, we show here that these NLSs bind importin alpha and are both necessary and sufficient for the nuclear import of LXRs. Imaging, transactivation, and electro-mobility shift experiments show that RXR rescues the nuclear import of the LXRalpha NLS mutant yet does not restore its transcriptional activity despite intact DNA binding. In contrast, RXR partially rescues the import of the LXRbeta NLS mutant, but has no effect on its transcriptional activity due to the loss of DNA binding. Experiments with NLS mutant RXR confirmed that RXR may dominate the nuclear import of the RXR/LXRalpha heterodimer, whereas LXRbeta dominates the nuclear import of the RXR/LXRbeta heterodimer. Intriguingly, our data indicate differences between LXRalpha and LXRbeta in their interaction with RXR and in the role their NLSs play in transactivating functions independent of nuclear import.

  20. Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

    SciTech Connect

    Kakuk, Annamaria; Friedlaender, Elza; Vereb, Gyoergy; Lisboa, Duarte; Bagossi, Peter; Toth, Gabor; Gergely, Pal; Vereb, Gyoergy

    2008-08-01

    PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence {sup 916}NFNHIHKRIRRVADKYLSG{sup 934} comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin {alpha}/{beta} mechanism employing importins {alpha}1 and {alpha}3 but not {alpha}5. This transport was inhibited by wheat germ agglutinin and GTP{gamma}S. The sequence {sup 1414}SKKTNRGSQLHKYYMKRRTL{sup 1433}, a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166-1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin {alpha}1/{beta} or {alpha}3/{beta} complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.

  1. Transcription-dependent nucleolar cap localization and possible nuclear function of DExH RNA helicase RHAU

    SciTech Connect

    Iwamoto, Fumiko; Stadler, Michael; Chalupnikova, Katerina; Oakeley, Edward; Nagamine, Yoshikuni

    2008-04-01

    RHAU (RNA helicase associated with AU-rich element) is a DExH protein originally identified as a factor accelerating AU-rich element-mediated mRNA degradation. The discovery that RHAU is predominantly localized in the nucleus, despite mRNA degradation occurring in the cytoplasm, prompted us to consider the nuclear functions of RHAU. In HeLa cells, RHAU was found to be localized throughout the nucleoplasm with some concentrated in nuclear speckles. Transcriptional arrest altered the localization to nucleolar caps, where RHAU is closely localized with RNA helicases p68 and p72, suggesting that RHAU is involved in transcription-related RNA metabolism in the nucleus. To see whether RHAU affects global gene expression transcriptionally or posttranscriptionally, we performed microarray analysis using total RNA from RHAU-depleted HeLa cell lines, measuring both steady-state mRNA levels and mRNA half-lives by actinomycin D chase. There was no change in the half-lives of most transcripts whose steady-state levels were affected by RHAU knockdown, suggesting that these transcripts are subjected to transcriptional regulation. We propose that RHAU has a dual function, being involved in both the synthesis and degradation of mRNA in different subcellular compartments.

  2. Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

    PubMed Central

    Don-Salu-Hewage, Ayesha S.; Chan, Siu Yuen; McAndrews, Kathleen M.; Chetram, Mahandranauth A.; Dawson, Michelle R.; Bethea, Danaya A.; Hinton, Cimona V.

    2013-01-01

    The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), ‘RPRK’, within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinβ1 and Transportinβ1-binding to CXCR4 promoted its nuclear translocation. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4. PMID:23468933

  3. Phosphorylation controls a dual-function polybasic nuclear localization sequence in the adapter protein SH2B1β to regulate its cellular function and distribution.

    PubMed

    Maures, Travis J; Su, Hsiao-Wen; Argetsinger, Lawrence S; Grinstein, Sergio; Carter-Su, Christin

    2011-05-01

    An intriguing question in cell biology is what targets proteins to, and regulates their translocation between, specific cellular locations. Here we report that the polybasic nuclear localization sequence (NLS) required for nuclear entry of the adapter protein and candidate human obesity gene product SH2B1β, also localizes SH2B1β to the plasma membrane (PM), most probably via electrostatic interactions. Binding of SH2B1β to the PM also requires its dimerization domain. Phosphorylation of serine residues near this polybasic region, potentially by protein kinase C, releases SH2B1β from the PM and enhances nuclear entry. Release of SH2B1β from the PM and/or nuclear entry appear to be required for SH2B1β enhancement of nerve growth factor (NGF)-induced expression of urokinase plasminogen activator receptor gene and neurite outgrowth of PC12 cells. Taken together, our results provide strong evidence that the polybasic NLS region of SH2B1 serves the dual function of localizing SH2B1 to both the nucleus and the PM, the latter most probably through electrostatic interactions that are enhanced by SH2B1β dimerization. Cycling between the different cellular compartments is a consequence of the phosphorylation and dephosphorylation of serine residues near the NLS and is important for physiological effects of SH2B1, including NGF-induced gene expression and neurite outgrowth.

  4. Nuclear Localization of the DNA Repair Scaffold XRCC1: Uncovering the Functional Role of a Bipartite NLS.

    PubMed

    Kirby, Thomas W; Gassman, Natalie R; Smith, Cassandra E; Pedersen, Lars C; Gabel, Scott A; Sobhany, Mack; Wilson, Samuel H; London, Robert E

    2015-08-25

    We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs of the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior provides a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.

  5. Nuclear localization of the DNA repair scaffold XRCC1: Uncovering the functional role of a bipartite NLS

    DOE PAGES

    Kirby, Thomas W.; Gassman, Natalie R.; Smith, Cassandra E.; ...

    2015-08-25

    We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs ofmore » the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior gives a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.« less

  6. Nuclear localization of the DNA repair scaffold XRCC1: Uncovering the functional role of a bipartite NLS

    SciTech Connect

    Kirby, Thomas W.; Gassman, Natalie R.; Smith, Cassandra E.; Pedersen, Lars C.; Gabel, Scott A.; Sobhany, Mack; Wilson, Samuel H.; London, Robert E.

    2015-08-25

    We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs of the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior gives a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.

  7. Functional characterization of nuclear localization and export signals in hepatitis C virus proteins and their role in the membranous web.

    PubMed

    Levin, Aviad; Neufeldt, Christopher J; Pang, Daniel; Wilson, Kristen; Loewen-Dobler, Darci; Joyce, Michael A; Wozniak, Richard W; Tyrrell, D Lorne J

    2014-01-01

    The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.

  8. Functional Characterization of Nuclear Localization and Export Signals in Hepatitis C Virus Proteins and Their Role in the Membranous Web

    PubMed Central

    Levin, Aviad; Neufeldt, Christopher J.; Pang, Daniel; Wilson, Kristen; Loewen-Dobler, Darci; Joyce, Michael A.; Wozniak, Richard W.; Tyrrell, D. Lorne J

    2014-01-01

    The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication. PMID:25485706

  9. Identification of a highly conserved, functional nuclear localization signal within the N-terminal region of herpes simplex virus type 1 VP1-2 tegument protein.

    PubMed

    Abaitua, F; O'Hare, P

    2008-06-01

    VP1-2 is a large structural protein assembled into the tegument compartment of the virion, conserved across the herpesviridae, and essential for virus replication. In herpes simplex virus (HSV) and pseudorabies virus, VP1-2 is tightly associated with the capsid. Studies of its assembly and function remain incomplete, although recent data indicate that in HSV, VP1-2 is recruited onto capsids in the nucleus, with this being required for subsequent recruitment of additional structural proteins. Here we have developed an antibody to characterize VP1-2 localization, observing the protein in both cytoplasmic and nuclear compartments, frequently in clusters in both locations. Within the nucleus, a subpopulation of VP1-2 colocalized with VP26 and VP5, though VP1-2-positive foci devoid of these components were observed. We note a highly conserved basic motif adjacent to the previously identified N-terminal ubiquitin hydrolase domain (DUB). The DUB domain in isolation exhibited no specific localization, but when extended to include the adjacent motif, it efficiently accumulated in the nucleus. Transfer of the isolated motif to a test protein, beta-galactosidase, conferred specific nuclear localization. Substitution of a single amino acid within the motif abolished the nuclear localization function. Deletion of the motif from intact VP1-2 abrogated its nuclear localization. Moreover, in a functional assay examining the ability of VP1-2 to complement growth of a VP1-2-ve mutant, deletion of the nuclear localization signal abolished complementation. The nuclear localization signal may be involved in transport of VP1-2 early in infection or to late assembly sites within the nucleus or, considering the potential existence of VP1-2 cleavage products, in selective localization of subdomains to different compartments.

  10. A Balance Between Two Nuclear Localization Sequences and a Nuclear Export Sequence Governs Extradenticle Subcellular Localization

    PubMed Central

    Stevens, Katherine E.; Mann, Richard S.

    2007-01-01

    During animal development, transcription factor activities are modulated by several means, including subcellular localization. The Hox cofactor Extradenticle (Exd) has a dynamic subcellular localization, such that Exd is cytoplasmic by default, but is nuclear when complexed with another homeodomain protein, Homothorax (Hth). These observations raise the question of whether dimerization with Hth simply induces Exd's nuclear localization or, alternatively, if Hth is also necessary for Exd activity. To address this question, we analyzed the nuclear transport signals in Exd, including a divergent nuclear export signal (NES) and two nuclear localization signals (NLSs). We show that, although these signals are weak compared to canonical signals, they balance each other in Exd. We also provide evidence that Exd contains an NLS mask that contributes to its cytoplasmic localization. With these signals characterized, we generated forms of Exd that are nuclear localized in the absence of Hth. Surprisingly, although these Exd forms are functional, they do not phenocopy Hth overexpression. These findings suggest that Hth is required for Exd activity, not simply for inducing its nuclear localization. PMID:17277370

  11. Nuclear Parton Distribution Functions

    SciTech Connect

    Schienbein, I.; Yu, J.-Y.; Keppel, Cynthia; Morfin, Jorge; Olness, F.; Owens, J.F.

    2009-01-01

    We study nuclear effects of charged current deep inelastic neutrino-iron scattering in the framework of a chi^2 analysis of parton distribution functions (PDFs). We extract a set of iron PDFs which are used to compute x_Bj-dependent and Q^2-dependent nuclear correction factors for iron structure functions which are required in global analyses of free nucleon PDFs. We compare our results with nuclear correction factors from neutrino-nucleus scattering models and correction factors for charged-lepton--iron scattering. We find that, except for very high x_Bj, our correction factors differ in both shape and magnitude from the correction factors of the models and charged-lepton scattering.

  12. Nuclear Parton Distribution Functions

    SciTech Connect

    I. Schienbein, J.Y. Yu, C. Keppel, J.G. Morfin, F. Olness, J.F. Owens

    2009-06-01

    We study nuclear effects of charged current deep inelastic neutrino-iron scattering in the framework of a {chi}{sup 2} analysis of parton distribution functions (PDFs). We extract a set of iron PDFs which are used to compute x{sub Bj}-dependent and Q{sup 2}-dependent nuclear correction factors for iron structure functions which are required in global analyses of free nucleon PDFs. We compare our results with nuclear correction factors from neutrino-nucleus scattering models and correction factors for charged-lepton--iron scattering. We find that, except for very high x{sub Bj}, our correction factors differ in both shape and magnitude from the correction factors of the models and charged-lepton scattering.

  13. Nuclear localization of tetrahydrobiopterin biosynthetic enzymes.

    PubMed

    Elzaouk, Lina; Laufs, Stephanie; Heerklotz, Dirk; Leimbacher, Walter; Blau, Nenad; Résibois, Annette; Thöny, Beat

    2004-01-05

    Biosynthesis of the tetrahydrobiopterin (BH(4)) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed beta-galactosidase in the nucleus of neurons. In contrast, PTPS-beta-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-beta-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH(4)-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5-10% of total GTPCH and PTPS and approximately 1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH(4)-biosynthetic proteins with yet unknown biological function.

  14. Nuclear functions of prefoldin

    PubMed Central

    Millán-Zambrano, Gonzalo; Chávez, Sebastián

    2014-01-01

    Prefoldin is a cochaperone, present in all eukaryotes, that cooperates with the chaperonin CCT. It is known mainly for its functional relevance in the cytoplasmic folding of actin and tubulin monomers during cytoskeleton assembly. However, both canonical and prefoldin-like subunits of this heterohexameric complex have also been found in the nucleus, and are functionally connected with nuclear processes in yeast and metazoa. Plant prefoldin has also been detected in the nucleus and physically associated with a gene regulator. In this review, we summarize the information available on the involvement of prefoldin in nuclear phenomena, place special emphasis on gene transcription, and discuss the possibility of a global coordination between gene regulation and cytoplasmic dynamics mediated by prefoldin. PMID:25008233

  15. The Arginine/Lysine-Rich Element within the DNA-Binding Domain Is Essential for Nuclear Localization and Function of the Intracellular Pathogen Resistance 1

    PubMed Central

    Yao, Kezhen; Wu, Yongyan; Chen, Qi; Zhang, Zihan; Chen, Xin; Zhang, Yong

    2016-01-01

    The mouse intracellular pathogen resistance 1 (Ipr1) gene plays important roles in mediating host immunity and previous work showed that it enhances macrophage apoptosis upon mycobacterium infection. However, to date, little is known about the regulation pattern of Ipr1 action. Recent studies have investigated the protein-coding genes and microRNAs regulated by Ipr1 in mouse macrophages, but the structure and the functional motif of the Ipr1 protein have yet to be explored. In this study, we analyzed the domains and functional motif of the Ipr1 protein. The resulting data reveal that Ipr1 protein forms a homodimer and that the Sp100-like domain mediates the targeting of Ipr1 protein to nuclear dots (NDs). Moreover, we found that an Ipr1 mutant lacking the classic nuclear localization signal (cNLS) also translocated into the nuclei, suggesting that the cNLS is not the only factor that directs Ipr1 nuclear localization. Additionally, mechanistic studies revealed that an arginine/lysine-rich element within the DNA-binding domain (SAND domain) is critical for Ipr1 binding to the importin protein receptor NPI-1, demonstrating that this element plays an essential role in mediating the nuclear localization of Ipr1 protein. Furthermore, our results show that this arginine/lysine-rich element contributes to the transcriptional regulation and apoptotic activity of Ipr1. These findings highlight the structural foundations of Ipr1 action and provide new insights into the mechanism of Ipr1-mediated resistance to mycobacterium. PMID:27622275

  16. Phenotype of HIV-1 lacking a functional nuclear localization signal in matrix protein of gag and Vpr is comparable to wild-type HIV-1 in primary macrophages.

    PubMed

    Kootstra, N A; Schuitemaker, H

    1999-01-20

    Human immunodeficiency virus type 1 (HIV-1) is considered to infect nondividing cells because nuclear localization signals (NLS) in matrix (MA, p17(Gag)) and Vpr allow active nuclear transport of the preintegration complex. Previous studies demonstrated that HIV-1 reverse transcription is successful only in cells with proliferative potential, thus restricting HIV-1 replication to cycling cells. To sort out this apparent discrepancy we compared the phenotype of a chimeric HIV-1 variant lacking a functional Vpr and MA-NLS (R7. deltaVpr.deltaNLS), and previously described to lack replicative capacity in macrophages and growth-arrested cells, with a chimera lacking a functional Vpr (R7.deltaVpr). Both variants replicated efficiently in primary macrophages, with only minimal differences in the kinetics of reverse transcription, integration, or p24 production. In agreement with our previous observation, elongation of reverse transcription was restricted to the proliferating subpopulation of macrophages. Replication of R7.deltaVpr and R7.deltaVpr.deltaNLS could also be demonstrated in aphidicolin-treated macrophages, indicating efficient nuclear transport in G1/S phase-arrested cells. In conclusion, our results confirm the dependency of the process of HIV-1 reverse transcriptase on cell proliferation in primary macrophages and exclude an important role of MA-NLS and Vpr in macrophage infection.

  17. One pot synthesis of highly luminescent polyethylene glycol anchored carbon dots functionalized with a nuclear localization signal peptide for cell nucleus imaging.

    PubMed

    Yang, Lei; Jiang, Weihua; Qiu, Lipeng; Jiang, Xuewei; Zuo, Daiying; Wang, Dongkai; Yang, Li

    2015-04-14

    Strong blue fluorescent polyethylene glycol (PEG) anchored carbon nitride dots (CDs@PEG) with a high quantum yield (QY) of 75.8% have been synthesized by a one step hydrothermal treatment. CDs with a diameter of ca. 6 nm are well dispersed in water and present a graphite-like structure. Photoluminescence (PL) studies reveal that CDs display excitation-dependent behavior and are stable under various test conditions. Based on the as-prepared CDs, we designed novel cell nucleus targeting imaging carbon dots functionalized with a nuclear localization signal (NLS) peptide. The favourable biocompatibilities of CDs and NLS modified CDs (NLS-CDs) are confirmed by in vitro cytotoxicity assays. Importantly, intracellular localization experiments in MCF7 and A549 cells demonstrate that NLS-CDs could be internalized in the nucleus and show blue light, which indicates that CDs may serve as cell nucleus imaging probes.

  18. One pot synthesis of highly luminescent polyethylene glycol anchored carbon dots functionalized with a nuclear localization signal peptide for cell nucleus imaging

    NASA Astrophysics Data System (ADS)

    Yang, Lei; Jiang, Weihua; Qiu, Lipeng; Jiang, Xuewei; Zuo, Daiying; Wang, Dongkai; Yang, Li

    2015-03-01

    Strong blue fluorescent polyethylene glycol (PEG) anchored carbon nitride dots (CDs@PEG) with a high quantum yield (QY) of 75.8% have been synthesized by a one step hydrothermal treatment. CDs with a diameter of ca. 6 nm are well dispersed in water and present a graphite-like structure. Photoluminescence (PL) studies reveal that CDs display excitation-dependent behavior and are stable under various test conditions. Based on the as-prepared CDs, we designed novel cell nucleus targeting imaging carbon dots functionalized with a nuclear localization signal (NLS) peptide. The favourable biocompatibilities of CDs and NLS modified CDs (NLS-CDs) are confirmed by in vitro cytotoxicity assays. Importantly, intracellular localization experiments in MCF7 and A549 cells demonstrate that NLS-CDs could be internalized in the nucleus and show blue light, which indicates that CDs may serve as cell nucleus imaging probes.Strong blue fluorescent polyethylene glycol (PEG) anchored carbon nitride dots (CDs@PEG) with a high quantum yield (QY) of 75.8% have been synthesized by a one step hydrothermal treatment. CDs with a diameter of ca. 6 nm are well dispersed in water and present a graphite-like structure. Photoluminescence (PL) studies reveal that CDs display excitation-dependent behavior and are stable under various test conditions. Based on the as-prepared CDs, we designed novel cell nucleus targeting imaging carbon dots functionalized with a nuclear localization signal (NLS) peptide. The favourable biocompatibilities of CDs and NLS modified CDs (NLS-CDs) are confirmed by in vitro cytotoxicity assays. Importantly, intracellular localization experiments in MCF7 and A549 cells demonstrate that NLS-CDs could be internalized in the nucleus and show blue light, which indicates that CDs may serve as cell nucleus imaging probes. Electronic supplementary information (ESI) available: The formulation of PEGylation CD optimization procedure, Table S1 and Fig. S1-S7. See DOI: 10.1039/c5nr01080

  19. Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription

    PubMed Central

    Tuand, Krizia; Stijnen, Pieter; Volders, Karolien; Declercq, Jeroen; Nuytens, Kim; Meulemans, Sandra; Creemers, John

    2016-01-01

    Background Neurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed. Methods Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression. Results Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated. Conclusion Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for

  20. Dissection of a nuclear localization signal.

    PubMed

    Hodel, M R; Corbett, A H; Hodel, A E

    2001-01-12

    The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS ( approximately 3 kcal/mol) as well as the energy of inhibition of the importin alpha importin beta-binding domain ( approximately 3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha-importin beta complex. On this scale, a functional NLS has a binding constant of approximately 10 nm, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 microm. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.

  1. Nuclear co-localization and functional interaction of COX-2 and HIF-1α characterize bone metastasis of human breast carcinoma.

    PubMed

    Maroni, Paola; Matteucci, Emanuela; Luzzati, Alessandro; Perrucchini, Giuseppe; Bendinelli, Paola; Desiderio, Maria Alfonsina

    2011-09-01

    The aim of this article is to identify nuclear co-localization of COX-2 and HIF-1α in human-bone metastasis of breast cancer, index of transcriptionally activated cells and functional for gene expression. In particular, we verified whether hypoxia exerted a direct role on metastasis-gene expression or through COX-2 signaling, due to the relevance for clinical implications to individuate molecular targets for diagnosis and therapy. The experiments were performed in vitro with two metastatic clones, 1833 and MDA-231BO, and the parental MDA-MB231 cells, in vivo (1833-xenograft model), and in human-bone metastasis specimens. In 1833 cells in vitro, COX-2 signaling pathway was critical for nuclear HIF-1α-protein expression/translocation, mechanisms determining HIF-1 activity and gene expression. The data were corroborated by immunohistochemistry in human-bone metastasis specimens. COX-2 and HIF-1α showed wide co-localization in the nucleus, indicative of COX-2-nuclear import in transcriptionally activated metastatic cells and consistent with COX-2-HIF-1α functional interaction. A network of microenvironmental signals controlled COX-2 induction and HIF-1 activation downstream. In fact, hypoxia through HGF and TGF-β1 autoregulatory loops triggered a specific array of transcription factors responsible for COX-2 transactivation. The novelty was that HGF and TGF-β1 biological signals were produced by hypoxic metastatic cells and, therefore, the microenvironment seemed to be modified by metastatic-cell engraftment in the bone. In agreement, HIF-1α expression in bone marrow supportive cells occurred in metastasis-bearing animals. Altogether, the data supported the pre-metastatic-niche theory. Our observations might be useful to design therapies against bone metastasis, by affecting the phenotype changes of metastatic cells occurring at the secondary growth site through COX-2-HIF-1 interaction.

  2. Espin actin-cytoskeletal proteins are in rat type I spiral ganglion neurons and include splice-isoforms with a functional nuclear localization signal.

    PubMed

    Sekerková, Gabriella; Zheng, Lili; Mugnaini, Enrico; Bartles, James R

    2008-08-20

    The espins are Ca(2+)-resistant actin-bundling proteins that are enriched in hair cell stereocilia and sensory cell microvilli. Here, we report a novel localization of espins to a large proportion of rat type I spiral ganglion neurons (SGNs) and their projections to the cochlear nucleus (CN). Moreover, we show that a fraction of these espins is in the nucleus of SGNs owing to the presence of splice-isoforms that contain a functional nuclear localization signal (NLS). Espin antibody labeled approximately 83% of type I SGNs, and the labeling intensity increased dramatically during early postnatal development. Type II SGNs and vestibular ganglion neurons were unlabeled. In the CN, espin-positive auditory nerve fibers showed a projection pattern typical of type I SGNs, with intense labeling in the nerve root region and posteroventral CN (PVCN). The anteroventral CN (AVCN) showed moderate labeling, whereas the dorsal CN showed weak labeling that was restricted to the deep layer. Espin-positive synaptic terminals were enriched around nerve root neurons and octopus cells in the PVCN and were also found on globular bushy cells and multipolar neurons in the PVCN and AVCN. SGNs expressed multiple espin transcripts and proteins, including splice-isoforms that contain a nonapeptide, which is rich in positively charged amino acids and creates a bipartite NLS. The nonapeptide was necessary to target espin isoforms to the nucleus and was sufficient to target an unrelated protein to the nucleus when joined with the upstream di-arginine-containing octapeptide. The presence of cytoplasmic and nuclear espins in SGNs suggests additional roles for espins in auditory neuroscience.

  3. Distinctive Nuclear Localization Signals in the Oomycete Phytophthora sojae.

    PubMed

    Fang, Yufeng; Jang, Hyo Sang; Watson, Gregory W; Wellappili, Dulani P; Tyler, Brett M

    2017-01-01

    To date, nuclear localization signals (NLSs) that target proteins to nuclei in oomycetes have not been defined, but have been assumed to be the same as in higher eukaryotes. Here, we use the soybean pathogen Phytophthora sojae as a model to investigate these sequences in oomycetes. By establishing a reliable in vivo NLS assay based on confocal microscopy, we found that many canonical monopartite and bipartite classical NLSs (cNLSs) mediated nuclear import poorly in P. sojae. We found that efficient localization of P. sojae nuclear proteins by cNLSs requires additional basic amino acids at distal sites or collaboration with other NLSs. We found that several representatives of another well-characterized NLS, proline-tyrosine NLS (PY-NLS) also functioned poorly in P. sojae. To characterize PY-NLSs in P. sojae, we experimentally defined the residues required by functional PY-NLSs in three P. sojae nuclear-localized proteins. These results showed that functional P. sojae PY-NLSs include an additional cluster of basic residues for efficient nuclear import. Finally, analysis of several highly conserved P. sojae nuclear proteins including ribosomal proteins and core histones revealed that these proteins exhibit a similar but stronger set of sequence requirements for nuclear targeting compared with their orthologs in mammals or yeast.

  4. Distinctive Nuclear Localization Signals in the Oomycete Phytophthora sojae

    PubMed Central

    Fang, Yufeng; Jang, Hyo Sang; Watson, Gregory W.; Wellappili, Dulani P.; Tyler, Brett M.

    2017-01-01

    To date, nuclear localization signals (NLSs) that target proteins to nuclei in oomycetes have not been defined, but have been assumed to be the same as in higher eukaryotes. Here, we use the soybean pathogen Phytophthora sojae as a model to investigate these sequences in oomycetes. By establishing a reliable in vivo NLS assay based on confocal microscopy, we found that many canonical monopartite and bipartite classical NLSs (cNLSs) mediated nuclear import poorly in P. sojae. We found that efficient localization of P. sojae nuclear proteins by cNLSs requires additional basic amino acids at distal sites or collaboration with other NLSs. We found that several representatives of another well-characterized NLS, proline-tyrosine NLS (PY-NLS) also functioned poorly in P. sojae. To characterize PY-NLSs in P. sojae, we experimentally defined the residues required by functional PY-NLSs in three P. sojae nuclear-localized proteins. These results showed that functional P. sojae PY-NLSs include an additional cluster of basic residues for efficient nuclear import. Finally, analysis of several highly conserved P. sojae nuclear proteins including ribosomal proteins and core histones revealed that these proteins exhibit a similar but stronger set of sequence requirements for nuclear targeting compared with their orthologs in mammals or yeast. PMID:28210240

  5. Nuclear overlap functions

    SciTech Connect

    Eskola, K.J.; Vogt, R.; Wang, X.N.

    1995-07-01

    A three parameter Wood-Saxon shape is used to describe the nuclear density distribution, which R{sub A} is the nuclear radius, {approx} is the surface thickness, and {omega} allows for central irregularities. The electron scattering data is used where available for R{sub A}, z, and {omega}. When data is unavailable, the parameters {omega} = O, z = 0.54 fm and R{sub A} = 1.19 A{sup 1/3} - 1.61 A{sup -1/3} fm are used. The central density {rho}{sub 0} is found from the normalization {infinity} d{sup 3}r{rho}{sub A}(r) = A.

  6. Fragmentation functions in nuclear media

    NASA Astrophysics Data System (ADS)

    Sassot, Rodolfo; Stratmann, Marco; Zurita, Pia

    2010-03-01

    We perform a detailed phenomenological analysis of how well hadronization in nuclear environments can be described in terms of effective fragmentation functions. The medium modified fragmentation functions are assumed to factorize from the partonic scattering cross sections and evolve in the hard scale in the same way as the standard or vacuum fragmentation functions. Based on precise data on semi-inclusive deep-inelastic scattering off nuclei and hadron production in deuteron-gold collisions, we extract sets of effective fragmentation functions for pions and kaons at next-to-leading order accuracy. The obtained sets provide a rather accurate description of the kinematical dependence of the analyzed cross sections and are found to differ significantly from standard fragmentation functions both in shape and magnitude. Our results support the notion of factorization and universality in the studied nuclear environments, at least in an effective way and within the precision of the available data.

  7. Nuclear tropomyosin and troponin in striated muscle: new roles in a new locale?

    PubMed

    Chase, P Bryant; Szczypinski, Mark P; Soto, Elliott P

    2013-08-01

    Tropomyosin and troponin have well known Ca(2+)-regulatory functions in the striated muscle sarcomere. In this review, we summarize experimental evidence that tropomyosin and troponin are localized, with as yet unidentified functional roles, in the striated muscle cell nucleus. We also apply bioinformatics approaches that predict localization of some tropomyosin and troponin to the nucleus, and that SUMOylation could be a covalent modification that modulates their nuclear localization and function. Further, we provide examples of cardiomyopathy mutations that alter the predicted likelihood of nuclear localization and SUMOylation of tropomyosin. These observations suggest novel mechanisms by which cardiomyopathy mutations in tropomyosin and troponin might alter not only cardiac contractility but also nuclear function.

  8. Screening of nuclear targeting proteins in Acinetobacter baumannii based on nuclear localization signals.

    PubMed

    Moon, Dong Chan; Gurung, Mamata; Lee, Jung Hwa; Lee, Yong Seok; Choi, Chi Won; Kim, Seung Il; Lee, Je Chul

    2012-05-01

    Nuclear targeting of bacterial proteins is an emerging pathogenic mechanism in bacteria. However, due to the absence of an appropriate screening system for nuclear targeting proteins, systematic approaches to nuclear targeting of bacterial proteins and subsequent host cell pathology are limited. In this study, we developed a screening system for nuclear targeting proteins in Acinetobacter baumannii using a combination of bioinformatic analysis based on nuclear localization signal (NLS) and the Gateway(®) recombinational cloning system. Among 3367 open reading frames of A. baumannii ATCC 17978, 34 functional or hypothetical proteins were predicted to carry the putative NLS sequences. Of the 29 clones generated by the Gateway(®) recombinational cloning system, 14 proteins tagged with green fluorescent protein (GFP) were targeted to nuclei of host cells. Among the 14 nuclear targeting proteins, S21, L20, and L32 ribosomal proteins and transposase carried putative nuclear export signal (NES) sequences, but only transposase harbored the functional NES. After translocation to nuclei of host cells, four A. baumannii proteins induced cytotoxicity. In conclusion, we have developed a screening system for nuclear targeting proteins in A. baumannii. This system may open the way to a new field of bacterial pathogenesis.

  9. HMG Modifications and Nuclear Function

    PubMed Central

    Zhang, Qingchun; Wang, Yinsheng

    2009-01-01

    High mobility group (HMG) proteins assume important roles in regulating chromatin dynamics, transcriptional activities of genes and other cellular processes. Post-translational modifications of HMG proteins can alter their interactions with DNA and proteins, and consequently, affect their biological activities. Although the mechanisms through which these modifications are involved in regulating biological processes in different cellular contexts are not fully understood, new insights into these modification “codes” have emerged from the increasing appreciation of the functions of these proteins. In this review, we focus on the chemical modifications of mammalian HMG proteins and highlight their roles in nuclear functions. PMID:20123066

  10. Cellular stress stimulates nuclear localization signal (NLS) independent nuclear transport of MRJ

    PubMed Central

    Andrews, Joel F.; Sykora, Landon J.; Barik-Letostak, Tiasha; Menezes, Mitchell E.; Mitra, Aparna; Barik, Sailen; Shevde, Lalita A.; Samant, Rajeev S.

    2012-01-01

    HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntington’s, Parkinson’s diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S). PMID:22504047

  11. Cellular stress stimulates nuclear localization signal (NLS) independent nuclear transport of MRJ

    SciTech Connect

    Andrews, Joel F.; Sykora, Landon J.; Barik Letostak, Tiasha; Menezes, Mitchell E.; Mitra, Aparna; Barik, Sailen; Shevde, Lalita A.; Samant, Rajeev S.

    2012-06-10

    HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntington's, Parkinson's diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S).

  12. Chitosan Nanoparticles for Nuclear Targeting: The Effect of Nanoparticle Size and Nuclear Localization Sequence Density.

    PubMed

    Tammam, Salma N; Azzazy, Hassan M E; Breitinger, Hans G; Lamprecht, Alf

    2015-12-07

    Many recently discovered therapeutic proteins exert their main function in the nucleus, thus requiring both efficient uptake and correct intracellular targeting. Chitosan nanoparticles (NPs) have attracted interest as protein delivery vehicles due to their biocompatibility and ability to escape the endosomes offering high potential for nuclear delivery. Molecular entry into the nucleus occurs through the nuclear pore complexes, the efficiency of which is dependent on NP size and the presence of nuclear localization sequence (NLS). Chitosan nanoparticles of different sizes (S-NPs ≈ 25 nm; L-NP ≈ 150 nm) were formulated, and they were modified with different densities of the octapeptide NLS CPKKKRKV (S-NPs, 0.25, 0.5, 2.0 NLS/nm(2); L-NPs, 0.6, 0.9, 2 NLS/nm(2)). Unmodified and NLS-tagged NPs were evaluated for their protein loading capacity, extent of cell association, cell uptake, cell surface binding, and finally nuclear delivery efficiency in L929 fibroblasts. To avoid errors generated with cell fractionation and nuclear isolation protocols, nuclear delivery was assessed in intact cells utilizing Förster resonance energy transfer (FRET) fluorometry and microscopy. Although L-NPs showed ≈10-fold increase in protein loading per NP when compared to S-NPs, due to higher cell association and uptake S-NPs showed superior protein delivery. NLS exerts a size and density dependent effect on nanoparticle uptake and surface binding, with a general reduction in NP cell surface binding and an increase in cell uptake with the increase in NLS density (up to 8.4-fold increase in uptake of High-NLS-L-NPs (2 NLS/nm(2)) compared to unmodified L-NPs). However, for nuclear delivery, unmodified S-NPs show higher nuclear localization rates when compared to NLS modified NPs (up to 5-fold by FRET microscopy). For L-NPs an intermediate NLS density (0.9 NLS/nm(2)) seems to provide highest nuclear localization (3.7-fold increase in nuclear delivery compared to High

  13. Expanding the definition of the classical bipartite nuclear localization signal.

    PubMed

    Lange, Allison; McLane, Laura M; Mills, Ryan E; Devine, Scott E; Corbett, Anita H

    2010-03-01

    Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best-characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin-alpha. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9-12 aa linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 aa linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 aa linker. Our studies show that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition.

  14. Characterization of a baculovirus nuclear localization signal domain in the late expression factor 3 protein

    SciTech Connect

    Au, Victoria; Yu Mei; Carstens, Eric B.

    2009-03-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) single-stranded DNA binding protein LEF-3 is a multi-functional protein that is required to transport the helicase protein P143 into the nucleus of infected cells where they function to replicate viral DNA. The N-terminal 56 amino acid region of LEF-3 is required for nuclear transport. In this report, we analyzed the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution. Fluorescence microscopy of expression plasmid-transfected cells demonstrated that the residues 28 to 32 formed the core nuclear localization signal, but other adjacent positively-charged residues augmented these sequences. Comparison with other group I Alphabaculoviruses suggested that this core region functionally duplicated residues including 18 and 19. This was demonstrated by the loss of nuclear localization when the equivalent residues (18 to 20) in Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) LEF-3 were mutated. The AcMNPV LEF-3 nuclear localization domain was also shown to drive nuclear transport in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. We also demonstrated by mutagenesis that two conserved cysteine residues located at 82 and 106 were not essential for nuclear localization or for interaction with P143. However, by using a modified construct of P143 that localized on its own to the nucleus, we demonstrated that a functional nuclear localization domain on LEF-3 was required for interaction between LEF-3 and P143.

  15. Nuclear Localization of Suppressor of Cytokine Signaling-1 Regulates Local Immunity in the Lung

    PubMed Central

    Zimmer, Jana; Weitnauer, Michael; Boutin, Sébastien; Küblbeck, Günter; Thiele, Sabrina; Walker, Patrick; Lasitschka, Felix; Lunding, Lars; Orinska, Zane; Vock, Christina; Arnold, Bernd; Wegmann, Michael; Dalpke, Alexander

    2016-01-01

    Suppressor of cytokine signaling 1 (SOCS1) is a negative feedback inhibitor of cytoplasmic Janus kinase and signal transducer and activator of transcription (STAT) signaling. SOCS1 also contains a nuclear localization sequence (NLS), yet, the in vivo importance of nuclear translocation is unknown. We generated transgenic mice containing mutated Socs1ΔNLS that fails to translocate in the cell nucleus (MGLtg mice). Whereas mice fully deficient for SOCS1 die within the first 3 weeks due to excessive interferon signaling and multiorgan inflammation, mice expressing only non-nuclear Socs1ΔNLS (Socs1−/−MGLtg mice) were rescued from early lethality. Canonical interferon gamma signaling was still functional in Socs1−/−MGLtg mice as shown by unaltered tyrosine phosphorylation of STAT1 and whole genome expression analysis. However, a subset of NFκB inducible genes was dysregulated. Socs1−/−MGLtg mice spontaneously developed low-grade inflammation in the lung and had elevated Th2-type cytokines. Upon ovalbumin sensitization and challenge, airway eosinophilia was increased in Socs1−/−MGLtg mice. Decreased transepithelial electrical resistance in trachea epithelial cells from Socs1−/−MGLtg mice suggests disrupted epithelial cell barrier. The results indicate that nuclear SOCS1 is a regulator of local immunity in the lung and unravel a so far unrecognized function for SOCS1 in the cell nucleus. PMID:27917175

  16. Identification of a nuclear localization signal in the polo box domain of Plk1.

    PubMed

    Lee, Moon-Sing; Huang, Yi-Han; Huang, Shu-Ping; Lin, Ru-Inn; Wu, Shu-Fen; Li, Chin

    2009-10-01

    Polo-like kinase 1 plays an essential role in mitosis and cytokinesis. Expression and nuclear localization of Plk1 during the S phase are necessary for its functions. Although it was reported that a bipartite nuclear localization signal located at the N-terminal kinase domain is required for nuclear import of Plk1, Plk1 carrying mutations in the polo box I of the polo box domain exhibited increased cytoplasmic accumulation. We further showed that the polo box domain was able to confer nuclear import of beta-galactosidase in vivo and GST-EGFP in vitro. The import carriers transportin and importin alpha were found to interact with the polo box domain directly in a Ran-GTP sensitive manner. These results indicate the presence of a nuclear localization signal in the polo box domain. A 38 amino acid sequence with the function of nuclear localization signal was identified to interact with transportin. Our findings demonstrated that a transportin-dependent nuclear localization signal is present in the polo box domain of Plk1, possibly required for efficient nuclear import. Showing little similarity to the M9 sequence, the 38 amino acid sequence identified here likely represents a novel nuclear localization signal.

  17. Regulation of Greatwall kinase by protein stabilization and nuclear localization

    PubMed Central

    Yamamoto, Tomomi M; Wang, Ling; Fisher, Laura A; Eckerdt, Frank D; Peng, Aimin

    2014-01-01

    Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. In this study we identified Hsp90, Cdc37 and members of the importin α and β families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. We found that Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. We identified KR456/457 as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, we showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry. PMID:25483093

  18. Localized Hartree product treatment of multiple protons in the nuclear-electronic orbital framework

    NASA Astrophysics Data System (ADS)

    Auer, Benjamin; Hammes-Schiffer, Sharon

    2010-02-01

    An approximation for treating multiple quantum nuclei within the nuclear-electronic orbital (NEO) framework for molecular systems is presented. In the approximation to NEO-Hartree-Fock, the nuclear wave function is represented by a Hartree product rather than a Slater determinant, corresponding to the neglect of the nuclear exchange interactions. In the approximation to NEO-density functional theory, the nuclear exchange-correlation functional is chosen to be the diagonal nuclear exchange interaction terms, thereby eliminating the nuclear self-interaction terms. To further enhance the simplicity and computational efficiency, the nuclear molecular orbitals or Kohn-Sham orbitals are expanded in terms of localized nuclear basis sets. These approximations are valid because of the inherent localization of the nuclear orbitals and the numerical insignificance of the nuclear exchange interactions in molecular systems. Moreover, these approximations lead to substantial computational savings due to the reduction in both the number of integrals that must be calculated and the size of the matrices that must be diagonalized. These nuclear Hartree product approximation (HPA) methods scale linearly with the number of quantum protons and are highly parallelizable. Applications to a water hexamer, glycine dimer, and 32-water cluster, where all hydrogen nuclei are treated quantum mechanically, illustrate the accuracy and computational efficiency of the nuclear HPA methods. These strategies will facilitate the implementation of explicitly correlated NEO methods for molecular systems with multiple quantum protons.

  19. PKCι regulates nuclear YAP1 localization and ovarian cancer tumorigenesis

    PubMed Central

    Wang, Yin; Justilien, Verline; Brennan, Katharyn I.; Jamieson, Lee; Murray, Nicole R.; Fields, Alan P.

    2016-01-01

    Atypical protein kinase Cι (PKCι) is an oncogene in lung and ovarian cancer. The PKCι gene PRKCI is targeted for frequent tumor-specific copy number gain (CNG) in both lung squamous cell carcinoma (LSCC) and ovarian serous carcinoma (OSC). We recently demonstrated that in LSCC cells PRKCI CNG functions to drive transformed growth and tumorigenicity by activating PKCι-dependent cell autonomous Hedgehog (Hh) signaling. Here, we assessed whether OSC cells harboring PRKCI CNG exhibit similar PKCι-dependent Hh signaling. Surprisingly, we find that whereas PKCι is required for the transformed growth for OSC cells harboring PRKCI CNG, these cells do not exhibit PKCι-dependent Hh signaling or Hh-dependent proliferation. Rather, transformed growth of OSC cells is regulated by PKCι-dependent nuclear localization of the oncogenic transcription factor, YAP1. Lentiviral shRNA-mediated knock down (KD) of PKCι leads to decreased nuclear YAP1 and increased YAP1 binding to angiomotin (AMOT), which sequesters YAP1 in the cytoplasm. Biochemical analysis reveals that PKCι directly phosphorylates AMOT at a unique site, Thr750, whose phosphorylation inhibits YAP1 binding. Pharmacologic inhibition of PKCι decreases YAP1 nuclear localization and blocks OSC tumor growth in vitro and in vivo. Immunohistochemical analysis reveals a strong positive correlation between tumor PKCι expression and nuclear YAP1 in primary OSC tumor samples, indicating the clinical relevance of PKCι-YAP1 signaling. Our results uncover a novel PKCι-AMOT-YAP1 signaling axis that promotes OSC tumor growth, and provide a rationale for therapeutic targeting of this pathway for treatment of OSC. PMID:27321186

  20. Constitutive and ligand-induced nuclear localization of oxytocin receptor.

    PubMed

    Kinsey, Conan G; Bussolati, Gianni; Bosco, Martino; Kimura, Tadashi; Pizzorno, Marie C; Chernin, Mitchell I; Cassoni, Paola; Novak, Josef F

    2007-01-01

    Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells. We report here that human osteosarcoma (U2OS, MG63, OS15 and SaOS2), breast cancer (MCF7), and primary human fibroblastic cells (HFF) all exhibit OTR not only on the cell membrane, but also in the various nuclear compartments including the nucleolus. Both an OTR-GFP fusion protein and the native OTR appear to be localized to the nucleus as detected by transfection and/or confocal immunofluorescence, respectively. Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus. Western blots indicate that OTR in the nucleus and on the plasma membrane are likely to be the same biochemical and immunological entities. It appears that OTR is first visible in the nucleoli and subsequently disperses within the nucleus into 4-20 spots while some of the OTR diffuses throughout the nucleoplasm. The behaviour and kinetics of OTR-GFP and OTR are different, indicating interference by GFP in both OTR entrance into the nucleus and subsequent relocalization of OTR within the nucleus. There are important differences among the tested cells, such as the requirement of a ligand for transfer of OTR in nuclei. A constitutive internalization of OTR was found only in osteosarcoma cells, while the nuclear localization in all other tested cells was dependent on ligand binding. The amount of OTR-positive material within and in the vicinity of the nucleus increased following a treatment with oxytocin in both constitutive and ligand-dependent type of cells. The evidence of OTR compartmentalization at the cell nucleus (either ligand-dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G-protein-coupled receptor to other heptahelical receptors displaying this atypical and unexpected

  1. Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

    SciTech Connect

    Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke; Ito, Hideki; Shimonohara, Nozomi; Tsuji, Takahiro; Nakajima, Noriko; Suzuki, Yoshio; Matsuo, Koma; Nakagawa, Hidemi; Sata, Tetsutaro; Katano, Harutaka

    2010-03-15

    To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

  2. Universal Nuclear Energy Density Functional

    SciTech Connect

    Carlson, Joseph; Furnstahl, Richard; Horoi, Mihai; Lusk, Rusty; Nazarewicz, Witold; Ng, Esmond; Thompson, Ian; Vary, James

    2012-12-01

    An understanding of the properties of atomic nuclei is crucial for a complete nuclear theory, for element formation, for properties of stars, and for present and future energy and defense applications. During the period of Dec. 1 2006 – Jun. 30, 2012, the UNEDF collaboration carried out a comprehensive study of all nuclei, based on the most accurate knowledge of the strong nuclear interaction, the most reliable theoretical approaches, the most advanced algorithms, and extensive computational resources, with a view towards scaling to the petaflop platforms and beyond. Until recently such an undertaking was hard to imagine, and even at the present time such an ambitious endeavor would be far beyond what a single researcher or a traditional research group could carry out.

  3. Sumoylation of SAE2 C terminus regulates SAE nuclear localization.

    PubMed

    Truong, Khue; Lee, Terry D; Li, Baozong; Chen, Yuan

    2012-12-14

    SUMOylation occurs predominantly in the nucleus, but non-nuclear proteins can also be SUMOylated. It is unclear how intracellular trafficking of the SUMOylation enzymes is regulated to catalyze SUMOylation in different cellular compartments. Here we report that the SAE2 subunit of human SUMO activation enzyme (SAE) underwent rapid nucleocytoplasmic shuttling and its nuclear accumulation depended on SUMO modification at the C terminus. The SUMOylation sites included three Lys residues on the bipartite nuclear localization sequence (NLS) and two Lys residues outside of but adjacent to the NLS, and their SUMOylation was catalyzed by Ubc9. Because SAE2 forms a tight heterodimer with SAE1 and it controls the trafficking of the heterodimer, this study has identified the mechanism used to localize SAE to the nucleus. Similar mechanisms are likely to exist for other proteins that depend on SUMOylation for nuclear localization.

  4. Characterization of a nuclear localization signal in the foot-and-mouth disease virus polymerase

    SciTech Connect

    Sanchez-Aparicio, Maria Teresa; Rosas, Maria Flora; Sobrino, Francisco

    2013-09-15

    We have experimentally tested whether the MRKTKLAPT sequence in FMDV 3D protein (residues 16 to 24) can act as a nuclear localization signal (NLS). Mutants with substitutions in two basic residues within this sequence, K18E and K20E, were generated. A decreased nuclear localization was observed in transiently expressed 3D and its precursor 3CD, suggesting a role of K18 and K20 in nuclear targeting. Fusion of MRKTKLAPT to the green fluorescence protein (GFP) increased the nuclear localization of GFP, which was not observed when GFP was fused to the 3D mutated sequences. These results indicate that the sequence MRKTKLAPT can be functionally considered as a NLS. When introduced in a FMDV full length RNA replacements K18E and K20E led to production of revertant viruses that replaced the acidic residues introduced (E) by K, suggesting that the presence of lysins at positions 18 and 20 of 3D is essential for virus multiplication. - Highlights: • The FMDV 3D polymerase contains a nuclear localization signal. • Replacements K18E and K20E decrease nuclear localization of 3D and its precursor 3CD. • Fusion of the MRKTKLAPT 3D motif to GFP increases the nuclear localization of GFP. • Replacements K18E and K20E abolish the ability of MRKTKLAPT to relocate GFP. • RNAs harboring replacements K18E and K20E lead to recovery of revertant FMDVs.

  5. Nuclear localization of the testis determining gene product SRY

    PubMed Central

    1995-01-01

    We have studied the expression of the human SRY protein (termed p27SRY) in two different cell lines by using specific antibodies. Confocal microscopy enabled us to localize p27SRY precisely in the nucleus in a discrete punctuate pattern. Furthermore, through microinjection experiments, we have demonstrated that the localization of the p27SRY protein into the nucleus was an event involving the NH2-terminal part of the high mobility group (HMG) domain. With the help of several synthetic peptides and various p27SRY mutants, we have characterized a bipartite basic motif in this part of the protein corresponding to a nuclear localization signal. This nuclear localization signal appears to be highly conserved in SRY box- and HMB box-containing proteins, suggesting common properties of nuclear targeting within the HMG box protein family. PMID:7876301

  6. Identification of a bipartite nuclear localization signal in the silkworm Masc protein.

    PubMed

    Sugano, Yudai; Kokusho, Ryuhei; Ueda, Masamichi; Fujimoto, Masaru; Tsutsumi, Nobuhiro; Shimada, Toru; Kiuchi, Takashi; Katsuma, Susumu

    2016-07-01

    The silkworm Masculinizer (Masc) gene encodes a CCCH-tandem zinc finger protein that controls both masculinization and dosage compensation. Masc protein is a nuclear protein, but the mechanism underlying the transport of this protein into the nucleus has not yet been elucidated. Here, we identified a functional bipartite nuclear localization signal (NLS) located between residues 274 and 290 of the Masc protein. Sequence comparison revealed that this bipartite NLS is evolutionarily conserved in Masc proteins from other lepidopteran insects. Furthermore, we showed that the degree of nuclear localization is not associated with the masculinizing activity of the Masc protein.

  7. Nuclear energy density functional and the nuclear α decay

    NASA Astrophysics Data System (ADS)

    Lim, Yeunhwan; Oh, Yongseok

    2017-03-01

    The nuclear α decay of heavy nuclei is investigated based on the nuclear energy density functional, which leads to the α potential inside the parent nucleus in terms of the proton and neutron density profiles of the daughter nucleus. We use the Skyrme force model, Gogny force model, and relativistic mean-field model to get the nucleon density profiles inside heavy nuclei. Once the nucleon density profiles are determined, the parameters of the nuclear α potential are fitted to the observed α decay half-lives of heavy nuclei. This approach is then applied to predict unknown α decay half-lives of heavy nuclei. To estimate the Q values of unobserved α decays, we make use of the liquid droplet model.

  8. LINCing complex functions at the nuclear envelope

    PubMed Central

    Rothballer, Andrea; Schwartz, Thomas U.; Kutay, Ulrike

    2013-01-01

    Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements. LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration, or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have recently revealed the molecular basis of nucleo-cytoskeletal coupling. They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. PMID:23324460

  9. Uncertainty Quantification for Nuclear Density Functional Theory

    NASA Astrophysics Data System (ADS)

    McDonnell, Jordan; Schunck, Nicolas; Nazarewicz, Witold; Higdon, Dave; Sarich, Jason; Wild, Stefan

    2014-09-01

    Nuclear density functional theory exhibits good overall agreement with measured nuclear masses for medium-mass to heavy nuclei. But the predictions of various models diverge substantially near the neutron and proton drip lines. Quantifying the theory's inherent uncertainty is essential for making reliable predictions. Through a Bayesian analysis, we calculate the theoretical uncertainty for nuclear masses obtained with a Skyrme-class energy density functional. We also assess whether a recent set of mass measurements of neutron-rich nuclei reduces the uncertainty in this model's predictions near the neutron drip line. Nuclear density functional theory exhibits good overall agreement with measured nuclear masses for medium-mass to heavy nuclei. But the predictions of various models diverge substantially near the neutron and proton drip lines. Quantifying the theory's inherent uncertainty is essential for making reliable predictions. Through a Bayesian analysis, we calculate the theoretical uncertainty for nuclear masses obtained with a Skyrme-class energy density functional. We also assess whether a recent set of mass measurements of neutron-rich nuclei reduces the uncertainty in this model's predictions near the neutron drip line. This work was supported by the US Department of Energy under Contracts No. DE-SC0008499 and No. DE-AC52-07NA27344.

  10. Reflection-Asymmetric Nuclear Deformations within the Density Functional Theory

    SciTech Connect

    Olsen, E; Erler, J; Nazarewicz, W.; Stoitsov, M

    2012-01-01

    Within the nuclear density functional theory (DFT) we study the effect of reflection- asymmetric shapes on ground-state binding energies and binding energy differences. To this end, we developed the new DFT solver axialhfb that uses an approximate second-order gradient to solve the Hartree-Fock-Bogoliubov equations of superconducting DFT with the quasi-local Skyrme energy density functionals. Illustrative calculations are carried out for even- even isotopes of radium and thorium.

  11. Coordinate nuclear targeting of the FANCD2 and FANCI proteins via a FANCD2 nuclear localization signal.

    PubMed

    Boisvert, Rebecca A; Rego, Meghan A; Azzinaro, Paul A; Mauro, Maurizio; Howlett, Niall G

    2013-01-01

    Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.

  12. Nuclear localization signal-dependent and -independent movements of Drosophila melanogaster dUTPase isoforms during nuclear cleavage

    SciTech Connect

    Muha, Villo; Zagyva, Imre; Venkei, Zsolt; Szabad, Janos; Vertessy, Beata G.

    2009-04-03

    Two dUTPase isoforms (23 kDa and 21 kDa) are present in the fruitfly with the sole difference of an N-terminal extension. In Drosophila embryo, both isoforms are detected inside the nucleus. Here, we investigated the function of the N-terminal segment using eYFP-dUTPase constructs. In Schneider 2 cells, only the 23 kDa construct showed nuclear localization arguing that it may contain a nuclear localization signal (NLS). Sequence comparisons identified a lysine-rich nonapeptide with similarity to the human c-myc NLS. In Drosophila embryos during nuclear cleavages, the 23 kDa isoform showed the expected localization shifts. Contrariwise, although the 21 kDa isoform was excluded from the nuclei during interphase, it was shifted to the nucleus during prophase and forthcoming mitotic steps. The observed dynamic localization character showed strict timing to the nuclear cleavage phases and explained how both isoforms can be present within the nuclear microenvironment, although at different stages of cell cycle.

  13. Immunological and biochemical evidence for nuclear localization of annexin in peas

    NASA Technical Reports Server (NTRS)

    Clark, G. B.; Dauwalder, M.; Roux, S. J.

    1998-01-01

    Immunofluorescent localization of annexins using an anti-pea annexin polyclonal antibody (anti-p35) in pea (Pisum sativum) leaf and stem epidermal peels showed staining of the nuclei and the cell periphery. Nuclear staining was also seen in cell teases prepared from pea plumules. The amount of nuclear stain was reduced both by fixation time and by dehydration and organic solvent treatment. Observation with confocal microscopy demonstrated that the anti-p35 stain was diffusely distributed throughout the nuclear structure. Immunoblots of purified nuclei, nuclear envelope matrix, nucleolar, and chromatin fractions showed a cross-reactive protein band of 35 kDa. These data are the first to show annexins localized in plant cell nuclei where they may play a role in nuclear function.

  14. Nuclear modifications of Parton Distribution Functions

    NASA Astrophysics Data System (ADS)

    Adeluyi, Adeola Adeleke

    This dissertation addresses a central question of modern nuclear physics: how does the behavior of fundamental degrees of freedom (quarks and gluons) change in the nuclear environment? This is an important aspect of experimental studies at current facilities such as the Relativistic Heavy Ion Collider (RHIC) at Brookhaven National Laboratory and the Continuous Electron Beam Accelerator Facility (CEBAF) at the Thomas Jefferson National Laboratory (JLAB). It is also highly relevant to planned experimental efforts at the Large Hadron Collider (LHC) and the future Electron Ion Collider (EIC). All these facilities probe matter via collisions involving nuclei; thus complications arise due to the presence of the attendant nuclear medium. Theoretical efforts to understand and interpret experimental results from such collisions are therefore largely dependent on the resolution of this question. The development of nuclear physics demonstrates that theoretical description is most efficient in terms of the effective degrees of freedom relevant to the scale (energy) being probed. Thus at low energies, nuclei are described as bound states of protons and neutrons (nucleons). At higher energies, the nucleons are no longer elementary, but are revealed to possess an underlying substructure: they are made up of quarks and gluons, collectively termed partons. The mometum distributions of these partons in the nucleon are referred to as Parton Distribution Functions (PDFs). Parton distributions can be determined from experimental measurements of structure functions. The ratio of nuclear structure functions to nucleon structure functions (generically referred to as nuclear ratio) is a measure of the nuclear modifications of the free nucleon PDFs. Thus a study of the nuclear ratio suffices to gain an understanding of nuclear modifications. In this dissertation we aim to describe theoretically nuclear modifications in a restricted region where the nuclear ratio is less than unity, the so

  15. Nuclear structure and dynamics with density functional theory

    NASA Astrophysics Data System (ADS)

    Stetcu, Ionel

    2015-10-01

    Even in the absence of ab initio methods capable of tackling heavy nuclei without restrictions, one can obtain an ab initio description of ground-state properties by means of the density functional theory (DFT), and its extension to superfluid systems in its local variant, the superfluid local density approximation (SLDA). Information about the properties of excited states can be obtained in the same framework by using an extension to the time-dependent (TD) phenomena. Unlike other approaches in which the nuclear structure information is used as a separate input into reaction models, the TD approach treats on the same footing the nuclear structure and dynamics, and is well suited to provide more reliable description for a large number of processes involving heavy nuclei, from the nuclear response to electroweak probes, to nuclear reactions, such as neutron-induced reactions, or nuclear fusion and fission. Such processes, sometimes part of integrated nuclear systems, have important applications in astrophysics, energy production, global security, etc. In this talk, I will present the simulation of a simple reaction, that is the Coulomb excitation of a 238U nucleus, and discuss the application of the TD-DFT formalism to the description of induced fission. I gratefully acknowledge partial support of the U.S. Department of Energy through an Early Career Award of the LANL/LDRD Program.

  16. Compartmentalization and Functionality of Nuclear Disorder: Intrinsic Disorder and Protein-Protein Interactions in Intra-Nuclear Compartments.

    PubMed

    Meng, Fanchi; Na, Insung; Kurgan, Lukasz; Uversky, Vladimir N

    2015-12-25

    The cell nucleus contains a number of membrane-less organelles or intra-nuclear compartments. These compartments are dynamic structures representing liquid-droplet phases which are only slightly denser than the bulk intra-nuclear fluid. They possess different functions, have diverse morphologies, and are typically composed of RNA (or, in some cases, DNA) and proteins. We analyzed 3005 mouse proteins localized in specific intra-nuclear organelles, such as nucleolus, chromatin, Cajal bodies, nuclear speckles, promyelocytic leukemia (PML) nuclear bodies, nuclear lamina, nuclear pores, and perinuclear compartment and compared them with ~29,863 non-nuclear proteins from mouse proteome. Our analysis revealed that intrinsic disorder is enriched in the majority of intra-nuclear compartments, except for the nuclear pore and lamina. These compartments are depleted in proteins that lack disordered domains and enriched in proteins that have multiple disordered domains. Moonlighting proteins found in multiple intra-nuclear compartments are more likely to have multiple disordered domains. Protein-protein interaction networks in the intra-nuclear compartments are denser and include more hubs compared to the non-nuclear proteins. Hubs in the intra-nuclear compartments (except for the nuclear pore) are enriched in disorder compared with non-nuclear hubs and non-nuclear proteins. Therefore, our work provides support to the idea of the functional importance of intrinsic disorder in the cell nucleus and shows that many proteins associated with sub-nuclear organelles in nuclei of mouse cells are enriched in disorder. This high level of disorder in the mouse nuclear proteins defines their ability to serve as very promiscuous binders, possessing both large quantities of potential disorder-based interaction sites and the ability of a single such site to be involved in a large number of interactions.

  17. Controlling Androgen receptor nuclear localization by dendrimer conjugates

    NASA Astrophysics Data System (ADS)

    Wang, Haoyu

    Androgen Receptor (AR) antagonists, such as bicalutamide and flutamide have been used widely in the treatment of prostate cancer. Although initial treatment is effective, prostate cancer cells often acquire antiandrogen resistance with prolonged treatment. AR over-expression and AR mutations contribute to the development of antiandrogen resistant cancer. Second generation antiandrogens such as enzalutamide are more effective and show reduced AR nuclear localization. In this study, derivatives of PAN52, a small molecule antiandrogen previously developed in our lab, were conjugated to the surface of generation 4 and generation 6 PAMAM dendrimers to obtain antiandrogen PAMAM dendrimer conjugates (APDC). APDCs readily enter cells and associate with AR in the cytoplasm. Due to their large size and positive charge, they can not enter the nucleus, thus retaining AR in the cytoplasm. In addition, APDCs are effective in decreasing AR mediated transcription and cell proliferation. APDC is the first AR antagonists that inhibit DHT-induced nuclear localization of AR. By inhibiting AR nuclear localization, APDC represents a new class of antiandrogens that offer an alternative approach to addressing antiandrogen-resistant prostate cancer. Lysine post-translational modification of AR Nuclear Localization Sequence (NLS) has great impact on AR cellular localization. It is of interest to understand which modifications modulate AR translocation into the nucleus. In this study, we prepared dendrimer-based acetyltransferase mimetic (DATM), DATM is able to catalytically acetylate AR in CWR22Rv1 cells, which will be a useful tool for studying AR modification effect on AR cellular localization. Derivatives of DATM, which transfer other chemical groups to AR, can be prepared similarly, and with more dendrimer based AR modification tools prepared in future, we will be able to understand and control AR cellular localization through AR modification.

  18. Functional Localization of Genetic Network Programming

    NASA Astrophysics Data System (ADS)

    Eto, Shinji; Hirasawa, Kotaro; Hu, Jinglu

    According to the knowledge of brain science, it is suggested that there exists cerebral functional localization, which means that a specific part of the cerebrum is activated depending on various kinds of information human receives. The aim of this paper is to build an artificial model to realize functional localization based on Genetic Network Programming (GNP), a new evolutionary computation method recently developed. GNP has a directed graph structure suitable for realizing functional localization. We studied the basic characteristics of the proposed system by making GNP work in a functionally localized way.

  19. MECHANICAL REGULATION OF NUCLEAR STRUCTURE AND FUNCTION

    PubMed Central

    Martins, Rui P.; Finan, John D.; Guilak, Farshid; Lee, David A.

    2013-01-01

    Mechanical loading induces both nuclear distortion and alterations in gene expression in a variety of cell types. Mechanotransduction is the process by which extracellular mechanical forces can activate a number of well-studied cytoplasmic signaling cascades. Inevitably such signals are transduced to the nucleus and induce transcription factor-mediated changes in gene expression. However, gene expression can be also regulated through alterations in nuclear architecture, providing direct control of genome function. One putative transduction mechanism for this phenomenon involves alterations in nuclear architecture that result from the mechanical perturbation of the cell. This perturbation is associated with direct mechanical strain or osmotic stress, which is transferred to the nucleus. This review describes the current state of knowledge relating the nuclear architecture and the transfer of mechanical forces to the nucleus mediated by the cytoskeleton, the nucleoskeleton, and the LINC (linker of the nucleoskeleton and cytoskeleton) complex. Moreover, remodeling of the nucleus induces alterations in nuclear stiffness, which may be associated with cell differentiation. These phenomena are discussed in relation to the potential influence of nuclear architecture-mediated mechanoregulation of transcription and cell fate. PMID:22655599

  20. Mechanical regulation of nuclear structure and function.

    PubMed

    Martins, Rui P; Finan, John D; Guilak, Farshid; Lee, David A

    2012-01-01

    Mechanical loading induces both nuclear distortion and alterations in gene expression in a variety of cell types. Mechanotransduction is the process by which extracellular mechanical forces can activate a number of well-studied cytoplasmic signaling cascades. Inevitably, such signals are transduced to the nucleus and induce transcription factor-mediated changes in gene expression. However, gene expression also can be regulated through alterations in nuclear architecture, providing direct control of genome function. One putative transduction mechanism for this phenomenon involves alterations in nuclear architecture that result from the mechanical perturbation of the cell. This perturbation is associated with direct mechanical strain or osmotic stress, which is transferred to the nucleus. This review describes the current state of knowledge relating the nuclear architecture and the transfer of mechanical forces to the nucleus mediated by the cytoskeleton, the nucleoskeleton, and the LINC (linker of the nucleoskeleton and cytoskeleton) complex. Moreover, remodeling of the nucleus induces alterations in nuclear stiffness, which may be associated with cell differentiation. These phenomena are discussed in relation to the potential influence of nuclear architecture-mediated mechanoregulation of transcription and cell fate.

  1. Nuclear localization of DMP1 proteins suggests a role in intracellular signaling

    SciTech Connect

    Siyam, Arwa; Wang, Suzhen; Qin, Chunlin; Mues, Gabriele; Stevens, Roy; D'Souza, Rena N.; Lu, Yongbo

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nuclear localization of DMP1 in various cell lines. Black-Right-Pointing-Pointer Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. Black-Right-Pointing-Pointer Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

  2. Nuclear cyclophilins affect spliceosome assembly and function in vitro.

    PubMed

    Adams, B M; Coates, Miranda N; Jackson, S RaElle; Jurica, Melissa S; Davis, Tara L

    2015-07-15

    Cyclophilins are ubiquitously expressed proteins that bind to prolines and can catalyse cis/trans isomerization of proline residues. There are 17 annotated members of the cyclophilin family in humans, ubiquitously expressed and localized variously to the cytoplasm, nucleus or mitochondria. Surprisingly, all eight of the nuclear localized cyclophilins are found associated with spliceosomal complexes. However, their particular functions within this context are unknown. We have therefore adapted three established assays for in vitro pre-mRNA splicing to probe the functional roles of nuclear cyclophilins in the context of the human spliceosome. We find that four of the eight spliceosom-associated cyclophilins exert strong effects on splicing in vitro. These effects are dose-dependent and, remarkably, uniquely characteristic of each cyclophilin. Using both qualitative and quantitative means, we show that at least half of the nuclear cyclophilins can act as regulatory factors of spliceosome function in vitro. The present work provides the first quantifiable evidence that nuclear cyclophilins are splicing factors and provides a novel approach for future work into small molecule-based modulation of pre-mRNA splicing.

  3. Nuclear cyclophilins affect spliceosome assembly and function in vitro

    PubMed Central

    Adams, B.M.; Coates, Miranda N.; Jackson, S. RaElle; Jurica, Melissa S.; Davis, Tara L.

    2015-01-01

    Cyclophilins are ubiquitously expressed proteins that bind to prolines and can catalyse cis/trans isomerization of proline residues. There are 17 annotated members of the cyclophilin family in humans, ubiquitously expressed and localized variously to the cytoplasm, nucleus or mitochondria. Surprisingly, all eight of the nuclear localized cyclophilins are found associated with spliceosomal complexes. However, their particular functions within this context are unknown. We have therefore adapted three established assays for in vitro pre-mRNA splicing to probe the functional roles of nuclear cyclophilins in the context of the human spliceosome. We find that four of the eight spliceosom-associated cyclophilins exert strong effects on splicing in vitro. These effects are dose-dependent and, remarkably, uniquely characteristic of each cyclophilin. Using both qualitative and quantitative means, we show that at least half of the nuclear cyclophilins can act as regulatory factors of spliceosome function in vitro. The present work provides the first quantifiable evidence that nuclear cyclophilins are splicing factors and provides a novel approach for future work into small molecule-based modulation of pre-mRNA splicing. PMID:25967372

  4. Regulation of Nuclear Localization of Signaling Proteins by Cytokinin

    SciTech Connect

    Kieber, J.J.

    2010-05-01

    Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

  5. Identification of the nuclear localization and export signals of high risk HPV16 E7 oncoprotein

    SciTech Connect

    Knapp, Alixandra A.; McManus, Patrick M.; Bockstall, Katy; Moroianu, Junona

    2009-01-05

    The E7 oncoprotein of high risk human papillomavirus type 16 (HPV16) binds and inactivates the retinoblastoma (RB) family of proteins. Our previous studies suggested that HPV16 E7 enters the nucleus via a novel Ran-dependent pathway independent of the nuclear import receptors (Angeline, M., Merle, E., and Moroianu, J. (2003). The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway. Virology 317(1), 13-23.). Here, analysis of the localization of specific E7 mutants revealed that the nuclear localization of E7 is independent of its interaction with pRB or of its phosphorylation by CKII. Fluorescence microscopy analysis of enhanced green fluorescent protein (EGFP) and 2xEGFP fusions with E7 and E7 domains in HeLa cells revealed that E7 contains a novel nuclear localization signal (NLS) in the N-terminal domain (aa 1-37). Interestingly, treatment of transfected HeLa cells with two specific nuclear export inhibitors, Leptomycin B and ratjadone, changed the localization of 2xEGFP-E7{sub 38-98} from cytoplasmic to mostly nuclear. These data suggest the presence of a leucine-rich nuclear export signal (NES) and a second NLS in the C-terminal domain of E7 (aa 38-98). Mutagenesis of critical amino acids in the putative NES sequence ({sub 76}IRTLEDLLM{sub 84}) changed the localization of 2xEGFP-E7{sub 38-98} from cytoplasmic to mostly nuclear suggesting that this is a functional NES. The presence of both NLSs and an NES suggests that HPV16 E7 shuttles between the cytoplasm and nucleus which is consistent with E7 having functions in both of these cell compartments.

  6. Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei.

    PubMed Central

    Rose, A M; Joyce, P B; Hopper, A K; Martin, N C

    1992-01-01

    The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization. Images PMID:1448094

  7. Building a Universal Nuclear Energy Density Functional

    SciTech Connect

    Carlson, Joe A.; Furnstahl, Dick; Horoi, Mihai; Lust, Rusty; Nazaewicc, Witek; Ng, Esmond; Thompson, Ian; Vary, James

    2012-12-30

    During the period of Dec. 1 2006 – Jun. 30, 2012, the UNEDF collaboration carried out a comprehensive study of all nuclei, based on the most accurate knowledge of the strong nuclear interaction, the most reliable theoretical approaches, the most advanced algorithms, and extensive computational resources, with a view towards scaling to the petaflop platforms and beyond. The long-term vision initiated with UNEDF is to arrive at a comprehensive, quantitative, and unified description of nuclei and their reactions, grounded in the fundamental interactions between the constituent nucleons. We seek to replace current phenomenological models of nuclear structure and reactions with a well-founded microscopic theory that delivers maximum predictive power with well-quantified uncertainties. Specifically, the mission of this project has been three-fold: First, to find an optimal energy density functional (EDF) using all our knowledge of the nucleonic Hamiltonian and basic nuclear properties; Second, to apply the EDF theory and its extensions to validate the functional using all the available relevant nuclear structure and reaction data; Third, to apply the validated theory to properties of interest that cannot be measured, in particular the properties needed for reaction theory.

  8. Local acceptance of a high-level nuclear waste repository.

    PubMed

    Sjöberg, Lennart

    2004-06-01

    The siting of nuclear waste facilities has been very difficult in all countries. Recent experience in Sweden indicates, however, that it may be possible, under certain circumstances, to gain local support for the siting of a high-level nuclear waste (HLNW) repository. The article reports on a study of attitudes and risk perceptions of people living in four municipalities in Sweden where HLNW siting was being intensely discussed at the political level, in media, and among the public. Data showed a relatively high level of consensus on acceptability of at least further investigation of the issue; in two cases local councils have since voted in favor of a go-ahead, and in one case only a very small majority defeated the issue. Models of policy attitudes showed that these were related to attitude to nuclear power, attributes of the perceived HLNW risk, and trust. Factors responsible for acceptance are discussed at several levels. One is the attitude to nuclear power, which is becoming more positive, probably because no viable alternatives are in sight. Other factors have to do with the extensive information programs conducted in these municipalities, and with the logical nature of the conclusion that they would be good candidates for hosting the national HLNW repository.

  9. Frequency-modulated nuclear localization bursts coordinate gene regulation

    PubMed Central

    Cai, Long; Dalal, Chiraj K.; Elowitz, Michael B.

    2008-01-01

    In yeast, the transcription factor Crz1 is dephosphorylated and translocates into the nucleus in response to extracellular calcium. Using time-lapse microscopy, we found that Crz1 exhibited short bursts of nuclear localization (∼2 minutes) that occurred stochastically in individual cells and propagated to the expression of downstream genes. Strikingly, calcium concentration controlled the frequency, but not duration, of localization bursts. Using an analytic model, we found that this frequency modulation (FM) of bursts ensures proportional expression of multiple target genes across a wide dynamic range of expression levels, independent of promoter characteristics. We experimentally confirmed this theory with natural and synthetic Crz1 target promoters. Another stress response transcription factor, Msn2, exhibits similar, but largely uncorrelated, localization bursts under calcium stress. These results suggest that FM regulation of localization bursts may be a general control strategy utilized by the cell to coordinate multi-gene responses to external signals. PMID:18818649

  10. Nucleon localization and fragment formation in nuclear fission

    NASA Astrophysics Data System (ADS)

    Zhang, C. L.; Schuetrumpf, B.; Nazarewicz, W.

    2016-12-01

    Background: An electron localization measure was originally introduced to characterize chemical bond structures in molecules. Recently, a nucleon localization based on Hartree-Fock densities has been introduced to investigate α -cluster structures in light nuclei. Compared to the local nucleonic densities, the nucleon localization function has been shown to be an excellent indicator of shell effects and cluster correlations. Purpose: Using the spatial nucleon localization measure, we investigate the emergence of fragments in fissioning heavy nuclei. Methods: To illustrate basic concepts of nucleon localization, we employ the self-consistent energy density functional method with a quantified energy density functional optimized for fission studies. Results: We study the particle densities and spatial nucleon localization distributions along the fission pathways of 264Fm, 232Th, and 240Pu. We demonstrate that the fission fragments are formed fairly early in the evolution, well before scission. We illustrate the usefulness of the localization measure by showing how the hyperdeformed state of 232Th can be understood in terms of a quasimolecular state made of 132Sn and 100Zr fragments. Conclusions: Compared to nucleonic distributions, the nucleon localization function more effectively quantifies nucleonic clustering: its characteristic oscillating pattern, traced back to shell effects, is a clear fingerprint of cluster/fragment configurations. This is of particular interest for studies of fragment formation and fragment identification in fissioning nuclei.

  11. Constitutive nuclear localization of an alternatively spliced sirtuin-2 isoform.

    PubMed

    Rack, Johannes G M; VanLinden, Magali R; Lutter, Timo; Aasland, Rein; Ziegler, Mathias

    2014-04-17

    Sirtuin-2 (SIRT2), the cytoplasmic member of the sirtuin family, has been implicated in the deacetylation of nuclear proteins. Although the enzyme has been reported to be located to the nucleus during G2/M phase, its spectrum of targets suggests functions in the nucleus throughout the cell cycle. While a nucleocytoplasmic shuttling mechanism has been proposed for SIRT2, recent studies have indicated the presence of a constitutively nuclear isoform. Here we report the identification of a novel splice variant (isoform 5) of SIRT2 that lacks a nuclear export signal and encodes a predominantly nuclear isoform. This novel isoform 5 fails to show deacetylase activity using several assays, both in vitro and in vivo, and we are led to conclude that this isoform is catalytically inactive. Nevertheless, it retains the ability to interact with p300, a known interaction partner. Moreover, changes in intrinsic tryptophan fluorescence upon denaturation indicate that the protein is properly folded. These data, together with computational analyses, confirm the structural integrity of the catalytic domain. Our results suggest an activity-independent nuclear function of the novel isoform.

  12. Error analysis in nuclear density functional theory

    NASA Astrophysics Data System (ADS)

    Schunck, Nicolas; McDonnell, Jordan D.; Sarich, Jason; Wild, Stefan M.; Higdon, Dave

    2015-03-01

    Nuclear density functional theory (DFT) is the only microscopic, global approach to the structure of atomic nuclei. It is used in numerous applications, from determining the limits of stability to gaining a deep understanding of the formation of elements in the Universe or the mechanisms that power stars and reactors. The predictive power of the theory depends on the amount of physics embedded in the energy density functional as well as on efficient ways to determine a small number of free parameters and solve the DFT equations. In this article, we discuss the various sources of uncertainties and errors encountered in DFT and possible methods to quantify these uncertainties in a rigorous manner.

  13. [Structure and Function of the Nuclear Receptor Constitutive Androstane Receptor].

    PubMed

    Inouye, Yoshio

    2016-01-01

    Animal defense mechanisms against both endogenous and exogenous toxic compounds function mainly through receptor-type transcription factors, including the constitutive androstane receptor (CAR). Following xenobiotic stimulation, CAR translocates into the nucleus and transactivates its target genes including oxygenic and conjugative enzymes and transporters in hepatocytes. We identified subcellular localization signals in the rat CAR: two nuclear localization signals (NLS1 and 2); two nuclear export signals (NES1 and 2); and a cytoplasmic retention region. The nuclear import of CAR is regulated by the importin-Ran system and microtubule network. Five splice variants (SV1-5) were identified in rat liver in addition to wild-type CAR. When expressed in immortalized cells, their artificial transcripts were inactive as transcription factors. A CAR mutant with three consecutive alanine residues inserted into the ligand-binding domain of CAR showed ligand-dependent activation of target genes in immortalized cells, which is in marked contrast to the constitutive transactivating nature of wild-type CAR. Using this assay system, androstenol and clotrimazole, both of which are inverse agonists of CAR, were classified as an antagonist and weak agonist, respectively. A member of the DEAD box DNA/RNA helicase family (DP97) and protein arginine methyltransferase 5 (PRMT5) were found to be gene (or promotor)-specific coactivators of CAR. The expression of the CAR gene might be under the control of clock genes mediated by the nuclear receptor Rev-erb-α.

  14. The defective nuclear lamina in Hutchinson-gilford progeria syndrome disrupts the nucleocytoplasmic Ran gradient and inhibits nuclear localization of Ubc9.

    PubMed

    Kelley, Joshua B; Datta, Sutirtha; Snow, Chelsi J; Chatterjee, Mandovi; Ni, Li; Spencer, Adam; Yang, Chun-Song; Cubeñas-Potts, Caelin; Matunis, Michael J; Paschal, Bryce M

    2011-08-01

    The mutant form of lamin A responsible for the premature aging disease Hutchinson-Gilford progeria syndrome (termed progerin) acts as a dominant negative protein that changes the structure of the nuclear lamina. How the perturbation of the nuclear lamina in progeria is transduced into cellular changes is undefined. Using patient fibroblasts and a variety of cell-based assays, we determined that progerin expression in Hutchinson-Gilford progeria syndrome inhibits the nucleocytoplasmic transport of several factors with key roles in nuclear function. We found that progerin reduces the nuclear/cytoplasmic concentration of the Ran GTPase and inhibits the nuclear localization of Ubc9, the sole E2 for SUMOylation, and of TPR, the nucleoporin that forms the basket on the nuclear side of the nuclear pore complex. Forcing the nuclear localization of Ubc9 in progerin-expressing cells rescues the Ran gradient and TPR import, indicating that these pathways are linked. Reducing nuclear SUMOylation decreases the nuclear mobility of the Ran nucleotide exchange factor RCC1 in vivo, and the addition of SUMO E1 and E2 promotes the dissociation of RCC1 and Ran from chromatin in vitro. Our data suggest that the cellular effects of progerin are transduced, at least in part, through reduced function of the Ran GTPase and SUMOylation pathways.

  15. Enzyme function is regulated by its localization.

    PubMed

    Gifford, Stacey M; Meyer, Pablo

    2015-12-01

    To better understand how enzyme localization affects enzyme activity we studied the cellular localization of the glycosyltransferase MurG, an enzyme necessary for cell wall synthesis at the spore during sporulation in the bacterium Bacillus subtilis. During sporulation MurG was gradually enriched to the membrane at the forespore and point mutations in a MurG helical domain disrupting its localization to the membrane caused severe sporulation defects, but did not affect localization nor caused detectable defects during exponential growth. We found that this localization is dependent on the phospholipid cardiolipin, as in strains where the cardiolipin-synthesizing genes were deleted, MurG levels were diminished at the forespore. Furthermore, in this cardiolipin-less strain, MurG localization during sporulation was rescued by external addition of purified cardiolipin. These results support localization as a critical factor in the regulation of proper enzyme function and catalysis.

  16. Green's function Monte Carlo in nuclear physics

    SciTech Connect

    Carlson, J.

    1990-01-01

    We review the status of Green's Function Monte Carlo (GFMC) methods as applied to problems in nuclear physics. New methods have been developed to handle the spin and isospin degrees of freedom that are a vital part of any realistic nuclear physics problem, whether at the level of quarks or nucleons. We discuss these methods and then summarize results obtained recently for light nuclei, including ground state energies, three-body forces, charge form factors and the coulomb sum. As an illustration of the applicability of GFMC to quark models, we also consider the possible existence of bound exotic multi-quark states within the framework of flux-tube quark models. 44 refs., 8 figs., 1 tab.

  17. Nuclear localization signals for four distinct karyopherin-β nuclear import systems.

    PubMed

    Soniat, Michael; Chook, Yuh Min

    2015-06-15

    The Karyopherin-β family of proteins mediates nuclear transport of macromolecules. Nuclear versus cytoplasmic localization of proteins is often suggested by the presence of NLSs (nuclear localization signals) or NESs (nuclear export signals). Import-Karyopherin-βs or Importins bind to NLSs in their protein cargos to transport them through nuclear pore complexes into the nucleus. Until recently, only two classes of NLS had been biochemically and structurally characterized: the classical NLS, which is recognized by the Importin-α/β heterodimer and the PY-NLS (proline-tyrosine NLS), which is recognized by Karyopherin-β2 or Transportin-1. Structures of two other Karyopherin-βs, Kap121 and Transportin-SR2, in complex with their respective cargos were reported for the first time recently, revealing two new distinct classes of NLSs. The present paper briefly describes the classical NLS, reviews recent literature on the PY-NLS and provides in-depth reviews of the two newly discovered classes of NLSs that bind Kap121p and Transportin-SR respectively.

  18. Meaning of the nuclear wave function

    NASA Astrophysics Data System (ADS)

    Terry, John D.; Miller, Gerald A.

    2016-07-01

    Background: The intense current experimental interest in studying the structure of the deuteron and using it to enable accurate studies of neutron structure motivate us to examine the four-dimensional space-time nature of the nuclear wave function and the various approximations used to reduce it to an object that depends only on three spatial variables. Purpose: The aim is to determine if the ability to understand and analyze measured experimental cross sections is compromised by making the reduction from four to three dimensions. Method: Simple, exactly calculable, covariant models of a bound-state wave-state wave function (a scalar boson made of two constituent-scalar bosons) with parameters chosen to represent a deuteron are used to investigate the accuracy of using different approximations to the nuclear wave function to compute the quasielastic scattering cross section. Four different versions of the wave function are defined (light-front-spectator, light-front, light-front with scaling, and nonrelativistic) and used to compute the cross sections as a function of how far off the mass shell (how virtual) is the struck constituent. Results: We show that making an exact calculation of the quasielastic scattering cross section involves using the light-front-spectator wave function. All of the other approaches fail to reproduce the model exact calculation if the value of Bjorken x differs from unity. The model is extended to consider an essential effect of spin to show that constituent nucleons cannot be treated as being on their mass shell even when taking the matrix element of a "good" current. Conclusions: Developing realistic light-front-spectator wave functions to meet the needs of current and planned experiments is a worthwhile activity.

  19. p150/Glued Modifies Nuclear Estrogen Receptor Function

    PubMed Central

    Lee, Soo Jung; Chae, Christina; Wang, Michael M.

    2009-01-01

    Estrogen modulates gene expression through interactions with estrogen receptors (ERs) that bind chromosomal target genes. Recent studies have suggested an interaction between the cytoskeletal system and estrogen signaling; these have implicated a role of cytoplasmic microtubules in scaffolding ERα and enhancing nongenomic function; in addition, other experiments demonstrate that dynein light chain 1 may chaperone ERα to the nucleus, indirectly increasing transcriptional potency. Actin/myosin and dynein light chain 1 are also required for estrogen-mediated chromosomal movement that is required for transcriptional up-regulation of ERα targets. We present evidence that the dynactin component, p150/glued, directly influences the potency of nuclear ER function. Increasing the stoichiometric ratio of p150/glued and ERα by overexpression enhances estrogen responses. ERα enhancement by p150/glued does not appear to be influenced by shifts in subcellular localization because microtubule disruption fails to increase nuclear ERα. Rather, we find that modest amounts of p150/glued reside in the nucleus of cells, suggesting that it plays a direct role in nuclear transcription. Notably, p150/glued is recruited to the pS2 promoter in the presence of hormone, and, in MCF-7 cells, knockdown of p150/glued levels reduces estrogen-dependent transcription. Our results suggest that p150/glued modulates estrogen sensitivity in cells through nuclear mechanisms. PMID:19228793

  20. Fission yeast Lem2 and Man1 perform fundamental functions of the animal cell nuclear lamina.

    PubMed

    Gonzalez, Yanira; Saito, Akira; Sazer, Shelley

    2012-01-01

    In animal cells the nuclear lamina, which consists of lamins and lamin-associated proteins, serves several functions: it provides a structural scaffold for the nuclear envelope and tethers proteins and heterochromatin to the nuclear periphery. In yeast, proteins and large heterochromatic domains including telomeres are also peripherally localized, but there is no evidence that yeast have lamins or a fibrous nuclear envelope scaffold. Nonetheless, we found that the Lem2 and Man1 proteins of the fission yeast Schizosaccharomyces pombe, evolutionarily distant relatives of the Lap2/Emerin/Man1 (LEM) sub-family of animal cell lamin-associated proteins, perform fundamental functions of the animal cell lamina. These integral inner nuclear membrane localized proteins, with nuclear localized DNA binding Helix-Extension-Helix (HEH) domains, impact nuclear envelope structure and integrity, are essential for the enrichment of telomeres at the nuclear periphery and by means of their HEH domains anchor chromatin, most likely transcriptionally repressed heterochromatin, to the nuclear periphery. These data indicate that the core functions of the nuclear lamina are conserved between fungi and animal cells and can be performed in fission yeast, without lamins or other intermediate filament proteins.

  1. Nuclear localization of Formyl-Peptide Receptor 2 in human cancer cells.

    PubMed

    Cattaneo, Fabio; Parisi, Melania; Fioretti, Tiziana; Sarnataro, Daniela; Esposito, Gabriella; Ammendola, Rosario

    2016-08-01

    Current models of G protein-coupled receptors (GPCRs) signaling describe binding of external agonists to cell surface receptors which, in turn, trigger several biological responses. New paradigms indicate that GPCRs localize to and signal at the nucleus, thus regulating distinct signaling cascades. The formyl-peptide receptor FPR2 belongs to the GPCR super-family and is coupled to PTX-sensitive Gi proteins. We show by western blot analysis, immunofluorescence experiments and radioligand binding assays that FPR2 is expressed at nuclear level in CaLu-6 and AGS cells. Nuclear FPR2 is a functional receptor, since it participates in intra-nuclear signaling, as assessed by decreased G protein-FPR2 association and enhanced ERK2, c-Jun and c-Myc phosphorylation upon stimulation of intact nuclei with the FPR2 agonist, WKYMVm. We analyzed FPR2 sequence for the search of a nuclear localization sequence (NLS) and we found a stretch of basic aminoacids (227-KIHKK-231) in the third cytoplasmic loop of the receptor. We performed single (K230A) and multiple (H229A/K230A/K231A) mutagenesis of NLS. The constructs were individually overexpressed in HEK293 cells and immunofluorescence and western blot analysis showed that nuclear localization or translocation of FPR2 depends on the integrity of the H(229) and K(231) residues within the NLS.

  2. Cloning, characterization and subcellular localization of Nuclear LIM interactor interacting factor gene from Leishmania donovani.

    PubMed

    Ravinder, R; Goyal, N

    2017-05-05

    LIM domains are zinc-binding motifs that mediate protein-protein interactions and are found in a wide variety of cytoplasmic and nuclear proteins. The nuclear LIM domain family members have a number of different functions including transcription factors, gene regulation, cell fate determination, organization of the cytoskeleton and tumour formation exerting their function through various LIM domain interacting protein partners/cofactors. Nuclear LIM domain interacting proteins/factors have not been reported in any protozoan parasites including Leishmania. Here, we report for the first time cloning, characterization and subcellular localization of nuclear LIM interactor-interacting factor (NLI) like protein from Leishmania donovani, the causative agent of Indian Kala-azar. Primary sequence analysis of LdNLI revealed presence of characteristic features of nuclear LIM interactor-interacting factor. However, leishmanial NLI represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. The sub-cellular distribution of LdNLI revealed the discreet localization in nucleus and kinetoplast only, suggesting that the gene may have a role in parasite gene expression.

  3. A proline-tyrosine nuclear localization signal (PY-NLS) is required for the nuclear import of fission yeast PAB2, but not of human PABPN1.

    PubMed

    Mallet, Pierre-Luc; Bachand, François

    2013-03-01

    Nuclear poly(A)-binding proteins (PABPs) are evolutionarily conserved proteins that play key roles in eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, the major nuclear PABP, Pab2, functions in the maturation of small nucleolar RNAs as well as in nuclear RNA decay. Despite knowledge about its nuclear functions, nothing is known about how Pab2 is imported into the nucleus. Here, we show that Pab2 contains a proline-tyrosine nuclear localization signal (PY-NLS) that is necessary and sufficient for its nuclear localization and function. Consistent with the role of karyopherin β2 (Kapβ2)-type receptors in the import of PY-NLS cargoes, we show that the fission yeast ortholog of human Kapβ2, Kap104, binds to recombinant Pab2 and is required for Pab2 nuclear localization. The absence of arginine methylation in a basic region N-terminal to the PY-core motif of Pab2 did not affect its nuclear localization. However, in the context of a sub-optimal PY-NLS, we found that Pab2 was more efficiently targeted to the nucleus in the absence of arginine methylation, suggesting that this modification can affect the import kinetics of a PY-NLS cargo. Although a sequence resembling a PY-NLS motif can be found in the human Pab2 ortholog, PABPN1, our results indicate that neither a functional PY-NLS nor Kapβ2 activity are required to promote entry of PABPN1 into the nucleus of human cells. Our findings describe the mechanism by which Pab2 is imported into the nucleus, providing the first example of a PY-NLS import system in fission yeast. In addition, this study suggests the existence of alternative or redundant nuclear import pathways for human PABPN1.

  4. Nuclear localization of platelet-activating factor receptor controls retinal neovascularization

    PubMed Central

    K Bhosle, Vikrant; Rivera, José Carlos; Zhou, Tianwei (Ellen); Omri, Samy; Sanchez, Melanie; Hamel, David; Zhu, Tang; Rouget, Raphael; Rabea, Areej Al; Hou, Xin; Lahaie, Isabelle; Ribeiro-da-Silva, Alfredo; Chemtob, Sylvain

    2016-01-01

    Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs. PMID:27462464

  5. Nuclear targeting of the maize R protein requires two nuclear localization sequences

    SciTech Connect

    Shieh, M.W.; Raikhel, N.V. ); Wessler, S.R. )

    1993-02-01

    Previous genetic and structural evidence indicates that the maize R gene encodes a nuclear transcriptional activating factor. In-frame carboxyl- and amino-terminal fusions of the R gene to the reporter gene encoding [beta]-glucuronidase (GUS) were sufficient to direct GUS to the nucleus of the transiently transformed onion (Allium cepa) epidermal cells. Further analysis of chimeric constructs containing regions of the R gene fused to the GUS cDNA revealed three specific nuclear localization sequences (NLSs) that were capable of redirecting the GUS protein to the nucleus. Amino-terminal NLS-A (amino acids 100-109, GDRRAAPARP) contained several arginine residues; a similar localization signal is found in only a few viral proteins. The medial NLS-M (amino acids 419-428, MSERKRREKL) is a simian virus 40 large T antigen-type NLS, and the carboxyl-terminal NLS-C (amino acids 598-610, MISESLRKAIGKR) is a mating type [alpha]2 type. NLSs M and C are independently sufficient to direct the GUS protein to the nucleus when it is fused at the amino terminus of GUS, whereas NLS-A fused to GUS partitioned between the nucleus and cytoplasm. Similar partitioning was observed when localization signals NLS-A and NLS-C were independently fused to the carboxy-terminal portion of GUS. A sequential deletion of the localization signals indicated that the amino-terminal and carboxyl-terminal fusions of R and GUS were redirected to the nucleus only when both NLS-A and -M, or NLS-C and -M, were present. These results indicate that multiple localization signals are necessary for nuclear targeting of this protein. The conservation of the localization signals within the alleles of R and similar proteins from other organisms is also discussed. 45 refs., 6 figs.

  6. Phosphatidylinositol 3-kinase/Akt signaling enhances nuclear localization and transcriptional activity of BRCA1

    SciTech Connect

    Hinton, Cimona V.; Fitzgerald, Latricia D.; Thompson, Marilyn E. . E-mail: methompson@mmc.edu

    2007-05-15

    Signaling pathways involved in regulating nuclear-cytoplasmic distribution of BRCA1 have not been previously reported. Here, we provide evidence that heregulin {beta}1-induced activation of the Akt pathway increases the nuclear content of BRCA1. First, treatment of T47D breast cancer cells with heregulin {beta}1 results in a two-fold increase in nuclear BRCA1 as assessed by FACS analysis, immunoblotting and immunofluorescence. This heregulin-induced increase in nuclear BRCA1 is blocked by siRNA-mediated down-regulation of Akt. Second, mutation of threonine 509 in BRCA1, the site of Akt phosphorylation, to an alanine, attenuates the ability of heregulin to induce BRCA1 nuclear accumulation. These data suggest that Akt-catalyzed phosphorylation of BRCA1 is required for the heregulin-regulated nuclear concentration of BRCA1. Because most functions ascribed to BRCA1 occur within the nucleus, we postulated that phosphorylation-dependent nuclear accumulation of BRCA1 would result in enhanced nuclear activity, specifically transcriptional activity, of BRCA1. This postulate is affirmed by our observation that the ability of BRCA1 to transactivate GADD45 promoter constructs was enhanced in T47D cells treated with heregulin {beta}1. Furthermore, the heterologous expression of BRCA1 in HCC1937 human breast cancer cells, which have constitutively active Akt, also induces GADD45 promoter activity, whereas the expression of BRCA1 in which threonine 509 has been mutated to an alanine is able to only minimally induce promoter activity. These findings implicate Akt in upstream events leading to BRCA1 nuclear localization and function.

  7. Troponin T nuclear localization and its role in aging skeletal muscle.

    PubMed

    Zhang, Tan; Birbrair, Alexander; Wang, Zhong-Min; Taylor, Jackson; Messi, María Laura; Delbono, Osvaldo

    2013-04-01

    Troponin T (TnT) is known to mediate the interaction between Tn complex and tropomyosin (Tm), which is essential for calcium-activated striated muscle contraction. This regulatory function takes place in the myoplasm, where TnT binds Tm. However, recent findings of troponin I and Tm nuclear translocation in Drosophila and mammalian cells imply other roles for the Tn-Tm complex. We hypothesized that TnT plays a nonclassical role through nuclear translocation. Immunoblotting with different antibodies targeting the NH2- or COOH-terminal region uncovered a pool of fast skeletal muscle TnT3 localized in the nuclear fraction of mouse skeletal muscle as either an intact or fragmented protein. Construction of TnT3-DsRed fusion proteins led to the further observation that TnT3 fragments are closely related to nucleolus and RNA polymerase activity, suggesting a role for TnT3 in regulating transcription. Functionally, overexpression of TnT3 fragments produced significant defects in nuclear shape and caused high levels of apoptosis. Interestingly, nuclear TnT3 and its fragments were highly regulated by aging, thus creating a possible link between the deleterious effects of TnT3 and sarcopenia. We propose that changes in nuclear TnT3 and its fragments cause the number of myonuclei to decrease with age, contributing to muscle damage and wasting.

  8. An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium

    SciTech Connect

    Myre, Michael A.; O'Day, Danton H. . E-mail: doday@utm.utoronto.ca

    2005-06-24

    Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD ({sup 171}EDVSRFIKGKLLQKQQKIYKDLERF{sup 195}) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues {sup 48}KKSYQDPEIIAHSRPRK{sup 64} that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to {sup 48}EF{sup 49} abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the {sup 48}EF{sup 49} construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium.

  9. DNA Ligase IV regulates XRCC4 nuclear localization.

    PubMed

    Francis, Dailia B; Kozlov, Mikhail; Chavez, Jose; Chu, Jennifer; Malu, Shruti; Hanna, Mary; Cortes, Patricia

    2014-09-01

    DNA Ligase IV, along with its interacting partner XRCC4, are essential for repairing DNA double strand breaks by non-homologous end joining (NHEJ). Together, they complete the final ligation step resolving the DNA break. Ligase IV is regulated by XRCC4 and XLF. However, the mechanism(s) by which Ligase IV control the NHEJ reaction and other NHEJ factor(s) remains poorly characterized. Here, we show that a C-terminal region of Ligase IV (aa 620-800), which encompasses a NLS, the BRCT I, and the XRCC4 interacting region (XIR), is essential for nuclear localization of its co-factor XRCC4. In Ligase IV deficient cells, XRCC4 showed deregulated localization remaining in the cytosol even after induction of DNA double strand breaks. DNA Ligase IV was also required for efficient localization of XLF into the nucleus. Additionally, human fibroblasts that harbor hypomorphic mutations within the Ligase IV gene displayed decreased levels of XRCC4 protein, implicating that DNA Ligase IV is also regulating XRCC4 stability. Our results provide evidence for a role of DNA Ligase IV in controlling the cellular localization and protein levels of XRCC4.

  10. DNA Ligase IV regulates XRCC4 nuclear localization

    PubMed Central

    Francis, Dailia B.; Kozlov, Mikhail; Chavez, Jose; Chu, Jennifer; Malu, Shruti; Hanna, Mary; Cortes, Patricia

    2014-01-01

    DNA Ligase IV, along with its interacting partner XRCC4, are essential for repairing DNA double strand breaks by non-homologous end joining (NHEJ). Together, they complete the final ligation step resolving the DNA break. Ligase IV is regulated by XRCC4 and XLF. However, the mechanism(s) by which Ligase IV control the NHEJ reaction and other NHEJ factor(s) remains poorly characterized. Here, we show that a C-terminal region of Ligase IV (aa 620 to 800), which encompasses a NLS, the BRCT I, and the XRCC4 interacting region (XIR), is essential for nuclear localization of its co-factor XRCC4. In Ligase IV deficient cells, XRCC4 showed deregulated localization remaining in the cytosol even after induction of DNA double strand breaks. DNA Ligase IV was also required for efficient localization of XLF into the nucleus. Additionally, human fibroblasts that harbor hypomorphic mutations within the Ligase IV gene displayed decreased levels of XRCC4 protein, implicating that DNA Ligase IV is also regulating XRCC4 stability. Our results provide evidence for a role of DNA Ligase IV in controlling the cellular localization and protein levels of XRCC4. PMID:24984242

  11. Ras-activated RSK1 phosphorylates EBP50 to regulate its nuclear localization and promote cell proliferation.

    PubMed

    Lim, Hooi Cheng; Jou, Tzuu-Shuh

    2016-03-01

    Differential subcellular localization of EBP50 leads to its controversial role in cancer biology either as a tumor suppressor when it resides at the membrane periphery, or a tumor facilitator at the nucleus. However, the mechanism behind nuclear localization of EBP50 remains unclear. A RNA interference screening identified the downstream effector of the Ras-ERK cascade, RSK1, as the molecule unique for nuclear transport of EBP50. RSK1 binds to EBP50 and phosphorylates it at a conserved threonine residue at position 156 (T156) under the regulation of growth factor. Mutagenesis experiments confirmed the significance of T156 residue in nuclear localization of EBP50, cellular proliferation, and oncogenic transformation. Our study sheds light on a possible therapeutic strategy targeting at this aberrant nuclear expression of EBP50 without affecting the normal physiological function of EBP50 at other subcellular localization.

  12. [The role of Piwi nuclear localization in the differentiation and proliferation of germline stem cells].

    PubMed

    Yakushev, E Y; Mikhaleva, E A; Abramov, Y A; Sokolova, O A; Zyrianova, I M; Gvozdev, V A; Klenov, M S

    2016-01-01

    The Piwi protein and its orthologs are considered as the key components of the piRNA machinery implicated in transcriptional silencing of transposons. Неre, we show that nuclear localization of the Piwi protein is required not only for transposon repression, but also for proper differentiation of germline stem cells (GSCs). piwi^(Nt) mutation that causes loss of nuclear Piwi and its retention in the cytoplasm leads to the accumulation of undifferentiated GSC-like cells. The analysis of piwi^(Nt) mutation in combination with a bam gene mutation blocking GSC differentiation shows that the loss of nuclear Piwi decreases GSC proliferation rate. This is accompanied by the accumulation of DNA double-strand breaks in GSCs that may be caused by transposition events. Here, for the first time a set of transposons repressed by Piwi in GSCs and surrounding niche cells has been identified. The present study together with our previous data show that nuclear and cytoplasmic Piwi can regulate different stages of the functioning of germinal cells: cytoplasmic Piwi is sufficient to maintain GSCs, while nuclear Piwi localization is necessary for their proper proliferation and differentiation.

  13. Automated local bright feature image analysis of nuclear proteindistribution identifies changes in tissue phenotype

    SciTech Connect

    Knowles, David; Sudar, Damir; Bator, Carol; Bissell, Mina

    2006-02-01

    The organization of nuclear proteins is linked to cell and tissue phenotypes. When cells arrest proliferation, undergo apoptosis, or differentiate, the distribution of nuclear proteins changes. Conversely, forced alteration of the distribution of nuclear proteins modifies cell phenotype. Immunostaining and fluorescence microscopy have been critical for such findings. However, there is an increasing need for quantitative analysis of nuclear protein distribution to decipher epigenetic relationships between nuclear structure and cell phenotype, and to unravel the mechanisms linking nuclear structure and function. We have developed imaging methods to quantify the distribution of fluorescently-stained nuclear protein NuMA in different mammary phenotypes obtained using three-dimensional cell culture. Automated image segmentation of DAPI-stained nuclei was generated to isolate thousands of nuclei from three-dimensional confocal images. Prominent features of fluorescently-stained NuMA were detected using a novel local bright feature analysis technique, and their normalized spatial density calculated as a function of the distance from the nuclear perimeter to its center. The results revealed marked changes in the distribution of the density of NuMA bright features as non-neoplastic cells underwent phenotypically normal acinar morphogenesis. In contrast, we did not detect any reorganization of NuMA during the formation of tumor nodules by malignant cells. Importantly, the analysis also discriminated proliferating non-neoplastic cells from proliferating malignant cells, suggesting that these imaging methods are capable of identifying alterations linked not only to the proliferation status but also to the malignant character of cells. We believe that this quantitative analysis will have additional applications for classifying normal and pathological tissues.

  14. Acetylation directs survivin nuclear localization to repress STAT3 oncogenic activity.

    PubMed

    Wang, Haijuan; Holloway, Michael P; Ma, Li; Cooper, Zachary A; Riolo, Matthew; Samkari, Ayman; Elenitoba-Johnson, Kojo S J; Chin, Y Eugene; Altura, Rachel A

    2010-11-12

    The multiple functions of the oncofetal protein survivin are dependent on its selective expression patterns within immunochemically distinct subcellular pools. The mechanism by which survivin localizes to these compartments, however, is only partly understood. Here we show that nuclear accumulation of survivin is promoted by CREB-binding protein (CBP)-dependent acetylation on lysine 129 (129K, Lys-129). We demonstrate a mechanism by which survivin acetylation at this position results in its homodimerization, while deacetylation promotes the formation of survivin monomers that heterodimerize with CRM1 and facilitate its nuclear export. Using proteomic analysis, we identified the oncogenic transcription factor STAT3 as a binding partner of nuclear survivin. We show that acetylated survivin binds to the N-terminal transcriptional activation domain of the STAT3 dimer and represses STAT3 transactivation of target gene promoters. Using multiplex PCR and DNA sequencing, we identified a single-nucleotide polymorphism (A → G) at Lys-129 that exists as a homozygous mutation in a neuroblastoma cell line and corresponds with a defect in survivin nuclear localization. Our results demonstrate that the dynamic equilibrium between survivin acetylation and deacetylation at amino acid 129 determines its interaction with CRM1, its subsequent subcellular localization, and its ability to inhibit STAT3 transactivation, providing a potential route for therapeutic intervention in STAT3-dependent tumors.

  15. Proteolytic cleavage, trafficking, and functions of nuclear receptor tyrosine kinases.

    PubMed

    Chen, Mei-Kuang; Hung, Mien-Chie

    2015-10-01

    Intracellular localization has been reported for over three-quarters of receptor tyrosine kinase (RTK) families in response to environmental stimuli. Internalized RTK may bind to non-canonical substrates and affect various cellular processes. Many of the intracellular RTKs exist as fragmented forms that are generated by γ-secretase cleavage of the full-length receptor, shedding, alternative splicing, or alternative translation initiation. Soluble RTK fragments are stabilized and intracellularly transported into subcellular compartments, such as the nucleus, by binding to chaperone or transcription factors, while membrane-bound RTKs (full-length or truncated) are transported from the plasma membrane to the ER through the well-established Rab- or clathrin adaptor protein-coated vesicle retrograde trafficking pathways. Subsequent nuclear transport of membrane-bound RTK may occur via two pathways, INFS or INTERNET, with the former characterized by release of receptors from the ER into the cytosol and the latter characterized by release of membrane-bound receptor from the ER into the nucleoplasm through the inner nuclear membrane. Although most non-canonical intracellular RTK signaling is related to transcriptional regulation, there may be other functions that have yet to be discovered. In this review, we summarize the proteolytic processing, intracellular trafficking and nuclear functions of RTKs, and discuss how they promote cancer progression, and their clinical implications.

  16. Maximally localized Wannier functions: Theory and applications

    NASA Astrophysics Data System (ADS)

    Marzari, Nicola; Mostofi, Arash A.; Yates, Jonathan R.; Souza, Ivo; Vanderbilt, David

    2012-10-01

    The electronic ground state of a periodic system is usually described in terms of extended Bloch orbitals, but an alternative representation in terms of localized “Wannier functions” was introduced by Gregory Wannier in 1937. The connection between the Bloch and Wannier representations is realized by families of transformations in a continuous space of unitary matrices, carrying a large degree of arbitrariness. Since 1997, methods have been developed that allow one to iteratively transform the extended Bloch orbitals of a first-principles calculation into a unique set of maximally localized Wannier functions, accomplishing the solid-state equivalent of constructing localized molecular orbitals, or “Boys orbitals” as previously known from the chemistry literature. These developments are reviewed here, and a survey of the applications of these methods is presented. This latter includes a description of their use in analyzing the nature of chemical bonding, or as a local probe of phenomena related to electric polarization and orbital magnetization. Wannier interpolation schemes are also reviewed, by which quantities computed on a coarse reciprocal-space mesh can be used to interpolate onto much finer meshes at low cost, and applications in which Wannier functions are used as efficient basis functions are discussed. Finally the construction and use of Wannier functions outside the context of electronic-structure theory is presented, for cases that include phonon excitations, photonic crystals, and cold-atom optical lattices.

  17. Multidimensional stochastic approximation using locally contractive functions

    NASA Technical Reports Server (NTRS)

    Lawton, W. M.

    1975-01-01

    A Robbins-Monro type multidimensional stochastic approximation algorithm which converges in mean square and with probability one to the fixed point of a locally contractive regression function is developed. The algorithm is applied to obtain maximum likelihood estimates of the parameters for a mixture of multivariate normal distributions.

  18. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization.

    PubMed

    Shiheido, Hirokazu; Shimizu, Jun

    2015-02-20

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND356-58, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3.

  19. Actin-related proteins localized in the nucleus: from discovery to novel roles in nuclear organization.

    PubMed

    Oma, Yukako; Harata, Masahiko

    2011-01-01

    The actin family consists of conventional actin and actin-related proteins (ARPs), and the members show moderate similarity and share the same basal structure. Following the finding of various ARPs in the cytoplasm in the 1990s, multiple subfamilies that are localized predominantly in the nucleus were identified. Consistent with these cytological observations, subsequent biochemical analyses revealed the involvement of the nuclear ARPs in ATP-dependent chromatin-remodeling and histone acetyltransferase complexes. In addition to their contribution to chromatin remodeling, recent studies have shown that nuclear ARPs have roles in the organization of the nucleus that are independent of the activity of the above-mentioned complexes. Therefore, nuclear ARPs are recognized as novel key regulators of genome function, and affect not only the remodeling of chromatin but also the spatial arrangement and dynamics of chromatin within the nucleus.

  20. Local and Global Comparison of Continuous Functions

    SciTech Connect

    Edelsbrunner, H; Harer, J; Natarajan, V; Pascucci, V

    2004-12-16

    We introduce local and global comparison measures for a collection of k {<=} d real-valued smooth functions on a common d-dimensional Riemannian manifold. For k = d = 2 we relate the measures to the set of critical points of one function restricted to the level sets of the other. The definition of the measures extends to piecewise linear functions for which they are easy to compute. The computation of the measures forms the centerpiece of a software tool which we use to study scientific datasets.

  1. Robust determination of maximally localized Wannier functions

    NASA Astrophysics Data System (ADS)

    Cancès, Éric; Levitt, Antoine; Panati, Gianluca; Stoltz, Gabriel

    2017-02-01

    We propose an algorithm to determine maximally localized Wannier functions (MLWFs). This algorithm, based on recent theoretical developments, does not require any physical input such as initial guesses for the Wannier functions, unlike popular schemes based on the projection method. We discuss how the projection method can fail on fine grids when the initial guesses are too far from MLWFs. We demonstrate that our algorithm is able to find localized Wannier functions through tests on two-dimensional systems, simplified models of semiconductors, and realistic DFT systems by interfacing with the wannier90 code. We also test our algorithm on the Haldane and Kane-Mele models to examine how it fails in the presence of topological obstructions.

  2. Nuclear but Not Cytosolic Phosphoinositide 3-Kinase Beta Has an Essential Function in Cell Survival ▿

    PubMed Central

    Kumar, Amit; Redondo-Muñoz, Javier; Perez-García, Vicente; Cortes, Isabel; Chagoyen, Monica; Carrera, Ana C.

    2011-01-01

    Class IA phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110α and p110β that have nonredundant functions in embryonic development and cell division. However, whereas p110α concentrates in the cytoplasm, p110β localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110β nuclear localization remain unknown. We describe here that association with the p85β regulatory subunit controls p110β nuclear localization. We identified a nuclear localization signal (NLS) in p110β C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85β. Deletion of p110β induced apoptosis, and complementation with the cytoplasmic C2-NLS p110β mutant was unable to restore cell survival. These studies show that p110β NLS and p85β NES regulate p85β/p110β nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110β controls cell survival. PMID:21383062

  3. Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

    SciTech Connect

    Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan

    2013-08-15

    The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP.

  4. Structure, dynamics and function of nuclear pore complexes

    PubMed Central

    D’Angelo, M. A.; Hetzer, M. W.

    2009-01-01

    Nuclear pore complexes are large aqueous channels that penetrate the nuclear envelope, connecting the nuclear interior with the cytoplasm. Until recently, these macromolecular complexes were viewed as static structures whose only function was to control the molecular trafficking between the two compartments. It has now become evident that this simplistic scenario is inaccurate and that nuclear pore complexes are highly dynamic multiprotein assemblies involved in diverse cellular processes ranging from the organization of the cytoskeleton to gene expression. In this review, we will discuss the most recent developments in the nuclear pore complex field, focusing in the assembly, disassembly, maintenance and function of this macromolecular structure. PMID:18786826

  5. The Local [CII] Emission Line Luminosity Function

    NASA Astrophysics Data System (ADS)

    Hemmati, Shoubaneh

    2017-01-01

    I present, for the first time, the local [CII]158 $\\mu$m emission line luminosity function measured using a sample of more than 500 galaxies from the RBGS. [CII] luminosities are measured from the Herschel PACS observations of the LIRGs in the GOALS survey and estimated for the rest of the sample based on the far-IR luminosity and color. The sample covers 91.3% of the sky and is complete at $S_{60\\mu m} > 5.24 Jy$. We calculated the completeness as a function of [CII] line luminosity and distance, based on the far-IR color and flux densities. The [CII] luminosity function is constrained in the range $\\sim 10^{7-9} \\ L_{\\odot}$ from both the 1/Vmax and the STY maximum likelihood methods. The shape of our derived [CII] emission line luminosity function agrees well with the IR luminosity function. For the CO(1-0) and [CII] luminosity functions to agree, we propose a varying ratio of [CII]/CO(1-0) as a function of CO luminosity, with larger ratios for fainter CO luminosities. Limited [CII] high redshift observations as well as estimates based on the IR and UV luminosity functions, are suggestive of an evolution in the [CII] luminosity function similar to the evolution trend of the cosmic star formation rate density. ALMA with full capability will be able to confirm this prediction.

  6. A nuclear export signal in the N-terminal regulatory domain of IκBα controls cytoplasmic localization of inactive NF-κB/IκBα complexes

    PubMed Central

    Huang, Tony T.; Kudo, Nobuaki; Yoshida, Minoru; Miyamoto, Shigeki

    2000-01-01

    Appropriate subcellular localization is crucial for regulation of NF-κB function. Herein, we show that latent NF-κB complexes can enter and exit the nucleus in preinduction states. The nuclear export inhibitor leptomycin B (LMB) sequestered NF-κB/IκBα complexes in the nucleus. Using deletion and site-directed mutagenesis, we identified a previously uncharacterized nuclear export sequence in residues 45–54 of IκBα that was required for cytoplasmic localization of inactive complexes. This nuclear export sequence also caused nuclear exclusion of heterologous proteins in a LMB-sensitive manner. Importantly, a LMB-insensitive CRM1 mutant (Crm1-K1) abolished LMB-induced nuclear accumulation of the inactive complexes. Moreover, a cell-permeable p50 NF-κB nuclear localization signal peptide also blocked these LMB effects. These results suggest that NF-κB/IκBα complexes shuttle between the cytoplasm and nucleus by a nuclear localization signal-dependent nuclear import and a CRM1-dependent nuclear export. The LMB-induced nuclear complexes could not bind DNA and were inaccessible to signaling events, because LMB inhibited NF-κB activation without affecting the subcellular localization of upstream kinases IKKβ and NIK. Our findings indicate that the dominant nuclear export over nuclear import contributes to the largely cytoplasmic localization of the inactive complexes to achieve efficient NF-κB activation by extracellular signals. PMID:10655476

  7. Morbillivirus nucleoprotein possesses a novel nuclear localization signal and a CRM1-independent nuclear export signal

    SciTech Connect

    Sato, Hiroki; Masuda, Munemitsu; Miura, Ryuichi; Yoneda, Misako; Kai, Chieko . E-mail: ckai@ims.u-tokyo.ac.jp

    2006-08-15

    Morbilliviruses, which belong to the Mononegavirales, replicate its RNA genome in the cytoplasm of the host cell. However, they also form characteristic intranuclear inclusion bodies, consisting of nucleoprotein (N), in infected cells. To analyze the mechanisms of nucleocytoplasmic transport of N protein, we characterized the nuclear localization (NLS) and nuclear export (NES) signals of canine distemper virus (CDV) N protein by deletion mutation and alanine substitution of the protein. The NLS has a novel leucine/isoleucine-rich motif (TGILISIL) at positions 70-77, whereas the NES is composed of a leucine-rich motif (LLRSLTLF) at positions 4-11. The NLS and NES of the N proteins of other morbilliviruses, that is, measles virus (MV) and rinderpest virus (RPV), were also analyzed. The NLS of CDV-N protein is conserved at the same position in MV-N protein, whereas the NES of MV-N protein is located in the C-terminal region. The NES of RPV-N protein is also located at the same position as CDV-N protein, whereas the NLS motif is present not only at the same locus as CDV-N protein but also at other sites. Interestingly, the nuclear export of all these N proteins appears to proceed via a CRM1-independent pathway.

  8. O-GlcNAc-glycosylation of {beta}-catenin regulates its nuclear localization and transcriptional activity

    SciTech Connect

    Sayat, Ria; Leber, Brian; Grubac, Vanja; Wiltshire, Lesley; Persad, Sujata

    2008-09-10

    {beta}-catenin plays a role in intracellular adhesion and regulating gene expression. The latter role is associated with its oncogenic properties. Phosphorylation of {beta}-catenin controls its intracellular expression but mechanism/s that regulates the nuclear localization of {beta}-catenin is unknown. We demonstrate that O-GlcNAc glycosylation (O-GlcNAcylation) of {beta}-catenin negatively regulates its levels in the nucleus. We show that normal prostate cells (PNT1A) have significantly higher amounts of O-GlcNAcylated {beta}-catenin compared to prostate cancer (CaP) cells. The total nuclear levels of {beta}-catenin are higher in the CaP cells than PNT1A but only a minimal fraction of the nuclear {beta}-catenin in the CaP cells are O-GlcNAcylated. Increasing the levels of O-GlcNAcylated {beta}-catenin in the CaP cells with PUGNAc (O- (2-acetamido-2-deoxy-D-gluco-pyranosylidene) amino-N-phenylcarbamate) treatment is associated with a progressive decrease in the levels of {beta}-catenin in the nucleus. TOPFlash reporter assay and mRNA expressions of {beta}-catenin's target genes indicate that O-GlcNAcylation of {beta}-catenin results in a decrease in its transcriptional activity. We define a novel modification of {beta}-catenin that regulates its nuclear localization and transcriptional function.

  9. Nuclear localization and shuttling of herpes simplex virus tegument protein VP13/14.

    PubMed

    Donnelly, M; Elliott, G

    2001-03-01

    The herpes simplex virus type 1 gene UL47 encodes the tegument proteins referred to collectively as VP13/14, which are believed to be differentially modified forms of the same protein. Here we show that the major product of the UL47 gene during transient expression is VP14, suggesting that some feature of virus infection is required to produce VP13. We have tagged VP13/14 with green fluorescent protein and have demonstrated that the protein is targeted efficiently to the nucleus, where it often localizes in numerous punctate domains. Furthermore, we show that removal of the N-terminal 127 residues of the protein abrogates nuclear accumulation, and we have identified a 14-amino-acid peptide from this region that is sufficient to function as a nuclear targeting signal and transport a heterologous protein to the nucleus. This short peptide contains two runs of four arginine residues, suggesting that the VP13/14 nuclear localization signal may behave in a manner similar to that of the arginine-rich nuclear localization signals of the retrovirus transactivator proteins Tat, Rev, and Rex. In addition, by using heterokaryon assays, we show that VP13/14 is capable of shuttling between the nucleus and cytoplasm of the cell, a property that may be attributed to three leucine-rich stretches in the C-terminal half of the protein that again bear similarity to the nuclear export signals of Rev and Rex. This is the first demonstration of a tegument protein that is specifically targeted to the nucleus, a feature which may be relevant both during virus entry, when VP13/14 enters the cell as a component of the tegument, and at later times, when large amounts of newly synthesized VP13/14 are present within the cell.

  10. A nuclear-localized histone-gene binding protein from rice (OsHBP1b) functions in salinity and drought stress tolerance by maintaining chlorophyll content and improving the antioxidant machinery.

    PubMed

    Lakra, Nita; Nutan, Kamlesh K; Das, Priyanka; Anwar, Khalid; Singla-Pareek, Sneh L; Pareek, Ashwani

    2015-03-15

    Plants have evolved a number of molecular strategies and regulatory mechanisms to cope with abiotic stresses. Among the various key factors/regulators, transcription factors (TFs) play critical role(s) towards regulating the gene expression patterns in response to stress conditions. Altering the expression of the key TFs can greatly influence plant stress tolerance. OsHBP1b (accession no. KM096571) is one such TF belonging to bZIP family, localized within the Saltol QTL, whose expression is induced upon salinity treatment in the rice seedlings. qRT-PCR based expression studies for OsHBP1b in seedlings of contrasting genotypes of rice showed its differential regulation in response to salinity stress. A GFP based in vivo study showed that the OsHBP1b protein is nuclear localized and possesses the trans-activation activity. As compared to the WT tobacco plants, the transgenic plants ectopically expressing OsHBP1b showed better survival and favourable osmotic parameters (such as germination and survival rate, membrane stability, K(+)/Na(+) ratio, lipid peroxidation, electrolyte leakage and proline contents) under salinity and drought stress. Under salinity conditions, the transgenic plants accumulated lower levels of reactive oxygen species as compared to the WT. It was also accompanied by higher activities of antioxidant enzymes (such as ascorbate peroxidase and superoxide dismutase), thereby demonstrating that transgenic plants are physiologically better adapted towards the oxidative damage. Taken together, our findings suggest that OsHBP1b contributes to abiotic stress tolerance through multiple physiological pathways and thus, may serve as a useful 'candidate gene' for improving multiple stress tolerance in crop plants.

  11. Nuclear localization of tricellulin promotes the oncogenic property of pancreatic cancer

    PubMed Central

    Takasawa, Akira; Murata, Masaki; Takasawa, Kumi; Ono, Yusuke; Osanai, Makoto; Tanaka, Satoshi; Nojima, Masanori; Kono, Tsuyoshi; Hirata, Koichi; Kojima, Takashi; Sawada, Norimasa

    2016-01-01

    Accumulating evidence has shown that dysregulation of tight junctions (TJs) is involved in tumor development and progression. In this study, we investigated the expression and subcellular distribution of tricellulin, which constitutes tricellular TJs, using human pancreatic adenocarcinomas. In well-differentiated pancreatic adenocarcinoma tissues, tricellulin immunostaining was prominent in the cytoplasm and the plasma membrane. In contrast, in poorly differentiated tissues, its immunostaining was predominantly observed in the nuclei and was almost absent in the plasma membrane. The distinct immunostaining of tricellulin successfully distinguished poorly differentiated adenocarcinoma from moderately and well-differentiated adenocarcinomas with high levels of sensitivity and specificity. Nuclear tricellulin expression significantly correlated with lymph node metastasis, lymphatic invasion and poor survival. In pancreatic cancer cell lines, tricellulin localization shifted from the membrane to nucleus with decreasing differentiation status. Nuclear localization of tricellulin promoted cell proliferation and invasiveness possibly in association with MAPK and PKC pathways in pancreatic cancers. Our results provide new insights into the function of tricellulin, and its nuclear localization may become a new prognostic factor for pancreatic cancers. PMID:27641742

  12. Identification and characterization of multiple conserved nuclear localization signals within adenovirus E1A

    SciTech Connect

    Marshall, Kris S.; Cohen, Michael J.; Fonseca, Greg J.; Todorovic, Biljana; King, Cason R.; Yousef, Ahmed F.; Zhang, Zhiying; Mymryk, Joe S.

    2014-04-15

    The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell. - Highlights: • HAdV E1A uses multiple mechanisms for nuclear import. • We identified an additional non-canonical NLS in the N-terminal/CR1 portion of E1A. • The new NLS does not contact importin-alpha directly. • All NLSs are functionally conserved in the E1A proteins of all 6 HAdV species.

  13. Influence of metallothionein-1 localization on its function.

    PubMed Central

    Levadoux-Martin, M; Hesketh, J E; Beattie, J H; Wallace, H M

    2001-01-01

    Metallothioneins (MTs) have a major role to play in metal metabolism, and may also protect DNA against oxidative damage. MT protein has been found localized in the nucleus during S-phase. The mRNA encoding the MT-1 isoform has a perinuclear localization, and is associated with the cytoskeleton; this targeting, due to signals within the 3'-untranslated region (3'-UTR), facilitates nuclear localization of MT-1 during S-phase [Levadoux, Mahon, Beattie, Wallace and Hesketh (1999) J. Biol. Chem. 274, 34961-34966]. Using cells transfected with MT gene constructs differing in their 3'-UTRs, the role of MT protein in the nucleus has been studied. Chinese hamster ovary cells were transfected with either the full MT gene (MTMT cells) or with the MT 5'-UTR and coding region linked to the 3'-UTR of glutathione peroxidase (MTGSH cells). Cell survival following exposure to oxidative stress and chemical agents was higher in cells expressing the native MT gene than in cells where MT localization was disrupted, or in untransfected cells. Also, MTMT cells showed less DNA damage than MTGSH cells in response to either hydrogen peroxide or mutagen. After exposure to UV light or mutagen, MTMT cells showed less apoptosis than MTGSH cells, as assessed by DNA fragmentation and flow cytometry. The data indicate that the perinuclear localization of MT mRNA is important for the function of MT in a protective role against DNA damage and apoptosis induced by external stress. PMID:11284736

  14. A highly organized structure mediating nuclear localization of a Myb2 transcription factor in the protozoan parasite Trichomonas vaginalis.

    PubMed

    Chu, Chien-Hsin; Chang, Lung-Chun; Hsu, Hong-Ming; Wei, Shu-Yi; Liu, Hsing-Wei; Lee, Yu; Kuo, Chung-Chi; Indra, Dharmu; Chen, Chinpan; Ong, Shiou-Jeng; Tai, Jung-Hsiang

    2011-12-01

    Nuclear proteins usually contain specific peptide sequences, referred to as nuclear localization signals (NLSs), for nuclear import. These signals remain unexplored in the protozoan pathogen, Trichomonas vaginalis. The nuclear import of a Myb2 transcription factor was studied here using immunodetection of a hemagglutinin-tagged Myb2 overexpressed in the parasite. The tagged Myb2 was localized to the nucleus as punctate signals. With mutations of its polybasic sequences, 48KKQK51 and 61KR62, Myb2 was localized to the nucleus, but the signal was diffusive. When fused to a C-terminal non-nuclear protein, the Myb2 sequence spanning amino acid (aa) residues 48 to 143, which is embedded within the R2R3 DNA-binding domain (aa 40 to 156), was essential and sufficient for efficient nuclear import of a bacterial tetracycline repressor (TetR), and yet the transport efficiency was reduced with an additional fusion of a firefly luciferase to TetR, while classical NLSs from the simian virus 40 T-antigen had no function in this assay system. Myb2 nuclear import and DNA-binding activity were substantially perturbed with mutation of a conserved isoleucine (I74) in helix 2 to proline that altered secondary structure and ternary folding of the R2R3 domain. Disruption of DNA-binding activity alone by point mutation of a lysine residue, K51, preceding the structural domain had little effect on Myb2 nuclear localization, suggesting that nuclear translocation of Myb2, which requires an ordered structural domain, is independent of its DNA binding activity. These findings provide useful information for testing whether myriad Mybs in the parasite use a common module to regulate nuclear import.

  15. Quantification of Uncertainties in Nuclear Density Functional Theory

    NASA Astrophysics Data System (ADS)

    Schunck, N.; McDonnell, J. D.; Higdon, D.; Sarich, J.; Wild, S.

    2015-01-01

    Reliable predictions of nuclear properties are needed as much to answer fundamental science questions as in applications such as reactor physics or data evaluation. Nuclear density functional theory is currently the only microscopic, global approach to nuclear structure that is applicable throughout the nuclear chart. In the past few years, a lot of effort has been devoted to setting up a general methodology to assess theoretical uncertainties in nuclear DFT calculations. In this paper, we summarize some of the recent progress in this direction. Most of the new material discussed here will be be published in separate articles.

  16. Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

    SciTech Connect

    Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka

    2015-07-31

    Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS{sup SV40}) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS{sup SV40} in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS{sup SV40} formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS{sup SV40} likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS{sup SV40} can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus.

  17. Cell cycle regulation of Greatwall kinase nuclear localization facilitates mitotic progression

    PubMed Central

    Wang, Peng; Galan, Jacob A.; Normandin, Karine; Bonneil, Éric; Hickson, Gilles R.; Roux, Philippe P.; Thibault, Pierre

    2013-01-01

    Cell division requires the coordination of critical protein kinases and phosphatases. Greatwall (Gwl) kinase activity inactivates PP2A-B55 at mitotic entry to promote the phosphorylation of cyclin B–Cdk1 substrates, but how Gwl is regulated is poorly understood. We found that the subcellular localization of Gwl changed dramatically during the cell cycle in Drosophila. Gwl translocated from the nucleus to the cytoplasm in prophase. We identified two critical nuclear localization signals in the central, poorly characterized region of Gwl, which are required for its function. The Polo kinase associated with and phosphorylated Gwl in this region, promoting its binding to 14-3-3ε and its localization to the cytoplasm in prophase. Our results suggest that cyclin B–Cdk1 phosphorylation of Gwl is also required for its nuclear exclusion by a distinct mechanism. We show that the nucleo-cytoplasmic regulation of Gwl is essential for its functions in vivo and propose that the spatial regulation of Gwl at mitotic entry contributes to the mitotic switch. PMID:23857770

  18. Global network influences on local functional connectivity

    PubMed Central

    Snyder, Adam C.; Morais, Michael J.; Willis, Cory M.; Smith, Matthew A.

    2015-01-01

    A central neuroscientific pursuit is understanding neuronal interactions that support computations underlying cognition and behavior. Although neurons interact across disparate scales – from cortical columns to whole-brain networks – research has been restricted to one scale at a time. We measured local interactions through multi-neuronal recordings while accessing global networks using scalp EEG in rhesus macaques. We measured spike count correlation, an index of functional connectivity with computational relevance, and EEG oscillations, which have been linked to various cognitive functions. We found a surprising non-monotonic relationship between EEG oscillation amplitude and spike count correlation, contrary to the intuitive expectation of a direct relationship. With a widely-used network model we replicated these findings by incorporating a private signal targeting inhibitory neurons, a common mechanism proposed for gain modulation. Finally, we report that spike count correlation explains nonlinearities in the relationship between EEG oscillations and response time in a spatial selective attention task. PMID:25799040

  19. Importin α1 Mediates Yorkie Nuclear Import via an N-terminal Non-canonical Nuclear Localization Signal.

    PubMed

    Wang, Shimin; Lu, Yi; Yin, Meng-Xin; Wang, Chao; Wu, Wei; Li, Jinhui; Wu, Wenqing; Ge, Ling; Hu, Lianxin; Zhao, Yun; Zhang, Lei

    2016-04-08

    The Hippo signaling pathway controls organ size by orchestrating cell proliferation and apoptosis. When the Hippo pathway was inactivated, the transcriptional co-activator Yorkie translocates into the nucleus and forms a complex with transcription factor Scalloped to promote the expression of Hippo pathway target genes. Therefore, the nuclear translocation of Yorkie is a critical step in Hippo signaling. Here, we provide evidence that the N-terminal 1-55 amino acids of Yorkie, especially Arg-15, were essential for its nuclear localization. By mass spectrometry and biochemical analyses, we found that Importin α1 can directly interact with the Yorkie N terminus and drive Yorkie into the nucleus. Further experiments show that the upstream component Hippo can inhibit Importin α1-mediated Yorkie nuclear import. Taken together, we identified a potential nuclear localization signal at the N-terminal end of Yorkie as well as a critical role for Importin α1 in Yorkie nuclear import.

  20. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells

    PubMed Central

    Niopek, Dominik; Benzinger, Dirk; Roensch, Julia; Draebing, Thomas; Wehler, Pierre; Eils, Roland; Di Ventura, Barbara

    2014-01-01

    The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light. PMID:25019686

  1. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells.

    PubMed

    Niopek, Dominik; Benzinger, Dirk; Roensch, Julia; Draebing, Thomas; Wehler, Pierre; Eils, Roland; Di Ventura, Barbara

    2014-07-14

    The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

  2. Nuclear localization of MBNL1: splicing-mediated autoregulation and repression of repeat-derived aberrant proteins.

    PubMed

    Kino, Yoshihiro; Washizu, Chika; Kurosawa, Masaru; Oma, Yoko; Hattori, Nobutaka; Ishiura, Shoichi; Nukina, Nobuyuki

    2015-02-01

    In some neurological diseases caused by repeat expansions such as myotonic dystrophy, the RNA-binding protein muscleblind-like 1 (MBNL1) accumulates in intranuclear inclusions containing mutant repeat RNA. The interaction between MBNL1 and mutant RNA in the nucleus is a key event leading to loss of MBNL function, yet the details of this effect have been elusive. Here, we investigated the mechanism and significance of MBNL1 nuclear localization. We found that MBNL1 contains two classes of nuclear localization signal (NLS), a classical bipartite NLS and a novel conformational NLS. Alternative splicing of exon 7 acts as a switch between these NLS types and couples MBNL1 activity and intracellular localization. Depending on its nuclear localization, MBNL1 promoted nuclear accumulation of mutant RNA containing a CUG or CAG repeat, some of which produced proteins containing homopolymeric tracts such as polyglutamine. Furthermore, MBNL1 repressed the expression of these homopolymeric proteins including those presumably produced through repeat-associated non-ATG (RAN) translation. These results suggest that nuclear retention of expanded RNA reflects a novel role of MBNL proteins in repressing aberrant protein expression and may provide pathological and therapeutic implications for a wide range of repeat expansion diseases associated with nuclear RNA retention and/or RAN translation.

  3. Nuclear F-actin enhances the transcriptional activity of β-catenin by increasing its nuclear localization and binding to chromatin.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; de Lanerolle, Primal; Harata, Masahiko

    2016-04-01

    Actin plays multiple roles both in the cytoplasm and in the nucleus. Cytoplasmic actin, in addition to its structural role in the cytoskeleton, also contributes to the subcellular localization of transcription factors by interacting with them or their partners. The transcriptional cofactor β-catenin, which acts as an intracellular transducer of canonical Wnt signaling, indirectly associates with the cytoplasmic filamentous actin (F-actin). Recently, it has been observed that F-actin is transiently formed within the nucleus in response to serum stimulation and integrin signaling, and also during gene reprogramming. Despite these earlier observations, information about the function of nuclear F-actin is poorly defined. Here, by facilitating the accumulation of nuclear actin artificially, we demonstrate that polymerizing nuclear actin enhanced the nuclear accumulation and transcriptional function of β-catenin. Our results also show that the nuclear F-actin colocalizes with β-catenin and enhances the binding of β-catenin to the downstream target genes of the Wnt/β-catenin signaling pathway, including the genes for the cell cycle regulators c-myc and cyclin D, and the OCT4 gene. Nuclear F-actin itself also associated with these genes. Since Wnt/β-catenin signaling has important roles in cell differentiation and pluripotency, our observations suggest that nuclear F-actin formed during these biological processes is involved in regulating Wnt/β-catenin signaling.

  4. Interaction between a plasma membrane-localized ankyrin-repeat protein ITN1 and a nuclear protein RTV1

    SciTech Connect

    Sakamoto, Hikaru; Sakata, Keiko; Kusumi, Kensuke; Kojima, Mikiko; Sakakibara, Hitoshi; Iba, Koh

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer ITN1, a plasma membrane ankyrin protein, interacts with a nuclear DNA-binding protein RTV1. Black-Right-Pointing-Pointer The nuclear transport of RTV1 is partially inhibited by interaction with ITN1. Black-Right-Pointing-Pointer RTV1 can promote the nuclear localization of ITN1. Black-Right-Pointing-Pointer Both overexpression of RTV1 and the lack of ITN1 increase salicylic acids sensitivity in plants. -- Abstract: The increased tolerance to NaCl 1 (ITN1) protein is a plasma membrane (PM)-localized protein involved in responses to NaCl stress in Arabidopsis. The predicted structure of ITN1 is composed of multiple transmembrane regions and an ankyrin-repeat domain that is known to mediate protein-protein interactions. To elucidate the molecular functions of ITN1, we searched for interacting partners using a yeast two-hybrid assay, and a nuclear-localized DNA-binding protein, RTV1, was identified as a candidate. Bimolecular fluorescence complementation analysis revealed that RTV1 interacted with ITN1 at the PM and nuclei in vivo. RTV1 tagged with red fluorescent protein localized to nuclei and ITN1 tagged with green fluorescent protein localized to PM; however, both proteins localized to both nuclei and the PM when co-expressed. These findings suggest that RTV1 and ITN1 regulate the subcellular localization of each other.

  5. Tissue specificity in the nuclear envelope supports its functional complexity.

    PubMed

    de Las Heras, Jose I; Meinke, Peter; Batrakou, Dzmitry G; Srsen, Vlastimil; Zuleger, Nikolaj; Kerr, Alastair Rw; Schirmer, Eric C

    2013-01-01

    Nuclear envelope links to inherited disease gave the conundrum of how mutations in near-ubiquitous proteins can yield many distinct pathologies, each focused in different tissues. One conundrum-resolving hypothesis is that tissue-specific partner proteins mediate these pathologies. Such partner proteins may have now been identified with recent proteome studies determining nuclear envelope composition in different tissues. These studies revealed that the majority of the total nuclear envelope proteins are tissue restricted in their expression. Moreover, functions have been found for a number these tissue-restricted nuclear envelope proteins that fit with mechanisms proposed to explain how the nuclear envelope could mediate disease, including defects in mechanical stability, cell cycle regulation, signaling, genome organization, gene expression, nucleocytoplasmic transport, and differentiation. The wide range of functions to which these proteins contribute is consistent with not only their involvement in tissue-specific nuclear envelope disease pathologies, but also tissue evolution.

  6. Tissue specificity in the nuclear envelope supports its functional complexity

    PubMed Central

    de las Heras, Jose I; Meinke, Peter; Batrakou, Dzmitry G; Srsen, Vlastimil; Zuleger, Nikolaj; Kerr, Alastair RW; Schirmer, Eric C

    2013-01-01

    Nuclear envelope links to inherited disease gave the conundrum of how mutations in near-ubiquitous proteins can yield many distinct pathologies, each focused in different tissues. One conundrum-resolving hypothesis is that tissue-specific partner proteins mediate these pathologies. Such partner proteins may have now been identified with recent proteome studies determining nuclear envelope composition in different tissues. These studies revealed that the majority of the total nuclear envelope proteins are tissue restricted in their expression. Moreover, functions have been found for a number these tissue-restricted nuclear envelope proteins that fit with mechanisms proposed to explain how the nuclear envelope could mediate disease, including defects in mechanical stability, cell cycle regulation, signaling, genome organization, gene expression, nucleocytoplasmic transport, and differentiation. The wide range of functions to which these proteins contribute is consistent with not only their involvement in tissue-specific nuclear envelope disease pathologies, but also tissue evolution. PMID:24213376

  7. Biological significance of nuclear localization of mitogen-activated protein kinase Pmk1 in fission yeast.

    PubMed

    Sánchez-Mir, Laura; Franco, Alejandro; Madrid, Marisa; Vicente-Soler, Jero; Villar-Tajadura, M Antonia; Soto, Teresa; Pérez, Pilar; Gacto, Mariano; Cansado, José

    2012-07-27

    Mitogen-activated protein kinase (MAPK) signaling pathways play a fundamental role in the response of eukaryotic cells to environmental changes. Also, much evidence shows that the stimulus-dependent nuclear targeting of this class of regulatory kinases is crucial for adequate regulation of distinct cellular events. In the fission yeast Schizosaccharomyces pombe, the cell integrity MAPK pathway, whose central element is the MAPK Pmk1, regulates multiple processes such as cell wall integrity, vacuole fusion, cytokinesis, and ionic homeostasis. In non-stressed cells Pmk1 is constitutively localized in both cytoplasm and nucleus, and its localization pattern appears unaffected by its activation status or in response to stress, thus questioning the biological significance of the presence of this MAPK into the nucleus. We have addressed this issue by characterizing mutants expressing Pmk1 versions excluded from the cell nucleus and anchored to the plasma membrane in different genetic backgrounds. Although nuclear Pmk1 partially regulates cell wall integrity at a transcriptional level, membrane-tethered Pmk1 performs many of the biological functions assigned to wild type MAPK like regulation of chloride homeostasis, vacuole fusion, and cellular separation. However, we found that down-regulation of nuclear Pmk1 by MAPK phosphatases induced by the stress activated protein kinase pathway is important for the fine modulation of extranuclear Pmk1 activity. These results highlight the importance of the control of MAPK activity at subcellular level.

  8. Biological Significance of Nuclear Localization of Mitogen-activated Protein Kinase Pmk1 in Fission Yeast*

    PubMed Central

    Sánchez-Mir, Laura; Franco, Alejandro; Madrid, Marisa; Vicente-Soler, Jero; Villar-Tajadura, M. Antonia; Soto, Teresa; Pérez, Pilar; Gacto, Mariano; Cansado, José

    2012-01-01

    Mitogen-activated protein kinase (MAPK) signaling pathways play a fundamental role in the response of eukaryotic cells to environmental changes. Also, much evidence shows that the stimulus-dependent nuclear targeting of this class of regulatory kinases is crucial for adequate regulation of distinct cellular events. In the fission yeast Schizosaccharomyces pombe, the cell integrity MAPK pathway, whose central element is the MAPK Pmk1, regulates multiple processes such as cell wall integrity, vacuole fusion, cytokinesis, and ionic homeostasis. In non-stressed cells Pmk1 is constitutively localized in both cytoplasm and nucleus, and its localization pattern appears unaffected by its activation status or in response to stress, thus questioning the biological significance of the presence of this MAPK into the nucleus. We have addressed this issue by characterizing mutants expressing Pmk1 versions excluded from the cell nucleus and anchored to the plasma membrane in different genetic backgrounds. Although nuclear Pmk1 partially regulates cell wall integrity at a transcriptional level, membrane-tethered Pmk1 performs many of the biological functions assigned to wild type MAPK like regulation of chloride homeostasis, vacuole fusion, and cellular separation. However, we found that down-regulation of nuclear Pmk1 by MAPK phosphatases induced by the stress activated protein kinase pathway is important for the fine modulation of extranuclear Pmk1 activity. These results highlight the importance of the control of MAPK activity at subcellular level. PMID:22685296

  9. Local people's understanding of risk from civil nuclear power in the Chinese context.

    PubMed

    Fang, Xiang

    2014-04-01

    This paper analyses how people understand civil nuclear risk in the local context in China. The findings of the paper are based on six months of fieldwork research on a potential inland nuclear power project in Dapu townland in 2007 and 2008. Understanding varies greatly depending on local context, with economic, geographic and social factors influencing the way people view risks and benefits. I argue that when local people do not have enough 'scientific knowledge' to understand risk from nuclear power, they can still use their experience of everyday life to reflect rationally on the risks and benefits that they face. I conclude that when local people trust in nuclear technology and 'the government', and are unaware of nuclear risk it is partly because of their over-dependence on institutions and experts. However, despite their lack of agency, local people rationally calculate risk and benefit in accordance with their social identity and geographical location.

  10. Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

    PubMed

    Rogers, Jason V; Rose, Mark D

    2014-12-02

    During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p's functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion.

  11. Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

    SciTech Connect

    Inagaki, Yuichi; Mitsutake, Susumu; Igarashi, Yasuyuki . E-mail: yigarash@pharm.hokudai.ac.jp

    2006-05-12

    Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide First evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.

  12. Linear response of homogeneous nuclear matter with energy density functionals

    NASA Astrophysics Data System (ADS)

    Pastore, A.; Davesne, D.; Navarro, J.

    2015-03-01

    Response functions of infinite nuclear matter with arbitrary isospin asymmetry are studied in the framework of the random phase approximation. The residual interaction is derived from a general nuclear Skyrme energy density functional. Besides the usual central, spin-orbit and tensor terms it could also include other components as new density-dependent terms or three-body terms. Algebraic expressions for the response functions are obtained from the Bethe-Salpeter equation for the particle-hole propagator. Applications to symmetric nuclear matter, pure neutron matter and asymmetric nuclear matter are presented and discussed. Spin-isospin strength functions are analyzed for varying conditions of density, momentum transfer, isospin asymmetry, and temperature for some representative Skyrme functionals. Particular attention is paid to the discussion of instabilities, either real or unphysical, which could manifest in finite nuclei.

  13. Monodisperse Magnetite Nanoparticles Coupled with Nuclear Localization Signal Peptide for Cell-Nucleus Targeting

    PubMed Central

    Xu, Chenjie; Xie, Jin; Kohler, Nathan; Walsh, Edward G.; Chin, Y. Eugene; Sun, Shouheng

    2009-01-01

    Functionalization of monodisperse superparamagnetic magnetite (Fe3O4) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe3O4 nanoparticles functionalized with protein and nuclear localization signal (NLS) peptide. These NLS-coated nanoparticles were introduced into the HeLa cell cytoplasm and nucleus, where the particles were monodispersed and non-aggregated. The success of labeling was examined and identified by fluorescence microscopy and MRI. The work demonstrates that monodisperse magnetic nanoparticles can be readily functionalized and stabilized for potential diagnostic and therapeutic applications. PMID:18080259

  14. Molecular and functional characterization of a Trypanosoma cruzi nuclear adenylate kinase isoform.

    PubMed

    Cámara, María de los Milagros; Bouvier, León A; Canepa, Gaspar E; Miranda, Mariana R; Pereira, Claudio A

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N-terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 3' UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes.

  15. Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform

    PubMed Central

    Cámara, María de los Milagros; Bouvier, León A.; Canepa, Gaspar E.; Miranda, Mariana R.; Pereira, Claudio A.

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N-terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 3′ UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes. PMID:23409202

  16. Role of zinc finger structure in nuclear localization of transcription factor Sp1

    SciTech Connect

    Ito, Tatsuo; Azumano, Makiko; Uwatoko, Chisana; Itoh, Kohji Kuwahara, Jun

    2009-02-27

    Transcription factor Sp1 is localized in the nucleus and regulates gene expression. Our previous study demonstrated that the carboxyl terminal region of Sp1 containing 3-zinc finger region as DNA binding domain can also serve as nuclear localization signal (NLS). However, the nuclear transport mechanism of Sp1 has not been well understood. In this study, we performed a gene expression study on mutant Sp1 genes causing a set of amino acid substitutions in zinc finger domains to elucidate nuclear import activity. Nuclear localization of the GFP-fused mutant Sp1 proteins bearing concomitant substitutions in the first and third zinc fingers was highly inhibited. These mutant Sp1 proteins had also lost the binding ability as to the GC box sequence. The results suggest that the overall tertiary structure formed by the three zinc fingers is essential for nuclear localization of Sp1 as well as dispersed basic amino acids within the zinc fingers region.

  17. Towards a Density Functional Theory Exchange-Correlation Functional able to describe localization/delocalization

    NASA Astrophysics Data System (ADS)

    Mattsson, Ann E.; Wills, John M.

    2013-03-01

    The inability to computationally describe the physics governing the properties of actinides and their alloys is the poster child of failure of existing Density Functional Theory exchange-correlation functionals. The intricate competition between localization and delocalization of the electrons, present in these materials, exposes the limitations of functionals only designed to properly describe one or the other situation. We will discuss the manifestation of this competition in real materials and propositions on how to construct a functional able to accurately describe properties of these materials. I addition we will discuss both the importance of using the Dirac equation to describe the relativistic effects in these materials, and the connection to the physics of transition metal oxides. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

  18. Analysis of the localization and topology of nurim, a polytopic protein tightly associated with the inner nuclear membrane.

    PubMed

    Hofemeister, Helmut; O'Hare, Peter

    2005-01-28

    Nurim is an inner nuclear membrane (INM) protein that was first isolated in a visual screen for nuclear envelope-localizing proteins. Nurim lacks an N-terminal domain characteristic of other INM proteins examined to date and may represent a class of proteins that localize to the INM by a distinct mechanism. To further characterize this protein, we constructed nurim-green fluorescent protein fusions and analyzed aspects of localization, biochemistry, and membrane topology. Results from immunoprobing and protease protection assays together with other analyses indicate that nurim (total length of 262 residues) is a six transmembrane-spanning protein and contains a hairpin turn in its C-terminal transmembrane domain, resulting in the N and C termini residing on the same side of the membrane. A loop region between the fourth and fifth transmembrane domains is exposed toward the nucleoplasm and contains a region accessible for site-specific endoproteinase cleavage. In biochemical fractionation, nurim remained extremely tightly bound to nuclear fractions and was released in significant quantities only in the presence of 4 m urea. Under conditions in which nuclear lamins were completely extracted, a significant population of nurim remained resistant to solubilization. This tight binding requires the C-terminal region of the protein. DNase treatment only marginally influenced its retention characteristics in nuclei. Results from consideration of sequence alignments and identification of specific topological features of nurim indicate that it may possess enzymic function. These results are discussed with reference to the retention mechanism and possible nuclear function of nurim.

  19. Specific deletion of CMF1 nuclear localization domain causes incomplete cell cycle withdrawal and impaired differentiation in avian skeletal myoblasts

    SciTech Connect

    Dees, Ellen . E-mail: ellen.dees@vanderbilt.edu; Robertson, J. Brian; Zhu, Tianli; Bader, David

    2006-10-01

    CMF1 is a protein expressed in embryonic striated muscle with onset of expression preceding that of contractile proteins. Disruption of CMF1 in myoblasts disrupts muscle-specific protein expression. Preliminary studies indicate both nuclear and cytoplasmic distribution of CMF1 protein, suggesting functional roles in both cellular compartments. Here we examine the nuclear function of CMF1, using a newly characterized antibody generated against the CMF1 nuclear localization domain and a CMF1 nuclear localization domain-deleted stable myocyte line. The antibody demonstrates nuclear distribution of the CMF1 protein both in vivo and in cell lines, with clustering of CMF1 protein around chromatin during mitosis. In more differentiated myocytes, the protein shifts to the cytoplasm. The CMF1 NLS-deleted cell lines have markedly impaired capacity to differentiate. Specifically, these cells express less contractile protein than wild-type or full-length CMF1 stably transfected cells, and do not fuse properly into multinucleate syncytia with linear nuclear alignment. In response to low serum medium, a signal to differentiate, CMF1 NLS-deleted cells enter G0, but continue to express proliferation markers and will reenter the cell cycle when stimulated by restoring growth medium. These data suggest that CMF1 is involved in regulation the transition from proliferation to differentiation in embryonic muscle.

  20. Functional Characterization of Nuclear Trafficking Signals in Pseudorabies Virus pUL31

    PubMed Central

    Paßvogel, Lars; Klupp, Barbara G.; Granzow, Harald; Fuchs, Walter

    2014-01-01

    ABSTRACT The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-ΔNLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes. IMPORTANCE The molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that

  1. Factorized molecular wave functions: Analysis of the nuclear factor

    SciTech Connect

    Lefebvre, R.

    2015-06-07

    The exact factorization of molecular wave functions leads to nuclear factors which should be nodeless functions. We reconsider the case of vibrational perturbations in a diatomic species, a situation usually treated by combining Born-Oppenheimer products. It was shown [R. Lefebvre, J. Chem. Phys. 142, 074106 (2015)] that it is possible to derive, from the solutions of coupled equations, the form of the factorized function. By increasing artificially the interstate coupling in the usual approach, the adiabatic regime can be reached, whereby the wave function can be reduced to a single product. The nuclear factor of this product is determined by the lowest of the two potentials obtained by diagonalization of the potential matrix. By comparison with the nuclear wave function of the factorized scheme, it is shown that by a simple rectification, an agreement is obtained between the modified nodeless function and that of the adiabatic scheme.

  2. Relations among several nuclear and electronic density functional reactivity indexes

    NASA Astrophysics Data System (ADS)

    Torrent-Sucarrat, Miquel; Luis, Josep M.; Duran, Miquel; Toro-Labbé, Alejandro; Solà, Miquel

    2003-11-01

    An expansion of the energy functional in terms of the total number of electrons and the normal coordinates within the canonical ensemble is presented. A comparison of this expansion with the expansion of the energy in terms of the total number of electrons and the external potential leads to new relations among common density functional reactivity descriptors. The formulas obtained provide explicit links between important quantities related to the chemical reactivity of a system. In particular, the relation between the nuclear and the electronic Fukui functions is recovered. The connection between the derivatives of the electronic energy and the nuclear repulsion energy with respect to the external potential offers a proof for the "Quantum Chemical le Chatelier Principle." Finally, the nuclear linear response function is defined and the relation of this function with the electronic linear response function is given.

  3. Phenosafranin inhibits nuclear localization of transglutaminase 2 without affecting its transamidase activity.

    PubMed

    Furutani, Yutaka; Toguchi, Mariko; Shrestha, Rajan; Kojima, Soichi

    2017-03-01

    Transglutaminase 2 (TG2) localizes to the nucleus and induces apoptosis through a crosslinking inactivation of Sp1 in JHH-7 cells treated with acyclic retinoid. We screened an inhibitor suppressing transamidase activity in the nucleus without affecting transamidase activity itself. Phenosafranin was found to inhibit nuclear localization of EGFP-tagged TG2 and dose-dependently reduce nuclear transamidase activity without affecting the activity in a tube. We concluded that phenosafranin was a novel TG2 inhibitor capable of suppressing its nuclear localization.

  4. Nuclear charge radii: density functional theory meets Bayesian neural networks

    NASA Astrophysics Data System (ADS)

    Utama, R.; Chen, Wei-Chia; Piekarewicz, J.

    2016-11-01

    The distribution of electric charge in atomic nuclei is fundamental to our understanding of the complex nuclear dynamics and a quintessential observable to validate nuclear structure models. The aim of this study is to explore a novel approach that combines sophisticated models of nuclear structure with Bayesian neural networks (BNN) to generate predictions for the charge radii of thousands of nuclei throughout the nuclear chart. A class of relativistic energy density functionals is used to provide robust predictions for nuclear charge radii. In turn, these predictions are refined through Bayesian learning for a neural network that is trained using residuals between theoretical predictions and the experimental data. Although predictions obtained with density functional theory provide a fairly good description of experiment, our results show significant improvement (better than 40%) after BNN refinement. Moreover, these improved results for nuclear charge radii are supplemented with theoretical error bars. We have successfully demonstrated the ability of the BNN approach to significantly increase the accuracy of nuclear models in the predictions of nuclear charge radii. However, as many before us, we failed to uncover the underlying physics behind the intriguing behavior of charge radii along the calcium isotopic chain.

  5. Nuclear matrix - structure, function and pathogenesis.

    PubMed

    Wasąg, Piotr; Lenartowski, Robert

    2016-12-20

    The nuclear matrix (NM), or nuclear skeleton, is the non-chromatin, ribonucleoproteinaceous framework that is resistant to high ionic strength buffers, nonionic detergents, and nucleolytic enzymes. The NM fulfills a structural role in eukaryotic cells and is responsible for maintaining the shape of the nucleus and the spatial organization of chromatin. Moreover, the NM participates in several cellular processes, such as DNA replication/repair, gene expression, RNA transport, cell signaling and differentiation, cell cycle regulation, apoptosis and carcinogenesis. Short nucleotide sequences called scaffold/matrix attachment regions (S/MAR) anchor the chromatin loops to the NM proteins (NMP). The NMP composition is dynamic and depends on the cell type and differentiation stage or metabolic activity. Alterations in the NMP composition affect anchoring of the S/MARs and thus alter gene expression. This review aims to systematize information about the skeletal structure of the nucleus, with particular emphasis on the organization of the NM and its role in selected cellular processes. We also discuss several diseases that are caused by aberrant NM structure or dysfunction of individual NM elements.

  6. A nuclear localization domain in the hnRNP A1 protein

    PubMed Central

    1995-01-01

    The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta- galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2. PMID:7730395

  7. Comparative analyses of nuclear proteome: extending its function

    PubMed Central

    Narula, Kanika; Datta, Asis; Chakraborty, Niranjan; Chakraborty, Subhra

    2013-01-01

    Organeller proteomics is an emerging technology that is critical in determining the cellular signal transduction pathways. Nucleus, the regulatory hub of the eukaryotic cell is a dynamic system and a repository of various macromolecules that serve as modulators of such signaling that dictate cell fate decisions. Nuclear proteins (NPs) are predicted to comprise about 10–20% of the total cellular proteins, suggesting the involvement of the nucleus in a number of diverse functions. Indeed, NPs constitute a highly organized but complex network that plays diverse roles during development and physiological processes. In plants, relatively little is known about the nature of the molecular components and mechanisms involved in coordinating NP synthesis, their action and function. Proteomic study hold promise to understand the molecular basis of nuclear function using an unbiased comparative and differential approach. We identified a few hundred proteins that include classical and non-canonical nuclear components presumably associated with variety of cellular functions impinging on the complexity of nuclear proteome. Here, we review the nuclear proteome based on our own findings, available literature, and databases focusing on detailed comparative analysis of NPs and their functions in order to understand how plant nucleus works. The review also shed light on the current status of plant nuclear proteome and discusses the future prospect. PMID:23637696

  8. Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies.

    PubMed

    Dury, Alain Y; El Fatimy, Rachid; Tremblay, Sandra; Rose, Timothy M; Côté, Jocelyn; De Koninck, Paul; Khandjian, Edouard W

    2013-10-01

    Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.

  9. Atypical nuclear localization of VIP receptors in glioma cell lines and patients

    SciTech Connect

    Barbarin, Alice; Séité, Paule; Godet, Julie; Bensalma, Souheyla; Muller, Jean-Marc; Chadéneau, Corinne

    2014-11-28

    Highlights: • The VIP receptor VPAC1 contains a putative NLS signal. • VPAC1 is predominantly nuclear in GBM cell lines but not VPAC2. • Non-nuclear VPAC1/2 protein expression is correlated with glioma grade. • Nuclear VPAC1 is observed in 50% of stage IV glioma (GBM). - Abstract: An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.

  10. A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris

    PubMed Central

    Weninger, Astrid; Glieder, Anton; Vogl, Thomas

    2015-01-01

    Nuclear localization sequences (NLSs) are required for the import of proteins in the nucleus of eukaryotes. However many proteins from bacteria or bacteriophages are used for basic studies in molecular biology, to generate synthetic genetic circuits or for genome editing applications. Prokaryotic recombinases, CRISPR-associated proteins such as Cas9 or bacterial and viral polymerases require efficient NLSs to function in eukaryotes. The yeast Pichia pastoris is a widely used expression platform for heterologous protein production, but molecular tools such as NLSs are limited. Here we have characterized a set of 10 NLSs for P. pastoris, including the first endogenous NLSs (derived from P. pastoris proteins) and commonly used heterologous NLSs. The NLSs were evaluated by fusing them in N- and C-terminal position to an enhanced green fluorescent protein showing pronounced differences in fluorescence levels and nuclear targeting. Thereby we provide a set of different NLSs that can be applied to optimize the nuclear import of heterologous proteins in P. pastoris, paving the way for the establishment of intricate synthetic biology applications. PMID:26347503

  11. A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris.

    PubMed

    Weninger, Astrid; Glieder, Anton; Vogl, Thomas

    2015-11-01

    Nuclear localization sequences (NLSs) are required for the import of proteins in the nucleus of eukaryotes. However many proteins from bacteria or bacteriophages are used for basic studies in molecular biology, to generate synthetic genetic circuits or for genome editing applications. Prokaryotic recombinases, CRISPR-associated proteins such as Cas9 or bacterial and viral polymerases require efficient NLSs to function in eukaryotes. The yeast Pichia pastoris is a widely used expression platform for heterologous protein production, but molecular tools such as NLSs are limited. Here we have characterized a set of 10 NLSs for P. pastoris, including the first endogenous NLSs (derived from P. pastoris proteins) and commonly used heterologous NLSs. The NLSs were evaluated by fusing them in N- and C-terminal position to an enhanced green fluorescent protein showing pronounced differences in fluorescence levels and nuclear targeting. Thereby we provide a set of different NLSs that can be applied to optimize the nuclear import of heterologous proteins in P. pastoris, paving the way for the establishment of intricate synthetic biology applications.

  12. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization

    SciTech Connect

    Shiheido, Hirokazu Shimizu, Jun

    2015-02-20

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND3{sub 56–58}, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. - Highlights: • BEND3 localizes to the nucleus. • The N-terminal 60 amino acids region of BEND3 contains NLS. • Amino acids located between 56 and 58 of BEND3 (KRK) are part of NLS. • KRK motif is highly conserved among BEND3 homologs.

  13. Function of nuclear membrane proteins in shaping the nuclear envelope integrity during closed mitosis.

    PubMed

    Yang, Hui-Ju; Iwamoto, Masaaki; Hiraoka, Yasushi; Haraguchi, Tokuko

    2017-04-08

    The nuclear envelope (NE) not only protects the genome from being directly accessed by detrimental agents but also regulates genome organization. Breaches in NE integrity threaten genome stability and impede cellular function. Nonetheless, the NE constantly remodels, and NE integrity is endangered in dividing or differentiating cells. Specifically, in unicellular eukaryotes undergoing closed mitosis, the NE expands instead of breaking down during chromosome segregation. The newly assembling nuclear pore complexes (NPCs) penetrate the existing NE in interphase. A peculiar example of NE remodeling during nuclear differentiation in Tetrahymena involves formation of the redundant NE and clustered NPCs. Even under these conditions, the NE remains intact. Many recent studies on unicellular organisms have revealed that nuclear membrane proteins, such as LEM-domain proteins, play a role in maintaining NE integrity. This review summarizes and discusses how nuclear membrane proteins participate in NE integrity.

  14. Identification of an unconventional nuclear localization signal in human ribosomal protein S2

    SciTech Connect

    Antoine, M.; Reimers, K.; Wirz, W.; Gressner, A.M.; Mueller, R.; Kiefer, P. . E-Mail: pkiefer@ukaachen.de

    2005-09-16

    Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-{beta}-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-{beta}-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric {beta}-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importin{beta} binding site fused to VP22 blocks nuclear import of rpS2-{beta}-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importin{alpha}/{beta} and transportin.

  15. Nuclear Localization of Schizosaccharomyces pombe Mcm2/Cdc19p Requires MCM Complex Assembly

    PubMed Central

    Pasion, Sally G.; Forsburg, Susan L.

    1999-01-01

    The minichromosome maintenance (MCM) proteins MCM2–MCM7 are conserved eukaryotic replication factors that assemble in a heterohexameric complex. In fission yeast, these proteins are nuclear throughout the cell cycle. In studying the mechanism that regulates assembly of the MCM complex, we analyzed the cis and trans elements required for nuclear localization of a single subunit, Mcm2p. Mutation of any single mcm gene leads to redistribution of wild-type MCM subunits to the cytoplasm, and this redistribution depends on an active nuclear export system. We identified the nuclear localization signal sequences of Mcm2p and showed that these are required for nuclear targeting of other MCM subunits. In turn, Mcm2p must associate with other MCM proteins for its proper localization; nuclear localization of MCM proteins thus requires assembly of MCM proteins in a complex. We suggest that coupling complex assembly to nuclear targeting and retention ensures that only intact heterohexameric MCM complexes remain nuclear. PMID:10588642

  16. Hairless is a nuclear receptor corepressor essential for skin function

    PubMed Central

    Thompson, Catherine C.

    2009-01-01

    The activity of nuclear receptors is modulated by numerous coregulatory factors. Corepressors can either mediate the ability of nuclear receptors to repress transcription, or can inhibit transactivation by nuclear receptors. As we learn more about the mechanisms of transcriptional repression, the importance of repression by nuclear receptors in development and disease has become clear. The protein encoded by the mammalian Hairless (Hr) gene was shown to be a corepressor by virtue of its functional similarity to the well-established corepressors N-CoR and SMRT. Mutation of the Hr gene results in congenital hair loss in both mice and men. Investigation of Hairless function both in vitro and in mouse models in vivo has revealed a critical role in maintaining skin and hair by regulating the differentiation of epithelial stem cells, as well as a putative role in regulating gene expression via chromatin remodeling. PMID:20087431

  17. Differentiating plant cells switched to proliferation remodel the functional organization of nuclear domains.

    PubMed

    Testillano, P S; González-Melendi, P; Coronado, M J; Seguí-Simarro, J M; Moreno-Risueño, M A; Risueño, M C

    2005-01-01

    The immature pollen grain, the microspore, under stress conditions can switch its developmental program towards proliferation and embryogenesis. The comparison between the gametophytic and sporophytic pathways followed by the microspore permitted us to analyse the nuclear changes in plant differentiating cells when switched to proliferation. The nucleus is highly dynamic, the architecture of its well organised functional domains--condensed chromatin, interchromatin region, nuclear bodies and nucleolus--changing in response to DNA replication, RNA transcription, processing and transport. In the present work, the rearrangements of the nuclear domains during the switch to proliferation have been determined by in situ molecular identification methods for the subcellular localization of chromatin at different functional states, rDNA, elements of the nuclear machinery (PCNA, splicing factors), signalling and stress proteins. The study of the changes in the nuclear domains was determined by a correlative approach at confocal and electron microscopy levels. The results showed that the switch of the developmental program and the activation of the proliferative activity affected the functional organization of the nuclear domains, which accordingly changed their architecture and functional state. A redistribution of components, among them various signalling molecules which targeted structures within the interchromatin region upon translocation from the cytoplasm, was also observed.

  18. Functional renormalization group study of nuclear and neutron matter

    SciTech Connect

    Drews, Matthias; Weise, Wolfram

    2016-01-22

    A chiral model based on nucleons interacting via boson exchange is investigated. Fluctuation effects are included consistently beyond the mean-field approximation in the framework of the functional renormalization group. The liquid-gas phase transition of symmetric nuclear matter is studied in detail. No sign of a chiral restoration transition is found up to temperatures of about 100 MeV and densities of at least three times the density of normal nuclear matter. Moreover, the model is extended to asymmetric nuclear matter and the constraints from neutron star observations are discussed.

  19. ERK5 and Cell Proliferation: Nuclear Localization Is What Matters.

    PubMed

    Gomez, Nestor; Erazo, Tatiana; Lizcano, Jose M

    2016-01-01

    ERK5, the last MAP kinase family member discovered, is activated by the upstream kinase MEK5 in response to growth factors and stress stimulation. MEK5-ERK5 pathway has been associated to different cellular processes, playing a crucial role in cell proliferation in normal and cancer cells by mechanisms that are both dependent and independent of its kinase activity. Thus, nuclear ERK5 activates transcription factors by either direct phosphorylation or acting as co-activator thanks to a unique transcriptional activation TAD domain located at its C-terminal tail. Consequently, ERK5 has been proposed as an interesting target to tackle different cancers, and either inhibitors of ERK5 activity or silencing the protein have shown antiproliferative activity in cancer cells and to block tumor growth in animal models. Here, we review the different mechanisms involved in ERK5 nuclear translocation and their consequences. Inactive ERK5 resides in the cytosol, forming a complex with Hsp90-Cdc37 superchaperone. In a canonical mechanism, MEK5-dependent activation results in ERK5 C-terminal autophosphorylation, Hsp90 dissociation, and nuclear translocation. This mechanism integrates signals such as growth factors and stresses that activate the MEK5-ERK5 pathway. Importantly, two other mechanisms, MEK5-independent, have been recently described. These mechanisms allow nuclear shuttling of kinase-inactive forms of ERK5. Although lacking kinase activity, these forms activate transcription by interacting with transcription factors through the TAD domain. Both mechanisms also require Hsp90 dissociation previous to nuclear translocation. One mechanism involves phosphorylation of the C-terminal tail of ERK5 by kinases that are activated during mitosis, such as Cyclin-dependent kinase-1. The second mechanism involves overexpression of chaperone Cdc37, an oncogene that is overexpressed in cancers such as prostate adenocarcinoma, where it collaborates with ERK5 to promote cell proliferation

  20. ERK5 and Cell Proliferation: Nuclear Localization Is What Matters

    PubMed Central

    Gomez, Nestor; Erazo, Tatiana; Lizcano, Jose M.

    2016-01-01

    ERK5, the last MAP kinase family member discovered, is activated by the upstream kinase MEK5 in response to growth factors and stress stimulation. MEK5-ERK5 pathway has been associated to different cellular processes, playing a crucial role in cell proliferation in normal and cancer cells by mechanisms that are both dependent and independent of its kinase activity. Thus, nuclear ERK5 activates transcription factors by either direct phosphorylation or acting as co-activator thanks to a unique transcriptional activation TAD domain located at its C-terminal tail. Consequently, ERK5 has been proposed as an interesting target to tackle different cancers, and either inhibitors of ERK5 activity or silencing the protein have shown antiproliferative activity in cancer cells and to block tumor growth in animal models. Here, we review the different mechanisms involved in ERK5 nuclear translocation and their consequences. Inactive ERK5 resides in the cytosol, forming a complex with Hsp90-Cdc37 superchaperone. In a canonical mechanism, MEK5-dependent activation results in ERK5 C-terminal autophosphorylation, Hsp90 dissociation, and nuclear translocation. This mechanism integrates signals such as growth factors and stresses that activate the MEK5-ERK5 pathway. Importantly, two other mechanisms, MEK5-independent, have been recently described. These mechanisms allow nuclear shuttling of kinase-inactive forms of ERK5. Although lacking kinase activity, these forms activate transcription by interacting with transcription factors through the TAD domain. Both mechanisms also require Hsp90 dissociation previous to nuclear translocation. One mechanism involves phosphorylation of the C-terminal tail of ERK5 by kinases that are activated during mitosis, such as Cyclin-dependent kinase-1. The second mechanism involves overexpression of chaperone Cdc37, an oncogene that is overexpressed in cancers such as prostate adenocarcinoma, where it collaborates with ERK5 to promote cell proliferation

  1. Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis.

    PubMed

    Bryant, Nicole; Lloyd, Johnny; Sweeney, Colleen; Myouga, Fumiyoshi; Meinke, David

    2011-04-01

    We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function.

  2. New Generation Nuclear Plant -- High Level Functions and Requirements

    SciTech Connect

    J. M. Ryskamp; E. J. Gorski; E. A. Harvego; S. T. Khericha; G. A. Beitel

    2003-09-01

    This functions and requirements (F&R) document was prepared for the Next Generation Nuclear Plant (NGNP) Project. The highest-level functions and requirements for the NGNP preconceptual design are identified in this document, which establishes performance definitions for what the NGNP will achieve. NGNP designs will be developed based on these requirements by commercial vendor(s).

  3. US Nuclear Regulatory Commission organization charts and functional statements

    SciTech Connect

    1997-11-01

    This document contains organization charts for the U.S. Nuclear Regulatory Commission (NRC) and for the five offices of the NRC. Function statements are provided delineating the major responsibilities and operations of each office. Organization and function are provided to the branch level. The head of each office, division, and branch is also listed.

  4. Subcellular Localization of RPB5-Mediating Protein and Its Putative Functional Partner

    PubMed Central

    Delgermaa, Luvsanjav; Hayashi, Naoyuki; Dorjsuren, Dorjbal; Nomura, Takahiro; Thuy, Le Thi-Thu; Murakami, Seishi

    2004-01-01

    We previously identified a novel cellular protein, RPB5-mediating protein (RMP), that retains corepressor activity and functionally antagonizes transcriptional modulation via hepatitis B virus X protein. The subcellular localization of RMP was examined using green fluorescent protein-fused protein forms. We found that a nuclear localization signal (NLS) and a coiled-coil (CC) domain functioning as a cytoplasmic localization signal (CLS) are important for the subcellular localization of RMP. The CLS apparently acts dominantly, since RMP was mostly localized in the cytoplasm with weak and diffuse signals in the nucleus, and the NLS was indispensable for the nuclear localization of RMP only in the absence of the CLS. Using a yeast two-hybrid method, we isolated a putative corepressor, DNA methyltransferase 1-associating protein (DMAP1), which was found to bind to the CC domain of RMP. DMAP1 facilitated the nuclear localization of RMP and the corepressor activity of RMP in a dose-dependent manner by interacting with the CC domain of RMP. These results are discussed in light of a recent paper showing a novel evolutionarily conserved role of URI in the TOR signaling pathway. PMID:15367675

  5. The nuclear pore complex: understanding its function through structural insight.

    PubMed

    Beck, Martin; Hurt, Ed

    2017-02-01

    Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes to form channels across the nuclear envelope. They are large macromolecular assemblies with a complex composition and diverse functions. Apart from facilitating nucleocytoplasmic transport, NPCs are involved in chromatin organization, the regulation of gene expression and DNA repair. Understanding the molecular mechanisms underlying these functions has been hampered by a lack of structural knowledge about the NPC. The recent convergence of crystallographic and biochemical in vitro analysis of nucleoporins (NUPs), the components of the NPC, with cryo-electron microscopic imaging of the entire NPC in situ has provided first pseudo-atomic view of its central core and revealed that an unexpected network of short linear motifs is an important spatial organization principle. These breakthroughs have transformed the way we understand NPC structure, and they provide an important base for functional investigations, including the elucidation of the molecular mechanisms underlying clinically manifested mutations of the nucleocytoplasmic transport system.

  6. A-dependence of weak nuclear structure functions

    SciTech Connect

    Haider, H.; Athar, M. Sajjad; Simo, I. Ruiz

    2015-05-15

    Effect of nuclear medium on the weak structure functions F{sub 2}{sup A}(x, Q{sup 2}) and F{sub 3}{sup A}(x, Q{sup 2}) have been studied using charged current (anti)neutrino deep inelastic scattering on various nuclear targets. Relativistic nuclear spectral function which incorporate Fermi motion, binding and nucleon correlations are used for the calculations. We also consider the pion and rho meson cloud contributions calculated from a microscopic model for meson-nucleus self-energies. Using these structure functions, F{sub i}{sup A}/F{sub i}{sup proton} and F{sub i}{sup A}/F{sub i}{sup deuteron}(i=2,3, A={sup 12}C, {sup 16}O, CH and H{sub 2}O) are obtained.

  7. Nuclear correlation functions in lattice QCD

    SciTech Connect

    Detmold, William; Orginos, Konstantinos

    2013-06-01

    We consider the problem of calculating the large number of Wick contractions necessary to compute states with the quantum numbers of many baryons in lattice QCD. We consider a constructive approach and a determinant-based approach and show that these methods allow the required contractions to be performed for certain choices of interpolating operators. Examples of correlation functions computed using these techniques are shown for the quantum numbers of the light nuclei, $^4$He, $^8$Be, $^{12}$C, $^{16}$O and $^{28}$Si.

  8. Pseudo-local Theories: A Functional Class Proposal

    NASA Astrophysics Data System (ADS)

    Taronna, Massimo

    In this article, using the language of jet space, we propose a functional class space for pseudo-local functionals. We test this functional class proposal in a number of examples ranging from string-field-theory to AdS/CFT dualities. Implications of the locality proposal at the quartic order are also discussed.

  9. To the non-local theory of cold nuclear fusion.

    PubMed

    Alexeev, Boris V

    2014-10-01

    In this paper, we revisit the cold fusion (CF) phenomenon using the generalized Bolzmann kinetics theory which can represent the non-local physics of this CF phenomenon. This approach can identify the conditions when the CF can take place as the soliton creation under the influence of the intensive sound waves. The vast mathematical modelling leads to affirmation that all parts of soliton move with the same velocity and with the small internal change of the pressure. The zone of the high density is shaped on the soliton's front. It means that the regime of the 'acoustic CF' could be realized from the position of the non-local hydrodynamics.

  10. Nuclear Localization of PRDM9 and Its Role in Meiotic Chromatin Modifications and Homologous Synapsis

    PubMed Central

    Sun, Fengyun; Fujiwara, Yasuhiro; Reinholdt, Laura G.; Hu, Jianjun; Saxl, Ruth L.; Baker, Christopher L.; Petkov, Petko M.; Paigen, Kenneth; Handel, Mary Ann

    2015-01-01

    Developmental progress of germ cells through meiotic phases is closely tied to ongoing meiotic recombination. In mammals, recombination preferentially occurs in genomic regions known as hotspots; the protein that activates these hotspots is PRDM9, containing a genetically variable zinc-finger domain and a PR-SET domain with histone H3K4 trimethyltransferase activity. PRDM9 is required for fertility in mice, but little is known about its localization and developmental dynamics. Application of spermatogenic stage-specific markers demonstrates that PRDM9 accumulates in male germ-cell nuclei at pre-leptonema to early leptonema, but is no longer detectable in nuclei by late zygonema. By the pachytene stage, PRDM9-dependent histone H3K4 trimethyl marks on hotspots also disappear. PRDM9 localizes to nuclei concurrently with the deposition of meiotic cohesin complexes, but is not required for incorporation of cohesin complex proteins into chromosomal axial elements, or accumulation of normal numbers of RAD51 foci on meiotic chromatin by late zygonema. Germ cells lacking PRDM9 exhibit inefficient homology recognition and synapsis, with aberrant repair of meiotic DNA double-strand breaks and transcriptional abnormalities characteristic of meiotic silencing of unsynapsed chromatin. Together, these results on the developmental time course for nuclear localization of PRDM9 establish its direct window of function, and demonstrate the independence of chromosome axial element formation from the concurrent PRDM9-mediated activation of recombination hotspots. PMID:25894966

  11. Nuclear localization of PRDM9 and its role in meiotic chromatin modifications and homologous synapsis.

    PubMed

    Sun, Fengyun; Fujiwara, Yasuhiro; Reinholdt, Laura G; Hu, Jianjun; Saxl, Ruth L; Baker, Christopher L; Petkov, Petko M; Paigen, Kenneth; Handel, Mary Ann

    2015-09-01

    Developmental progress of germ cells through meiotic phases is closely tied to ongoing meiotic recombination. In mammals, recombination preferentially occurs in genomic regions known as hotspots; the protein that activates these hotspots is PRDM9, containing a genetically variable zinc finger (ZNF) domain and a PR-SET domain with histone H3K4 trimethyltransferase activity. PRDM9 is required for fertility in mice, but little is known about its localization and developmental dynamics. Application of spermatogenic stage-specific markers demonstrates that PRDM9 accumulates in male germ cell nuclei at pre-leptonema to early leptonema but is no longer detectable in nuclei by late zygonema. By the pachytene stage, PRDM9-dependent histone H3K4 trimethyl marks on hotspots also disappear. PRDM9 localizes to nuclei concurrently with the deposition of meiotic cohesin complexes, but is not required for incorporation of cohesin complex proteins into chromosomal axial elements, or accumulation of normal numbers of RAD51 foci on meiotic chromatin by late zygonema. Germ cells lacking PRDM9 exhibit inefficient homology recognition and synapsis, with aberrant repair of meiotic DNA double-strand breaks and transcriptional abnormalities characteristic of meiotic silencing of unsynapsed chromatin. Together, these results on the developmental time course for nuclear localization of PRDM9 establish its direct window of function and demonstrate the independence of chromosome axial element formation from the concurrent PRDM9-mediated activation of recombination hotspots.

  12. Nuclear localization and phosphorylation of three 25-kilodalton rat stress proteins.

    PubMed Central

    Kim, Y J; Shuman, J; Sette, M; Przybyla, A

    1984-01-01

    The nuclear localization and phosphorylation of three 25-kilodalton rat myoblast stress proteins were examined. Data obtained in these analyses led to the following conclusions: (i) all three proteins become localized in the nucleus of stressed cells, (ii) two of the proteins are modified by phosphorylation, and (iii) phosphorylation occurs exclusively on serine residues. Images PMID:6717429

  13. Nuclear cardiology: Myocardial perfusion and function

    SciTech Connect

    Seldin, D.W. )

    1991-08-01

    Myocardial perfusion studies continue to be a major focus of research, with new investigations of the relationship of exercise-redistribution thallium imaging to diagnosis, prognosis, and case management. The redistribution phenomenon, which seemed to be fairly well understood a few years ago, is now recognized to be much more complex than originally thought, and various strategies have been proposed to clarify the meaning of persistent defects. Pharmacologic intervention with dipyridamole and adenosine has become available as an alternative to exercise, and comparisons with exercise imaging and catheterization results have been described. Thallium itself is no longer the sole single-photon perfusion radiopharmaceutical; two new technetium agents are now widely available. In addition to perfusion studies, advances in the study of ventricular function have been made, including reports of studies performed in conjunction with technetium perfusion studies, new insights into cardiac physiology, and the prognostic and case-management information that function studies provide. Finally, work has continued with monoclonal antibodies for the identification of areas of myocyte necrosis. 41 references.

  14. Remote Control of Gene Function by Local Translation

    PubMed Central

    Jung, Hosung; Gkogkas, Christos G.; Sonenberg, Nahum; Holt, Christine E.

    2014-01-01

    The subcellular position of a protein is a key determinant of its function. Mounting evidence indicates that RNA localization, where specific mRNAs are transported subcellularly and subsequently translated in response to localized signals, is an evolutionarily conserved mechanism to control protein localization. On-site synthesis confers novel signaling properties to a protein and helps to maintain local proteome homeostasis. Local translation plays particularly important roles in distal neuronal compartments, and dysregulated RNA localization and translation cause defects in neuronal wiring and survival. Here, we discuss key findings in this area and possible implications of this adaptable and swift mechanism for spatial control of gene function. PMID:24679524

  15. Self-association of Coilin Reveals a Common Theme in Nuclear Body Localization

    PubMed Central

    Hebert, Michael D.; Matera, A. Gregory

    2000-01-01

    We have found that coilin, the marker protein for Cajal bodies (coiled bodies, CBs), is a self-interacting protein, and we have mapped the domain responsible for this activity to the amino-terminus. Together with a nuclear localization signal, the self-interaction domain is necessary and sufficient for localization to CBs. Overexpression of various wild-type and mutant coilin constructs in HeLa cells results in disruption of both CBs and survival motor neurons (SMN) gems. Additionally, we have identified a cryptic nucleolar localization signal (NoLS), within the coilin protein, which may be exposed in specific coilin phospho-isoforms. The implications of these findings are discussed in light of the fact that other proteins known to localize within nuclear bodies (e.g., PML, SMN and Sam68) can also self-associate. Thus protein self-interaction appears to be a general feature of nuclear body marker proteins. PMID:11102515

  16. Uncertainty Quantification and Propagation in Nuclear Density Functional Theory

    SciTech Connect

    Schunck, N; McDonnell, J D; Higdon, D; Sarich, J; Wild, S M

    2015-03-17

    Nuclear density functional theory (DFT) is one of the main theoretical tools used to study the properties of heavy and superheavy elements, or to describe the structure of nuclei far from stability. While on-going eff orts seek to better root nuclear DFT in the theory of nuclear forces, energy functionals remain semi-phenomenological constructions that depend on a set of parameters adjusted to experimental data in fi nite nuclei. In this paper, we review recent eff orts to quantify the related uncertainties, and propagate them to model predictions. In particular, we cover the topics of parameter estimation for inverse problems, statistical analysis of model uncertainties and Bayesian inference methods. Illustrative examples are taken from the literature.

  17. Public meetings on nuclear waste management: their function and organization

    SciTech Connect

    Duvernoy, E.G.; Marcus, A.A.; Overcast, T.; Schilling, A.H.

    1981-05-01

    This report focuses on public meetings as a vehicle for public participation in nuclear waste management. The nature of public meetings is reviewed and the functions served by meetings highlighted. The range of participants and their concerns are addressed, including a review of the participants from past nuclear waste management meetings. A sound understanding of the expected participants allows DOE to tailor elements of the meeting, such as notification, format, and agenda to accommodate the attendees. Finally, the report discusses the organization of public meetings on nuclear waste management in order to enhance the DOE's functions for such meetings. Possible structures are suggested for a variety of elements that are relevant prior to, during and after the public meeting. These suggestions are intended to supplement the DOE Public Participation Manual.

  18. BUILDING A UNIVERSAL NUCLEAR ENERGY DENSITY FUNCTIONAL (UNEDF)

    SciTech Connect

    Nazarewicz, Witold

    2012-07-01

    The long-term vision initiated with UNEDF is to arrive at a comprehensive, quantitative, and unified description of nuclei and their reactions, grounded in the fundamental interactions between the constituent nucleons. We seek to replace current phenomenological models of nuclear structure and reactions with a well-founded microscopic theory that delivers maximum predictive power with well-quantified uncertainties. Specifically, the mission of this project has been three-fold: First, to find an optimal energy density functional (EDF) using all our knowledge of the nucleonic Hamiltonian and basic nuclear properties. Second, to apply the EDF theory and its extensions to validate the functional using all the available relevant nuclear structure and reaction data. Third, to apply the validated theory to properties of interest that cannot be measured, in particular the properties needed for reaction theory.

  19. To the non-local theory of cold nuclear fusion

    PubMed Central

    Alexeev, Boris V.

    2014-01-01

    In this paper, we revisit the cold fusion (CF) phenomenon using the generalized Bolzmann kinetics theory which can represent the non-local physics of this CF phenomenon. This approach can identify the conditions when the CF can take place as the soliton creation under the influence of the intensive sound waves. The vast mathematical modelling leads to affirmation that all parts of soliton move with the same velocity and with the small internal change of the pressure. The zone of the high density is shaped on the soliton's front. It means that the regime of the ‘acoustic CF’ could be realized from the position of the non-local hydrodynamics. PMID:26064528

  20. Local representation of the electronic dielectric response function

    DOE PAGES

    Lu, Deyu; Ge, Xiaochuan

    2015-12-11

    We present a local representation of the electronic dielectric response function, based on a spatial partition of the dielectric response into contributions from each occupied Wannier orbital using a generalized density functional perturbation theory. This procedure is fully ab initio, and therefore allows us to rigorously define local metrics, such as “bond polarizability,” on Wannier centers. We show that the locality of the bare response function is determined by the locality of three quantities: Wannier functions of the occupied manifold, the density matrix, and the Hamiltonian matrix. Furthermore, in systems with a gap, the bare dielectric response is exponentially localized,more » which supports the physical picture of the dielectric response function as a collection of interacting local responses that can be captured by a tight-binding model.« less

  1. Local representation of the electronic dielectric response function

    SciTech Connect

    Lu, Deyu; Ge, Xiaochuan

    2015-12-11

    We present a local representation of the electronic dielectric response function, based on a spatial partition of the dielectric response into contributions from each occupied Wannier orbital using a generalized density functional perturbation theory. This procedure is fully ab initio, and therefore allows us to rigorously define local metrics, such as “bond polarizability,” on Wannier centers. We show that the locality of the bare response function is determined by the locality of three quantities: Wannier functions of the occupied manifold, the density matrix, and the Hamiltonian matrix. Furthermore, in systems with a gap, the bare dielectric response is exponentially localized, which supports the physical picture of the dielectric response function as a collection of interacting local responses that can be captured by a tight-binding model.

  2. Nanotopographical Modulation of Cell Function through Nuclear Deformation

    PubMed Central

    Wang, Kai; Bruce, Allison; Mezan, Ryan; Kadiyala, Anand; Wang, Liying; Dawson, Jeremy; Rojanasakul, Yon; Yang, Yong

    2016-01-01

    Although nanotopography has been shown to be a potent modulator of cell behavior, it is unclear how the nanotopographical cue, through focal adhesions, affects the nucleus, eventually influencing cell phenotype and function. Thus, current methods to apply nanotopography to regulate cell behavior are basically empirical. We, herein, engineered nanotopographies of various shapes (gratings and pillars) and dimensions (feature size, spacing and height), and thoroughly investigated cell spreading, focal adhesion organization and nuclear deformation of human primary fibroblasts as the model cell grown on the nanotopographies. We examined the correlation between nuclear deformation and cell functions such as cell proliferation, transfection and extracellular matrix protein type I collagen production. It was found that the nanoscale gratings and pillars could facilitate focal adhesion elongation by providing anchoring sites, and the nanogratings could orient focal adhesions and nuclei along the nanograting direction, depending on not only the feature size but also the spacing of the nanogratings. Compared with continuous nanogratings, discrete nanopillars tended to disrupt the formation and growth of focal adhesions and thus had less profound effects on nuclear deformation. Notably, nuclear volume could be effectively modulated by the height of nanotopography. Further, we demonstrated that cell proliferation, transfection, and type I collagen production were strongly associated with the nuclear volume, indicating that the nucleus serves as a critical mechanosensor for cell regulation. Our study delineated the relationships between focal adhesions, nucleus and cell function and highlighted that the nanotopography could regulate cell phenotype and function by modulating nuclear deformation. This study provides insight into the rational design of nanotopography for new biomaterials and the cell–substrate interfaces of implants and medical devices. PMID:26844365

  3. Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV.

    PubMed

    Fukuchi, Mikoto; Wanotayan, Rujira; Liu, Sicheng; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa

    2015-06-12

    XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4(K271R) mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4(K210R) mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3'-untranslated region, and tested for the nuclear localization function by fluorescence microscopy. XRCC4(K271R) was defective in the nuclear localization of itself and LIG4, whereas XRCC4(K210R) was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4(K271R), but not M10-XRCC4(K210R), showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4(WT). The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4(K271R) than in M10-XRCC4(WT), whereas it was only marginally increased in M10-XRCC4(K210R) as compared to M10-XRCC4(WT). The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4.

  4. Goal Direction and Effectiveness, Emotional Maturity, and Nuclear Family Functioning

    ERIC Educational Resources Information Center

    Klever, Phillip

    2009-01-01

    Differentiation of self, a cornerstone concept in Bowen theory, has a profound influence over time on the functioning of the individual and his or her family unit. This 5-year longitudinal study tested this hypothesis with 50 developing nuclear families. The dimensions of differentiation of self that were examined were goal direction and…

  5. Local-basis-function approach to computed tomography

    NASA Astrophysics Data System (ADS)

    Hanson, K. M.; Wecksung, G. W.

    1985-12-01

    In the local basis-function approach, a reconstruction is represented as a linear expansion of basis functions, which are arranged on a rectangular grid and possess a local region of support. The basis functions considered here are positive and may overlap. It is found that basis functions based on cubic B-splines offer significant improvements in the calculational accuracy that can be achieved with iterative tomographic reconstruction algorithms. By employing repetitive basis functions, the computational effort involved in these algorithms can be minimized through the use of tabulated values for the line or strip integrals over a single-basis function. The local nature of the basis functions reduces the difficulties associated with applying local constraints on reconstruction values, such as upper and lower limits. Since a reconstruction is specified everywhere by a set of coefficients, display of a coarsely represented image does not require an arbitrary choice of an interpolation function.

  6. Association of Nuclear Localization of a Long Interspersed Nuclear Element-1 Protein in Breast Tumors with Poor Prognostic Outcomes

    PubMed Central

    Harris, Chris R.; Normart, Robin; Yang, Qifeng; Stevenson, Elizabeth; Haffty, Bruce G.; Ganesan, Shridar; Cordon-Cardo, Carlos; Levine, Arnold J.; Tang, Laura H.

    2010-01-01

    Within healthy human somatic cells, retrotransposition by long interspersed nuclear element-1 (also known as LINE-1 or L1) is thought to be held in check by a variety of mechanisms, including DNA methylation and RNAi. The expression of L1-ORF1 protein, which is rarely found in normal tissue, was assayed using antibodies with a variety of clinical cancer specimens and cancer cell lines. L1-ORF1p expression was detected in nearly all breast tumors that the authors examined, and the protein was also present in a high percentage of ileal carcinoids, bladder, and pancreatic neuroendocrine tumors, as well as in a smaller percentage of prostate and colorectal tumors. Tumors generally demonstrated cytoplasmic L1-ORF1p; however, in several breast cancers, L1-ORF1p was nuclear. Patients with breast tumors displaying nuclear L1-ORF1p had a greater incidence of both local recurrence and distal metastases and also showed poorer overall survival when compared with patients with tumors displaying cytoplasmic L1-ORF1p. These data suggest that expression of L1-ORF1p is widespread in many cancers and that redistribution from cytoplasm to nucleus could be a poor prognostic indicator during breast cancer. High expression and nuclear localization of L1-ORF1p may result in a higher rate of L1 retrotransposition, which could increase genomic instability. PMID:20948976

  7. Towards reconciling structure and function in the nuclear pore complex

    PubMed Central

    Aebi, Ueli; Fahrenkrog, Birthe

    2008-01-01

    The spatial separation between the cytoplasm and the cell nucleus necessitates the continuous exchange of macromolecular cargo across the double-membraned nuclear envelope. Being the only passageway in and out of the nucleus, the nuclear pore complex (NPC) has the principal function of regulating the high throughput of nucleocytoplasmic transport in a highly selective manner so as to maintain cellular order and function. Here, we present a retrospective review of the evidence that has led to the current understanding of both NPC structure and function. Looking towards the future, we contemplate on how various outstanding effects and nanoscopic characteristics ought to be addressed, with the goal of reconciling structure and function into a single unified picture of the NPC. PMID:18228033

  8. Functional renormalization group studies of nuclear and neutron matter

    NASA Astrophysics Data System (ADS)

    Drews, Matthias; Weise, Wolfram

    2017-03-01

    Functional renormalization group (FRG) methods applied to calculations of isospin-symmetric and asymmetric nuclear matter as well as neutron matter are reviewed. The approach is based on a chiral Lagrangian expressed in terms of nucleon and meson degrees of freedom as appropriate for the hadronic phase of QCD with spontaneously broken chiral symmetry. Fluctuations beyond mean-field approximation are treated solving Wetterich's FRG flow equations. Nuclear thermodynamics and the nuclear liquid-gas phase transition are investigated in detail, both in symmetric matter and as a function of the proton fraction in asymmetric matter. The equations of state at zero temperature of symmetric nuclear matter and pure neutron matter are found to be in good agreement with advanced ab-initio many-body computations. Contacts with perturbative many-body approaches (in-medium chiral perturbation theory) are discussed. As an interesting test case, the density dependence of the pion mass in the medium is investigated. The question of chiral symmetry restoration in nuclear and neutron matter is addressed. A stabilization of the phase with spontaneously broken chiral symmetry is found to persist up to high baryon densities once fluctuations beyond mean-field are included. Neutron star matter including beta equilibrium is discussed under the aspect of the constraints imposed by the existence of two-solar-mass neutron stars.

  9. Local hybrid functionals: an assessment for thermochemical kinetics.

    PubMed

    Kaupp, Martin; Bahmann, Hilke; Arbuznikov, Alexei V

    2007-11-21

    Local hybrid functionals with position-dependent exact-exchange admixture are a new class of exchange-correlation functionals in density functional theory that promise to advance the available accuracy in many areas of application. Local hybrids with different local mixing functions (LMFs) governing the position dependence are validated for the heats of formation of the extended G3/99 set, and for two sets of barriers of hydrogen-transfer and heavy-atom transfer reactions (HTBH38 and NHTBH38 databases). A simple local hybrid Lh-SVWN with only Slater and exact exchange plus local correlation and a one-parameter LMF, g(r)=b(tau(W)(r)tau(r)), performs best and provides overall mean absolute errors for thermochemistry and kinetics that are a significant improvement over standard state-of-the-art global hybrid functionals. In particular, this local hybrid functional does not suffer from the systematic deterioration that standard functionals exhibit for larger molecules. In contrast, local hybrids based on generalized gradient approximation exchange tend to give rise to nonintuitive LMFs, and no improved functionals have been obtained along this route. The LMF is a real-space function and thus can be analyzed in detail. We use, in particular, graphical analyses to rationalize the performance of different local hybrids for thermochemistry and reaction barriers.

  10. Local hybrid functionals: An assessment for thermochemical kinetics

    SciTech Connect

    Kaupp, Martin; Bahmann, Hilke; Arbuznikov, Alexei V.

    2007-11-21

    Local hybrid functionals with position-dependent exact-exchange admixture are a new class of exchange-correlation functionals in density functional theory that promise to advance the available accuracy in many areas of application. Local hybrids with different local mixing functions (LMFs) governing the position dependence are validated for the heats of formation of the extended G3/99 set, and for two sets of barriers of hydrogen-transfer and heavy-atom transfer reactions (HTBH38 and NHTBH38 databases). A simple local hybrid Lh-SVWN with only Slater and exact exchange plus local correlation and a one-parameter LMF, g(r)=b({tau}{sub W}(r)/{tau}(r)), performs best and provides overall mean absolute errors for thermochemistry and kinetics that are a significant improvement over standard state-of-the-art global hybrid functionals. In particular, this local hybrid functional does not suffer from the systematic deterioration that standard functionals exhibit for larger molecules. In contrast, local hybrids based on generalized gradient approximation exchange tend to give rise to nonintuitive LMFs, and no improved functionals have been obtained along this route. The LMF is a real-space function and thus can be analyzed in detail. We use, in particular, graphical analyses to rationalize the performance of different local hybrids for thermochemistry and reaction barriers.

  11. TOPICAL REVIEW: Spatial localization in nuclear magnetic resonance spectroscopy

    NASA Astrophysics Data System (ADS)

    Keevil, Stephen F.

    2006-08-01

    The ability to select a discrete region within the body for signal acquisition is a fundamental requirement of in vivo NMR spectroscopy. Ideally, it should be possible to tailor the selected volume to coincide exactly with the lesion or tissue of interest, without loss of signal from within this volume or contamination with extraneous signals. Many techniques have been developed over the past 25 years employing a combination of RF coil properties, static magnetic field gradients and pulse sequence design in an attempt to meet these goals. This review presents a comprehensive survey of these techniques, their various advantages and disadvantages, and implications for clinical applications. Particular emphasis is placed on the reliability of the techniques in terms of signal loss, contamination and the effect of nuclear relaxation and J-coupling. The survey includes techniques based on RF coil and pulse design alone, those using static magnetic field gradients, and magnetic resonance spectroscopic imaging. Although there is an emphasis on techniques currently in widespread use (PRESS, STEAM, ISIS and MRSI), the review also includes earlier techniques, in order to provide historical context, and techniques that are promising for future use in clinical and biomedical applications.

  12. Localization of P-glycoprotein at the nuclear envelope of rat brain cells

    SciTech Connect

    Babakhanian, Karlo; Bendayan, Moise; Bendayan, Reina . E-mail: r.bendayan@utoronto.ca

    2007-09-21

    P-Glycoprotein is a plasma membrane drug efflux protein implicated in extrusion of cytotoxic compounds out of a cell. There is now evidence that suggests expression of this transporter at several subcellular sites, including the nucleus, mitochondria, and Golgi apparatus. This study investigated the localization and expression of P-glycoprotein at the nuclear membrane of rat brain microvessel endothelial (RBE4) and microglial (MLS-9) cell lines. Immunocytochemistry at the light and electron microscope levels using P-glycoprotein monoclonals antibodies demonstrated the localization of the protein at the nuclear envelope of RBE4 and MLS-9 cells. Western blot analysis revealed a single band of 170-kDa in purified nuclear membranes prepared from isolated nuclei of RBE4 and MLS-9 cells. These findings indicate that P-glycoprotein is expressed at the nuclear envelope of rat brain cells and suggest a role in multidrug resistance at this subcellular site.

  13. Characterization of the bipartite nuclear localization signal of protein LANA2 from Kaposi's sarcoma-associated herpesvirus.

    PubMed Central

    Muñoz-Fontela, Cesar; Rodríguez, Estefanía; Nombela, Cesar; Arroyo, Javier; Rivas, Carmen

    2003-01-01

    LANA2 is a nuclear latent protein detected exclusively in Kaposi's sarcoma-associated herpesvirus-infected B cells. The protein inhibits p53-dependent transactivation and apoptosis, suggesting an important role in the transforming activity of the virus. To explore the molecular mechanisms of its nuclear localization, fusion proteins of green fluorescent protein (EGFP) and deletion constructs of LANA2 were expressed in HeLa cells. Only the fragment comprising amino acid residues 355-440 of LANA2 localized in the cell nucleus. This fragment contains two closely located basic domains and forms a putative bipartite nuclear localization signal (NLS). The putative LANA2 NLS was able to target EGFP to the nucleus consistently. Site-directed mutation analyses demonstrated that LANA2 contains a functional bipartite NLS between amino acid positions 367 and 384. In addition, analysis of cells transfected with a cytoplasmic LANA2 mutant revealed that an appropriate subcellular localization may be crucial to regulate p53 activity. PMID:12767255

  14. Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV

    SciTech Connect

    Fukuchi, Mikoto; Wanotayan, Rujira; Liu, Sicheng; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa

    2015-06-12

    XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4{sup K271R} mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4{sup K210R} mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3′-untranslated region, and tested for the nuclear localization function by fluorescence microscopy. XRCC4{sup K271R} was defective in the nuclear localization of itself and LIG4, whereas XRCC4{sup K210R} was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4{sup K271R}, but not M10-XRCC4{sup K210R}, showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4{sup WT}. The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4{sup K271R} than in M10-XRCC4{sup WT}, whereas it was only marginally increased in M10-XRCC4{sup K210R} as compared to M10-XRCC4{sup WT}. The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4. - Highlights: • XRCC4{sup K271R} is defective in the nuclear localization of itself and LIG4. • XRCC4{sup K210R} is competent for the nuclear localization of itself and LIG4. • XRCC4{sup K271R} is deficient in DSB repair function. • XRCC4{sup K210R} is mostly normal in DSB repair function.

  15. Localization and function of three monothiol glutaredoxins in Schizosaccharomyces pombe

    SciTech Connect

    Chung, Woo-Hyun; Kim, Kyoung-Dong; Roe, Jung-Hye . E-mail: jhroe@plaza.snu.ac.kr

    2005-05-06

    The fission yeast Schizosaccharomyces pombe contains two dithiol glutaredoxins (Grx1 and Grx2) and genes for three putative monothiol glutaredoxins (grx3, 4, and 5). We investigated the expression, sub-cellular localization, and functions of the three monothiol glutaredoxins. Fluorescence microscopy revealed that Grx3 is targeted to nuclear rim and endoplasmic reticulum, Grx4 primarily to the nucleus, and Grx5 to mitochondria. Null mutation of grx3 did not significantly affect growth and resistance against various oxidants, whereas grx5 mutation caused slow growth and sensitivity toward oxidants such as hydrogen peroxide, paraquat, and diamide. The grx2grx5 double mutation, deficient in all mitochondrial glutaredoxins, caused further retardation in growth and severe sensitivity toward all the oxidants tested. The grx4 mutation was not viable, suggesting a critical role of Grx4 for the physiology of S. pombe. Overproduction of Grx3 and Grx5, but not the truncated form of Grx5 without mitochondrial target sequence, severely retarded growth as Grx2 did, supporting the idea that Grx2, 3, and 5 are targeted to organellar compartments. Our results propose a distinct role for each glutaredoxin to maintain thiol redox balance, and hence the growth and stress resistance, of the fission yeast.

  16. Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein

    SciTech Connect

    Kutty, R. Krishnan . E-mail: kuttyk@nei.nih.gov; Chen, Shanyi; Samuel, William; Vijayasarathy, Camasamudram; Duncan, Todd; Tsai, Jen-Yue; Fariss, Robert N.; Carper, Deborah; Jaworski, Cynthia; Wiggert, Barbara

    2006-07-14

    NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P{sup 27}KKRKAP{sup 276}) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.

  17. Spin Density Matrices for Nuclear Density Functionals with Parity Violation

    NASA Astrophysics Data System (ADS)

    Barrett, Bruce; Giraud, Bertrand

    2010-11-01

    Within the context of the radial density functional [1], we apply the spin density matrix (SDM) used in atomic and molecular physics [2] to nuclear physics. The vector part of the SDM defines a ``hedgehog'' situation, which exists only if nuclear states contain some amount of parity violation. Thus, looking for the vector profile of the SDM could be used as a test for parity violation in nuclei. The difference between the scalar profile and the vector profile of the SDM will be illustrated by a toy model. [4pt] [1] B. G. Giraud, Phys. Rev. C 78, 014307 (2008).[0pt] [2] A. Goerling, Phys. Rev. A 47, 2783 (1993).

  18. Building A Universal Nuclear Energy Density Functional (UNEDF)

    SciTech Connect

    Joe Carlson; Dick Furnstahl; Mihai Horoi; Rusty Lusk; Witek Nazarewicz; Esmond Ng; Ian Thompson; James Vary

    2012-09-30

    During the period of Dec. 1 2006 - Jun. 30, 2012, the UNEDF collaboration carried out a comprehensive study of all nuclei, based on the most accurate knowledge of the strong nuclear interaction, the most reliable theoretical approaches, the most advanced algorithms, and extensive computational resources, with a view towards scaling to the petaflop platforms and beyond. The long-term vision initiated with UNEDF is to arrive at a comprehensive, quantitative, and unified description of nuclei and their reactions, grounded in the fundamental interactions between the constituent nucleons. We seek to replace current phenomenological models of nuclear structure and reactions with a well-founded microscopic theory that delivers maximum predictive power with well-quantified uncertainties. Specifically, the mission of this project has been three-fold: first, to find an optimal energy density functional (EDF) using all our knowledge of the nucleonic Hamiltonian and basic nuclear properties; second, to apply the EDF theory and its extensions to validate the functional using all the available relevant nuclear structure and reaction data; third, to apply the validated theory to properties of interest that cannot be measured, in particular the properties needed for reaction theory. The main physics areas of UNEDF, defined at the beginning of the project, were: ab initio structure; ab initio functionals; DFT applications; DFT extensions; reactions.

  19. Characterization of a nuclear localization signal in the C-terminus of the adeno-associated virus Rep68/78 proteins

    SciTech Connect

    Cassell, Geoffrey D.; Weitzman, Matthew D. . E-mail: weitzman@salk.edu

    2004-10-01

    Adeno-associated virus (AAV) replicates in the nucleus of infected cells, and therefore multiple nuclear import events are required for productive infection. We analyzed nuclear import of the viral Rep proteins and characterized a nuclear localization signal (NLS) in the C-terminus. We demonstrate that basic residues in this region constitute an NLS that is transferable and mediates interaction with the nuclear import receptor importin {alpha} in vitro. Mutant Rep proteins are predominantly cytoplasmic and are severely compromised for interactions with importin {alpha}, but retain their enzymatic functions in vitro. Interestingly, mutations of the NLS had significantly less effect on importin {alpha} interaction and replication in the context of Rep78 than when incorporated into the Rep68 protein. Together, our results demonstrate that a bipartite NLS exists in the shared part of Rep68 and Rep78, and suggest that an alternate entry mechanism may also contribute to nuclear localization of the Rep78 protein.

  20. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

  1. Dynamic localization of tripartite motif-containing 22 in nuclear and nucleolar bodies

    SciTech Connect

    Sivaramakrishnan, Gayathri; Sun, Yang; Tan, Si Kee; Lin, Valerie C.L.

    2009-05-01

    Tripartite motif-containing 22 (TRIM22) exhibits antiviral and growth inhibitory properties, but there has been no study on the localization and dynamics of the endogenous TRIM22 protein. We report here that TRIM22 is dramatically induced by progesterone in MDA-MB-231-derived ABC28 cells and T47D cells. This induction was associated with an increase in TRIM22 nuclear bodies (NB), and an even more prominent increase in nucleolar TRIM22 bodies. Distinct endogenous TRIM22 NB were also demonstrated in several other cell lines including MCF7 and HeLa cells. These TRIM22 NB resemble Cajal bodies, co-localized with these structures and co-immunoprecipitated with p80-coilin. However, IFN{gamma}-induced TRIM22 in HeLa and MCF7 cells did not form NB, implying the forms and distribution of TRIM22 are regulated by specific cellular signals. This notion is also supported by the observation that TRIM22 NB undergoes dynamic cell-cycle dependent changes in distribution such that TRIM22 NB started to form in early G0/G1 but became dispersed in the S-phase. In light of its potential antiviral and antitumor properties, the findings here provide an interesting gateway to study the relationship between the different forms and functions of TRIM22.

  2. [Immunochemical study of nuclear matrix proteins localization in the structure of perinucleolar chromatin].

    PubMed

    Murasheva, M I; Chentsov, Iu S

    2014-01-01

    Immunofluorescence labeling of proteins with molecular mass of 27, 38, 40, 50 and 65 kDa obtained from serum of patients with autoimmune disease demonstrated different patterns (small clusters or granules) in interphase nuclei of pig kidney cells. It was remarkable that there was no staining inside the nucleoli, but the proteins immunoreactivity was detected around them in the regions of perinucleolar chromatin. Moreover, expression of nucleolar proteins, such as fibrillarin and B23, was found only in nucleoli. After extraction of DNA, PNA and histones, the proteins with molecular mass 27 and 38 kDa were found in the periphery of residual nucleoli, and proteins with molecular mass 40, 50 and 65 kDa had similar localization and were also present in karyoplasm of cells as small clusters. According to our data, nucleolar protein, fibrillarin, was distributed regularly throughout the whole volume of residual nucleoli. At the same time, B23 protein was revealed only at their periphery, where perinucleolar chromatin had localized before treatment. Thus, it has been revealed that the proteins of nuclear matrix with molecular mass 27, 38, 40, 50 and 65 kDa, as well as nucleolar protein B23 are the parts of perinucleolar chromatin, which could be considered as special chromosomal domain associated with the functioning of the nucleolus.

  3. Many-body Green functions in nuclear physics

    NASA Astrophysics Data System (ADS)

    Speth, J.; Lyutorovich, N.

    Many-body Green functions are a very efficient formulation of the many-body problem. We review the application of this method to nuclear physics problems. The formulas which can be derived are of general applicability, e.g., in self-consistent as well as in nonself-consistent calculations. With the help of the Landau renormalization, one obtains relations without any approximations. This allows to apply conservation laws which lead to important general relations. We investigate the one-body and two-body Green functions as well as the three-body Green function and discuss their connection to nuclear observables. The generalization to systems with pair correlations are also presented. Numerical examples are compared with experimental data.

  4. Cardiac nuclear receptors: architects of mitochondrial structure and function.

    PubMed

    Vega, Rick B; Kelly, Daniel P

    2017-04-03

    The adult heart is uniquely designed and equipped to provide a continuous supply of energy in the form of ATP to support persistent contractile function. This high-capacity energy transduction system is the result of a remarkable surge in mitochondrial biogenesis and maturation during the fetal-to-adult transition in cardiac development. Substantial evidence indicates that nuclear receptor signaling is integral to dynamic changes in the cardiac mitochondrial phenotype in response to developmental cues, in response to diverse postnatal physiologic conditions, and in disease states such as heart failure. A subset of cardiac-enriched nuclear receptors serve to match mitochondrial fuel preferences and capacity for ATP production with changing energy demands of the heart. In this Review, we describe the role of specific nuclear receptors and their coregulators in the dynamic control of mitochondrial biogenesis and energy metabolism in the normal and diseased heart.

  5. Importin β2 Mediates the Spatio-temporal Regulation of Anillin through a Noncanonical Nuclear Localization Signal.

    PubMed

    Chen, Anan; Akhshi, Tara K; Lavoie, Brigitte D; Wilde, Andrew

    2015-05-22

    The compartmentalization of cell cycle regulators is a common mechanism to ensure the precise temporal control of key cell cycle events. For instance, many mitotic spindle assembly factors are known to be sequestered in the nucleus prior to mitotic onset. Similarly, the essential cytokinetic factor anillin, which functions at the cell membrane to promote the physical separation of daughter cells at the end of mitosis, is sequestered in the nucleus during interphase. To address the mechanism and role of anillin targeting to the nucleus in interphase, we identified the nuclear targeting motif. Here, we show that anillin is targeted to the nucleus by importin β2 in a Ran-dependent manner through an atypical basic patch PY nuclear localization signal motif. We show that although importin β2 binding does not regulate anillin's function in mitosis, it is required to prevent the cytosolic accumulation of anillin, which disrupts cellular architecture during interphase. The nuclear sequestration of anillin during interphase serves to restrict anillin's function at the cell membrane to mitosis and allows anillin to be rapidly available when the nuclear envelope breaks down to remodel the cellular architecture necessary for successful cell division.

  6. The black hole mass function derived from local spiral galaxies

    SciTech Connect

    Davis, Benjamin L.; Berrier, Joel C.; Shields, Douglas W.; Kennefick, Daniel; Kennefick, Julia; Seigar, Marc S.; Lacy, Claud H. S.; Hartley, Matthew T.

    2014-07-10

    We present our determination of the nuclear supermassive black hole (SMBH) mass function for spiral galaxies in the local universe, established from a volume-limited sample consisting of a statistically complete collection of the brightest spiral galaxies in the southern (δ < 0°) hemisphere. Our SMBH mass function agrees well at the high-mass end with previous values given in the literature. At the low-mass end, inconsistencies exist in previous works that still need to be resolved, but our work is more in line with expectations based on modeling of black hole evolution. This low-mass end of the spectrum is critical to our understanding of the mass function and evolution of black holes since the epoch of maximum quasar activity. The sample is defined by a limiting luminosity (redshift-independent) distance, D{sub L} = 25.4 Mpc (z = 0.00572) and a limiting absolute B-band magnitude, M{sub B}=−19.12. These limits define a sample of 140 spiral galaxies, with 128 measurable pitch angles to establish the pitch angle distribution for this sample. This pitch-angle distribution function may be useful in the study of the morphology of late-type galaxies. We then use an established relationship between the logarithmic spiral arm pitch angle and the mass of the central SMBH in a host galaxy in order to estimate the mass of the 128 respective SMBHs in this volume-limited sample. This result effectively gives us the distribution of mass for SMBHs residing in spiral galaxies over a lookback time, t{sub L} ≤ 82.1 h{sub 67.77}{sup −1} Myr and contained within a comoving volume, V{sub C} = 3.37 × 10{sup 4} h{sub 67.77}{sup −3} Mpc{sup 3}. We estimate that the density of SMBHs residing in spiral galaxies in the local universe is ρ=5.54{sub −2.73}{sup +6.55} × 10{sup 4} h{sub 67.77}{sup 3} M{sub ☉} Mpc{sup –3}. Thus, our derived cosmological SMBH mass density for spiral galaxies is Ω{sub BH}=4.35{sub −2.15}{sup +5.14} × 10{sup –7} h{sub 67.77}. Assuming that

  7. Living with nuclear power: A Q-method study of local community perceptions.

    PubMed

    Venables, Dan; Pidgeon, Nick; Simmons, Peter; Henwood, Karen; Parkhill, Karen

    2009-08-01

    The issue of new nuclear power is once again high up on the public policy agenda in many countries, and candidate sites for new civilian stations are likely to include those that have existing nuclear facilities. A common assumption is that existing nuclear communities will be more accepting of new build because of the direct economic and other benefits nuclear power already makes to a local area. Surprisingly, there is a dearth of contemporary data on perceptions of the risks, benefits, and values associated with nuclear power within such communities. This study uses Q-methodology to investigate the perspectives on living with nuclear risk among people (n = 84) drawn from communities near to two nuclear power stations in the United Kingdom. Both stations, at Bradwell-on-Sea and Oldbury-on-Severn, had been in operation for over 40 years. The Q-analysis identified four main perspectives, or points of view, accounting for 53% of total variance. These were interpreted as: Beneficial and Safe; Threat and Distrust; Reluctant Acceptance; and There's No Point Worrying. We conclude that the "landscape of beliefs" about nuclear power in such communities is both subtle and complex, avoiding simplistic bipolar dichotomies such as "for" or "against," and that there is a need for extensive and meaningful dialogue with such communities over any new build plans. The usefulness of Q-methodology for investigating the ways in which people live with risk is highlighted, as are the implications of the results for theories of risk and trust.

  8. Time-dependent density-functional description of nuclear dynamics

    NASA Astrophysics Data System (ADS)

    Nakatsukasa, Takashi; Matsuyanagi, Kenichi; Matsuo, Masayuki; Yabana, Kazuhiro

    2016-10-01

    The basic concepts and recent developments in the time-dependent density-functional theory (TDDFT) for describing nuclear dynamics at low energy are presented. The symmetry breaking is inherent in nuclear energy density functionals, which provides a practical description of important correlations at the ground state. Properties of elementary modes of excitation are strongly influenced by the symmetry breaking and can be studied with TDDFT. In particular, a number of recent developments in the linear response calculation have demonstrated their usefulness in the description of collective modes of excitation in nuclei. Unrestricted real-time calculations have also become available in recent years, with new developments for quantitative description of nuclear collision phenomena. There are, however, limitations in the real-time approach; for instance, it cannot describe the many-body quantum tunneling. Thus, the quantum fluctuations associated with slow collective motions are explicitly treated assuming that time evolution of densities is determined by a few collective coordinates and momenta. The concept of collective submanifold is introduced in the phase space associated with the TDDFT and used to quantize the collective dynamics. Selected applications are presented to demonstrate the usefulness and quality of the new approaches. Finally, conceptual differences between nuclear and electronic TDDFT are discussed, with some recent applications to studies of electron dynamics in the linear response and under a strong laser field.

  9. SURFACE SYMMETRY ENERGY OF NUCLEAR ENERGY DENSITY FUNCTIONALS

    SciTech Connect

    Nikolov, N; Schunck, N; Nazarewicz, W; Bender, M; Pei, J

    2010-12-20

    We study the bulk deformation properties of the Skyrme nuclear energy density functionals. Following simple arguments based on the leptodermous expansion and liquid drop model, we apply the nuclear density functional theory to assess the role of the surface symmetry energy in nuclei. To this end, we validate the commonly used functional parametrizations against the data on excitation energies of superdeformed band-heads in Hg and Pb isotopes, and fission isomers in actinide nuclei. After subtracting shell effects, the results of our self-consistent calculations are consistent with macroscopic arguments and indicate that experimental data on strongly deformed configurations in neutron-rich nuclei are essential for optimizing future nuclear energy density functionals. The resulting survey provides a useful benchmark for further theoretical improvements. Unlike in nuclei close to the stability valley, whose macroscopic deformability hangs on the balance of surface and Coulomb terms, the deformability of neutron-rich nuclei strongly depends on the surface-symmetry energy; hence, its proper determination is crucial for the stability of deformed phases of the neutron-rich matter and description of fission rates for r-process nucleosynthesis.

  10. Functional localization of the supplementary motor area.

    PubMed

    Hiroshima, Satoru; Anei, Ryogo; Murakami, Noboru; Kamada, Kyousuke

    2014-01-01

    The supplementary motor area (SMA) is a key structure involved in behavioral planning and execution. Although many reports have indicated that SMA is organized somatotopically, its exact organization remains still unclear. This study aimed to functionally map SMA using functional magnetic resonance imaging (fMRI) and validate the fMRI-SMA by electrocortical stimulation (ECS) and postsurgical symptoms. Total 32 healthy volunteers and 24 patients participated in this study. Motor tasks were right and left finger tapping and language tasks included simple reading, lexical decision for presented words, and verb generating tasks. SPM8 was used to conduct individual and group analyses. In all subjects, the lexical decision task induced the greatest number of active fMRI pixels in SMA. fMRI during the language tasks showed anterior part of SMA compared to finger tapping tasks. We found an overlap spot with all different tasks in posterior part of SMA, which we termed SMA core. Six patients underwent awake craniotomy for ECS mapping for primary regions and SMA. During awake craniotomy, ECS to posterior part of SMA, which might involve the possible SMA core consistently, evoked both speech arrest and flaccid hemiparesis. The SMA mapping suggested posterior part of SMA might play more important roles in any executions, which might involve the SMA core.

  11. Nuclear vs Cytoplasmic localization of Filamin A in Prostate Cancer: Immunohistochemical Correlation with Metastases

    PubMed Central

    Bedolla, Roble G.; Wang, Yu; Asuncion, Alfredo; Chamie, Karim; Siddiqui, Salma; Mudryj, Maria M.; Prihoda, Thomas J.; Siddiqui, Javed; Chinnaiyan, Arul M.; Mehra, Rohit; deVereWhite, Ralph W.; Ghosh, Paramita M.

    2009-01-01

    Purpose We previously showed that nuclear localization of the actin-binding protein FilaminA (FlnA) corresponded to hormone-dependence in prostate cancer (Oncogene, 2007, 26:6061-6070). Intact FlnA (280kDa, cytoplasmic) cleaved to a 90kDa fragment which translocated to the nucleus in hormone-naïve cells, whereas in hormone-refractory cells, FlnA was phosphorylated, preventing its cleavage and nuclear translocation. We now examined whether FlnA localization determines a propensity to metastasis in advanced androgen independent prostate cancer. Experimental Design We examined, by immunohistochemistry, FlnA localization in paraffin-embedded human prostate tissue representing different stages of progression. Results were correlated with in vitro studies in a cell model of prostate cancer. Results Nuclear FlnA was significantly higher in benign prostate (0.6612±0.5888), PIN (0.6024±0.4620) and clinically localized cancers (0.69134±0.5686), compared to metastatic prostate cancers (0.3719±0.4992, p=0.0007). Cytoplasmic FlnA increased from benign prostate (0.0833±0.2677), PIN (0.1409±0.2293), localized cancers (0.3008±0.3762, p=0.0150), to metastases (0.7632±0.4414, p<0.00001). Logistic regression of metastatic vs non-metastatic tissue yielded the area-under-ROC curve as 0.67 for nuclear-FlnA, 0.79 for cytoplasmic-FlnA and 0.82 for both, indicating that metastasis correlates with cytoplasmic-to-nuclear translocation. In vitro studies showed that cytoplasmic localization of FlnA induced cell invasion whereas nuclear translocation of the protein inhibited it. FlnA dephosphorylation with the PKA inhibitor H-89 facilitated FlnA nuclear translocation, resulting in decreased invasiveness and AR transcriptional activity, and induced sensitivity to androgen withdrawal in hormone-refractory cells. Conclusions The data presented in this study indicate that in prostate cancer, metastasis correlates with cytoplasmic localization of FlnA and may be prevented by cleavage and

  12. Sorghum DW1 positively regulates brassinosteroid signaling by inhibiting the nuclear localization of BRASSINOSTEROID INSENSITIVE 2.

    PubMed

    Hirano, Ko; Kawamura, Mayuko; Araki-Nakamura, Satoko; Fujimoto, Haruka; Ohmae-Shinohara, Kozue; Yamaguchi, Miki; Fujii, Akihiro; Sasaki, Hiroaki; Kasuga, Shigemitsu; Sazuka, Takashi

    2017-12-01

    Semi-dwarf traits have been widely introgressed into cereal crops to improve lodging resistance. In sorghum (Sorghum bicolor L. Moench), four major unlinked dwarfing genes, Dw1-Dw4, have been introduced to reduce plant height, and among them, Dw3 and Dw1 have been cloned. Dw3 encodes a gene involved in auxin transport, whereas, Dw1 was recently isolated and identified as a gene encoding a protein of unknown function. In this study, we show that DW1 is a novel component of brassinosteroid (BR) signaling. Sorghum possessing the mutated allele of Dw1 (dw1), showed similar phenotypes to rice BR-deficient mutants, such as reduced lamina joint bending, attenuated skotomorphogenesis, and insensitivity against feedback regulation of BR-related genes. Furthermore, DW1 interacted with a negative regulator of BR signaling, BRASSINOSTEROID INSENSITIVE 2 (BIN2), and inhibited its nuclear localization, indicating that DW1 positively regulates BR signaling by inhibiting the function of BIN2. In contrast to rice and wheat breeding which used gibberellin (GA) deficiency to reduce plant height, sorghum breeding modified auxin and BR signaling. This difference may result from GA deficiency in rice and wheat does not cause deleterious side effects on plant morphology, whereas in sorghum it leads to abnormal culm bending.

  13. "Sloppy" nuclear energy density functionals: Effective model reduction

    NASA Astrophysics Data System (ADS)

    Nikšić, Tamara; Vretenar, Dario

    2016-08-01

    Concepts from information geometry are used to analyze parameter sensitivity for a nuclear energy density functional, representative of a class of semiempirical functionals that start from a microscopically motivated ansatz for the density dependence of the energy of a system of protons and neutrons. It is shown that such functionals are "sloppy," namely, characterized by an exponential range of sensitivity to parameter variations. Responsive to only a few stiff parameter combinations, sloppy functionals exhibit an exponential decrease of sensitivity to variations of the remaining soft parameters. By interpreting the space of model predictions as a manifold embedded in the data space, with the parameters of the functional as coordinates on the manifold, it is also shown that the exponential distribution of model manifold widths corresponds to the range of parameter sensitivity. Using the manifold boundary approximation method, we illustrate how to systematically construct effective nuclear density functionals of successively lower dimension in parameter space until sloppiness is eventually eliminated and the resulting functional contains only stiff combinations of parameters.

  14. Semiconductor band gap localization via Gaussian function

    NASA Astrophysics Data System (ADS)

    Ullrich, B.; Brown, G. J.; Xi, H.

    2012-10-01

    To determine the band gap of bulk semiconductors with transmission spectroscopy alone is considered as an extremely difficult task because in the higher energy range, approaching and exceeding the band gap energy, the material is opaque yielding no useful data to be recorded. In this paper, by investigating the transmission of industrial GaSb wafers with a thickness of 500 µm, we demonstrate how these obstacles of transmission spectroscopy can be overcome. The key is the transmission spectrums’ derivative, which coincides with the Gaussian function. This understanding can be used to transfer Beers’ law in an integral form opening the pathway of band gap determinations based on mathematical parameters only. The work also emphasizes the correlation between the thermal band gap variation and Debye temperature.

  15. Neutralizing Interleukin-1β (IL-1β) Induces β-Cell Survival by Maintaining PDX1 Protein Nuclear Localization*

    PubMed Central

    Ardestani, Amin; Sauter, Nadine S.; Paroni, Federico; Dharmadhikari, Gitanjali; Cho, Jae-Hyoung; Lupi, Roberto; Marchetti, Piero; Oberholzer, José; Conte, Julie Kerr; Maedler, Kathrin

    2011-01-01

    The transcription factor PDX1 plays a critical role during β-cell development and in glucose-induced insulin gene transcription in adult β-cells. Acute glucose exposure leads to translocalization of PDX1 to the nucleoplasm, whereas under conditions of oxidative stress, PDX1 shuttles from the nucleus to the cytosol. Here we show that cytosolic PDX1 expression correlated with β-cell failure in diabetes. In isolated islets from patients with type 2 diabetes and from diabetic mice, we found opposite regulation of insulin and PDX1 mRNA; insulin was decreased in diabetes, but PDX1 was increased. This suggests that elevated PDX1 mRNA levels may be insufficient to regulate insulin. In diabetic islets, PDX1 protein was localized in the cytosol, whereas in non-diabetic controls, PDX1 was in the nucleus. In contrast, overexpression of either IL-1 receptor antagonist or shuttling-deficient PDX1 restored β-cell survival and function and PDX1 nuclear localization. Our results show that nuclear localization of PDX1 is essential for a functional β-cell and provides a novel mechanism of the protective effect of IL-1 receptor antagonist on β-cell survival and function. PMID:21393239

  16. Nuclear Localization and Cleavage of STAT6 Is Induced by Kaposi’s Sarcoma-Associated Herpesvirus for Viral Latency

    PubMed Central

    Zhang, Liming; Li, Yuhong; Feng, Yanling; Xu, Jianqing; Wang, Bin; Yuan, Zhenghong; Robertson, Erle S.; Cai, Qiliang

    2017-01-01

    Emerging evidence implies that STAT6 plays an important role in both the adaptive and innate immune responses to virus infection. Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus agent associated with several human malignancies, including Kaposi’s sarcoma (KS) and primary effusion lymphomas (PELs). Previously, we demonstrated that KSHV blocks IL-4-induced STAT6 phosphorylation and retains a basal IL-13/STAT6 constitutive activation for cell survival and proliferation. However, the mechanism by which KSHV regulates STAT6 remains largely unknown. Here, we found that KSHV-encoded LANA interacts with STAT6 and promotes nuclear localization of STAT6 independent of the tyrosine 641-phosphorylation state. Moreover, nuclear localization of STAT6 is also dramatically increased in KS tissue. The latent antigen LANA induces serine protease-mediated cleavage of STAT6 in the nucleus, where the cleaved STAT6 lacking transactivation domain functions as a dominant-negative regulator to repress transcription of Replication and Transcription Activator (RTA) and in turn shut off viral lytic replication. Blockade of STAT6 by small interference RNA dramatically enhances expression of RTA, and in turn reduces KSHV-infected endothelial cell growth and colony formation. Taken together, these results suggest that nuclear localization and cleavage of STAT6 is important for modulating the viral latency and pathogenesis of KSHV. PMID:28099521

  17. Nuclear Localization and Cleavage of STAT6 Is Induced by Kaposi's Sarcoma-Associated Herpesvirus for Viral Latency.

    PubMed

    Wang, Chong; Zhu, Caixia; Wei, Fang; Gao, Shujun; Zhang, Liming; Li, Yuhong; Feng, Yanling; Tong, Yin; Xu, Jianqing; Wang, Bin; Yuan, Zhenghong; Robertson, Erle S; Cai, Qiliang

    2017-01-01

    Emerging evidence implies that STAT6 plays an important role in both the adaptive and innate immune responses to virus infection. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus agent associated with several human malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphomas (PELs). Previously, we demonstrated that KSHV blocks IL-4-induced STAT6 phosphorylation and retains a basal IL-13/STAT6 constitutive activation for cell survival and proliferation. However, the mechanism by which KSHV regulates STAT6 remains largely unknown. Here, we found that KSHV-encoded LANA interacts with STAT6 and promotes nuclear localization of STAT6 independent of the tyrosine 641-phosphorylation state. Moreover, nuclear localization of STAT6 is also dramatically increased in KS tissue. The latent antigen LANA induces serine protease-mediated cleavage of STAT6 in the nucleus, where the cleaved STAT6 lacking transactivation domain functions as a dominant-negative regulator to repress transcription of Replication and Transcription Activator (RTA) and in turn shut off viral lytic replication. Blockade of STAT6 by small interference RNA dramatically enhances expression of RTA, and in turn reduces KSHV-infected endothelial cell growth and colony formation. Taken together, these results suggest that nuclear localization and cleavage of STAT6 is important for modulating the viral latency and pathogenesis of KSHV.

  18. Nucleosome functions in spindle assembly and nuclear envelope formation

    PubMed Central

    Zierhut, Christian; Funabiki, Hironori

    2016-01-01

    Summary Chromosomes are not only carriers of the genetic material, but also actively regulate the assembly of complex intracellular architectures. During mitosis, chromosome-induced microtubule polymerisation ensures spindle assembly in cells without centrosomes and plays a supportive role in centrosome-containing cells. Chromosomal signals also mediate post-mitotic nuclear envelope (NE) re-formation. Recent studies using novel approaches to manipulate histones in oocytes, where functions can be analysed in the absence of transcription, have established that nucleosomes, but not DNA alone, mediate the chromosomal regulation of spindle assembly and NE formation. Both processes require the generation of RanGTP by RCC1 recruited to nucleosomes but nucleosomes also acquire cell cycle stage specific regulators, Aurora B in mitosis and ELYS, the initiator of nuclear pore complex assembly, at mitotic exit. Here, we review the mechanisms by which nucleosomes control assembly and functions of the spindle and the NE, and discuss their implications for genome maintenance. PMID:26222742

  19. Local-hybrid functional based on the correlation length

    SciTech Connect

    Johnson, Erin R.

    2014-09-28

    Local-hybrid functionals involve position-dependent mixing of Hartree-Fock and density-functional exchange, which should allow improved performance relative to conventional hybrids by reducing the inherent delocalization error and improving the long-range behaviour. Herein, the same-spin correlation length, obtained from the Fermi-hole radius, is used as the mixing parameter. The performance of the resulting local-hybrid functional is assessed for standard thermochemical and kinetics benchmarks. The local hybrid is shown to perform significantly better than the corresponding global hybrid in almost all cases.

  20. Optogenetic Control of Nuclear Protein Import in Living Cells Using Light-Inducible Nuclear Localization Signals (LINuS).

    PubMed

    Wehler, Pierre; Niopek, Dominik; Eils, Roland; Di Ventura, Barbara

    2016-06-02

    Many biological processes are regulated by the timely import of specific proteins into the nucleus. The ability to spatiotemporally control the nuclear import of proteins of interest therefore allows study of their role in a given biological process as well as controlling this process in space and time. The light-inducible nuclear localization signal (LINuS) was developed based on a natural plant photoreceptor that reversibly triggers the import of proteins of interest into the nucleus with blue light. Each LINuS is a small, genetically encoded domain that is fused to the protein of interest at the N or C terminus. These protocols describe how to carry out initial microscopy-based screening to assess which LINuS variant works best with a protein of interest. © 2016 by John Wiley & Sons, Inc.

  1. Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif

    PubMed Central

    Hernández-Sánchez, Itzell E.; Maruri-López, Israel; Ferrando, Alejandro; Carbonell, Juan; Graether, Steffen P.; Jiménez-Bremont, Juan F.

    2015-01-01

    The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization. PMID:26442018

  2. Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif.

    PubMed

    Hernández-Sánchez, Itzell E; Maruri-López, Israel; Ferrando, Alejandro; Carbonell, Juan; Graether, Steffen P; Jiménez-Bremont, Juan F

    2015-01-01

    The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization.

  3. The tobamovirus Turnip Vein Clearing Virus 30-kilodalton movement protein localizes to novel nuclear filaments to enhance virus infection.

    PubMed

    Levy, Amit; Zheng, Judy Y; Lazarowitz, Sondra G

    2013-06-01

    Plant viruses overcome the barrier of the plant cell wall by encoding cell-to-cell movement proteins (MPs), which direct newly replicated viral genomes to, and across, the wall. The paradigm for how a single MP regulates and coordinates these activities is the Tobacco mosaic virus (TMV) 30-kDa protein (MP(TMV)). Detailed studies demonstrate that TMV multiplies exclusively in the cytoplasm and have documented associations of MP(TMV) with endoplasmic reticulum (ER) membrane, microtubules, and plasmodesmata throughout the course of infection. As TMV poorly infects Arabidopsis thaliana, Turnip vein clearing virus (TVCV) is the tobamovirus of choice for studies in this model plant. A key problem, which has contributed to confusion in the field, is the unproven assumption that the TVCV and TMV life cycles are identical. We engineered an infectious TVCV replicon that expressed a functional fluorescence-tagged MP(TVCV) and report here the unexpected discovery that MP(TVCV), beyond localizing to ER membrane and plasmodesmata, targeted to the nucleus in a nuclear localization signal (NLS)-dependent manner, where it localized to novel F-actin-containing filaments that associated with chromatin. The MP(TVCV) NLS appeared to be conserved in the subgroup 3 tobamoviruses, and our mutational analyses showed that nuclear localization of MP(TVCV) was necessary for efficient TVCV cell-to-cell movement and systemic infection in Nicotiana benthamiana and Arabidopsis thaliana. Our studies identify a novel nuclear stage in TVCV infection and suggest that nuclear MP encoded by TVCV and other subgroup 3 tobamoviruses interacts with F-actin and chromatin to modulate host defenses or cellular physiology to favor virus movement and infection.

  4. Description of induced nuclear fission with Skyrme energy functionals. II. Finite temperature effects

    NASA Astrophysics Data System (ADS)

    Schunck, N.; Duke, D.; Carr, H.

    2015-03-01

    Understanding the mechanisms of induced nuclear fission for a broad range of neutron energies could help resolve fundamental science issues, such as the formation of elements in the universe, but could have also a large impact on societal applications in energy production or nuclear waste management. The goal of this paper is to set up the foundations of a microscopic theory to study the static aspects of induced fission as a function of the excitation energy of the incident neutron, from thermal to fast neutrons. To account for the high excitation energy of the compound nucleus, we employ a statistical approach based on finite temperature nuclear density functional theory with Skyrme energy densities, which we benchmark on the 239Pu(n ,f ) reaction. We compute the evolution of the least-energy fission pathway across multidimensional potential energy surfaces with up to five collective variables as a function of the nuclear temperature and predict the evolution of both the inner and the outer fission barriers as a function of the excitation energy of the compound nucleus. We show that the coupling to the continuum induced by the finite temperature is negligible in the range of neutron energies relevant for many applications of neutron-induced fission. We prove that the concept of quantum localization introduced recently can be extended to T >0 , and we apply the method to study the interaction energy and total kinetic energy of fission fragments as a function of the temperature for the most probable fission. While large uncertainties in theoretical modeling remain, we conclude that a finite temperature nuclear density functional may provide a useful framework to obtain accurate predictions of fission fragment properties.

  5. Cultivation and differentiation change nuclear localization of chromosome centromeres in human mesenchymal stem cells.

    PubMed

    Voldgorn, Yana I; Adilgereeva, Elmira P; Nekrasov, Evgeny D; Lavrov, Alexander V

    2015-01-01

    Chromosome arrangement in the interphase nucleus is not accidental. Strong evidences support that nuclear localization is an important mechanism of epigenetic regulation of gene expression. The purpose of this research was to identify differences in the localization of centromeres of chromosomes 6, 12, 18 and X in human mesenchymal stem cells depending on differentiation and cultivating time. We analyzed centromere positions in more than 4000 nuclei in 19 mesenchymal stem cell cultures before and after prolonged cultivation and after differentiation into osteogenic and adipogenic directions. We found a centromere reposition of HSAX at late passages and after differentiation in osteogenic direction as well as of HSA12 and HSA18 after adipogenic differentiation. The observed changes of the nuclear structure are new nuclear characteristics of the studied cells which may reflect regulatory changes of gene expression during the studied processes.

  6. Cultivation and Differentiation Change Nuclear Localization of Chromosome Centromeres in Human Mesenchymal Stem Cells

    PubMed Central

    Voldgorn, Yana I.; Adilgereeva, Elmira P.; Nekrasov, Evgeny D.; Lavrov, Alexander V.

    2015-01-01

    Chromosome arrangement in the interphase nucleus is not accidental. Strong evidences support that nuclear localization is an important mechanism of epigenetic regulation of gene expression. The purpose of this research was to identify differences in the localization of centromeres of chromosomes 6, 12, 18 and X in human mesenchymal stem cells depending on differentiation and cultivating time. We analyzed centromere positions in more than 4000 nuclei in 19 mesenchymal stem cell cultures before and after prolonged cultivation and after differentiation into osteogenic and adipogenic directions. We found a centromere reposition of HSAX at late passages and after differentiation in osteogenic direction as well as of HSA12 and HSA18 after adipogenic differentiation. The observed changes of the nuclear structure are new nuclear characteristics of the studied cells which may reflect regulatory changes of gene expression during the studied processes. PMID:25775427

  7. An insertion in the methyltransferase domain of P. falciparum trimethylguanosine synthase harbors a classical nuclear localization signal.

    PubMed

    Babar, Prasad H; Dey, Vishakha; Jaiswar, Praveen; Patankar, Swati

    Many Plasmodium falciparum proteins do not share homology with, and are generally longer than their respective orthologs. This, to some extent, can be attributed to insertions. Here, we studied a P. falciparum RNA hypermethylase, trimethylguanosine synthase (PfTGS1) that harbors a 76 amino acid insertion in its methyltransferase domain. Bioinformatics analysis revealed that this insertion was present in TGS1 orthologs from other Plasmodium species as well. Interestingly, a classical nuclear localization signal (NLS) was predicted in the insertions of primate parasite TGS1 proteins. To check whether these predicted NLS are functional, we developed an in vivo heterologous system using S. cerevisiae. The predicted NLS when fused to dimeric GFP were able to localize the fusion protein to the nucleus in yeast indicating that it is indeed recognized by the yeast nuclear import machinery. We further showed that the PfTGS1 NLS binds to P. falciparum importin-α in vitro, confirming that the NLS is also recognized by the P. falciparum classical nuclear import machinery. Thus, in this study we report a novel function of the insertion in PfTGS1.

  8. A conserved C-terminal domain of the Aspergillus fumigatus developmental regulator MedA is required for nuclear localization, adhesion and virulence.

    PubMed

    Al Abdallah, Qusai; Choe, Se-In; Campoli, Paolo; Baptista, Stefanie; Gravelat, Fabrice N; Lee, Mark J; Sheppard, Donald C

    2012-01-01

    MedA is a developmental regulator that is conserved in the genome of most filamentous fungi. In the pathogenic fungus Aspergillus fumigatus MedA regulates conidiogenesis, adherence to host cells, and pathogenicity. The mechanism by which MedA governs these phenotypes remains unknown. Although the nuclear import of MedA orthologues has been reported in other fungi, no nuclear localization signal, DNA-binding domain or other conserved motifs have been identified within MedA. In this work, we performed a deletion analysis of MedA and identified a novel domain within the C-terminal region of the protein, designated MedA(346-557), that is necessary and sufficient for nuclear localization of MedA. We further demonstrate that MedA nuclear localization is required for the function of MedA. Surprisingly, expression of the minimal nuclear localization fragment MedA(346-557) alone was sufficient to restore conidogenesis, biofilm formation and virulence to the medA mutant strain. Collectively these results suggest that MedA functions in the regulation of transcription, and that the MedA(346-557) domain is both necessary and sufficient to mediate MedA function.

  9. A Conserved C-Terminal Domain of the Aspergillus fumigatus Developmental Regulator MedA Is Required for Nuclear Localization, Adhesion and Virulence

    PubMed Central

    Al Abdallah, Qusai; Choe, Se-In; Campoli, Paolo; Baptista, Stefanie; Gravelat, Fabrice N.; Lee, Mark J.; Sheppard, Donald C.

    2012-01-01

    MedA is a developmental regulator that is conserved in the genome of most filamentous fungi. In the pathogenic fungus Aspergillus fumigatus MedA regulates conidiogenesis, adherence to host cells, and pathogenicity. The mechanism by which MedA governs these phenotypes remains unknown. Although the nuclear import of MedA orthologues has been reported in other fungi, no nuclear localization signal, DNA-binding domain or other conserved motifs have been identified within MedA. In this work, we performed a deletion analysis of MedA and identified a novel domain within the C-terminal region of the protein, designated MedA346–557, that is necessary and sufficient for nuclear localization of MedA. We further demonstrate that MedA nuclear localization is required for the function of MedA. Surprisingly, expression of the minimal nuclear localization fragment MedA346–557 alone was sufficient to restore conidogenesis, biofilm formation and virulence to the medA mutant strain. Collectively these results suggest that MedA functions in the regulation of transcription, and that the MedA346–557 domain is both necessary and sufficient to mediate MedA function. PMID:23185496

  10. The N-terminal domain of the androgen receptor drives its nuclear localization in castration-resistant prostate cancer cells

    PubMed Central

    Dar, Javid A.; Masoodi, Khalid Z.; Eisermann, Kurtis; Isharwal, Sudhir; Ai, Junkui; Pascal, Laura E.; Nelson, Joel B.; Wang, Zhou

    2014-01-01

    Androgen-independent nuclear localization is required for androgen receptor (AR) transactivation in castration-resistant prostate cancer (CRPC) and should be a key step leading to castration resistance. However, mechanism(s) leading to androgen-independent AR nuclear localization are poorly understood. Since the N-terminal domain (NTD) of AR plays a role in transactivation under androgen-depleted conditions, we investigated the role of NTD in AR nuclear localization in CRPC. Deletion mutagenesis was used to identify amino acid sequences in the NTD essential for its androgen-independent nuclear localization in C4-2, a widely used CRPC cell line. Deletion mutants of AR tagged with green fluorescent protein (GFP) at the 5`-end were generated and their signal distribution was investigated in C4-2 cells by fluorescent microscopy. Our results showed that the region of a.a. 294–556 was required for androgen-independent AR nuclear localization whereas a.a. 1–293 mediates Hsp90 regulation of AR nuclear localization in CRPC cells. Although a.a. 294–556 does not contain a nuclear import signal, it was able to enhance DHT-induced import of the ligand binding domain (LBD). Also, transactivation of the NTD could be uncoupled from its modulation of AR nuclear localization in C4-2 cells. These observations suggest an important role of NTD in AR intracellular trafficking and androgen-independent AR nuclear localization in CRPC cells. PMID:24662325

  11. Structural basis for the regulation of nuclear import of Epstein-Barr virus nuclear antigen 1 (EBNA1) by phosphorylation of the nuclear localization signal.

    PubMed

    Nakada, Ryohei; Hirano, Hidemi; Matsuura, Yoshiyuki

    2017-02-26

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is expressed in every EBV-positive tumor and is essential for the maintenance, replication, and transcription of the EBV genome in the nucleus of host cells. EBNA1 is a serine phosphoprotein, and it has been shown that phosphorylation of S385 in the nuclear localization signal (NLS) of EBNA1 increases the binding affinity to the nuclear import adaptor importin-α1 as well as importin-α5, and stimulates nuclear import of EBNA1. To gain insights into how phosphorylation of the EBNA1 NLS regulates nuclear import, we have determined the crystal structures of two peptide complexes of importin-α1: one with S385-phosphorylated EBNA1 NLS peptide, determined at 2.0 Å resolution, and one with non-phosphorylated EBNA1 NLS peptide, determined at 2.2 Å resolution. The structures show that EBNA1 NLS binds to the major and minor NLS-binding sites of importin-α1, and indicate that the binding affinity of the EBNA1 NLS to the minor NLS-binding site could be enhanced by phosphorylation of S385 through electrostatic interaction between the phosphate group of phospho-S385 and K392 of importin-α1 (corresponding to R395 of importin-α5) on armadillo repeat 8.

  12. Mutational analysis of Mdm1p function in nuclear and mitochondrial inheritance.

    PubMed

    Fisk, H A; Yaffe, M P

    1997-08-11

    Nuclear and mitochondrial transmission to daughter buds of Saccharomyces cerevisiae depends on Mdm1p, an intermediate filament-like protein localized to numerous punctate structures distributed throughout the yeast cell cytoplasm. These structures disappear and organelle inheritance is disrupted when mdm1 mutant cells are incubated at the restrictive temperature. To characterize further the function of Mdm1p, new mutant mdm1 alleles that confer temperature-sensitive growth and defects in organelle inheritance but produce stable Mdm1p structures were isolated. Microscopic analysis of the new mdm1 mutants revealed three phenotypic classes: Class I mutants showed defects in both mitochondrial and nuclear transmission; Class II alleles displayed defective mitochondrial inheritance but had no effect on nuclear movement; and Class III mutants showed aberrant nuclear inheritance but normal mitochondrial distribution. Class I and II mutants also exhibited altered mitochondrial morphology, possessing primarily small, round mitochondria instead of the extended tubular structures found in wild-type cells. Mutant mdm1 alleles affecting nuclear transmission were of two types: Class Ia and IIIa mutants were deficient for nuclear movement into daughter buds, while Class Ib and IIIb mutants displayed a complete transfer of all nuclear DNA into buds. The mutations defining all three allelic classes mapped to two distinct domains within the Mdm1p protein. Genetic crosses of yeast strains containing different mdm1 alleles revealed complex genetic interactions including intragenic suppression, synthetic phenotypes, and intragenic complementation. These results support a model of Mdm1p function in which a network comprised of multimeric assemblies of the protein mediates two distinct cellular processes.

  13. Elevated copper impairs hepatic nuclear receptor function in Wilson's disease.

    PubMed

    Wooton-Kee, Clavia Ruth; Jain, Ajay K; Wagner, Martin; Grusak, Michael A; Finegold, Milton J; Lutsenko, Svetlana; Moore, David D

    2015-09-01

    Wilson's disease (WD) is an autosomal recessive disorder that results in accumulation of copper in the liver as a consequence of mutations in the gene encoding the copper-transporting P-type ATPase (ATP7B). WD is a chronic liver disorder, and individuals with the disease present with a variety of complications, including steatosis, cholestasis, cirrhosis, and liver failure. Similar to patients with WD, Atp7b⁻/⁻ mice have markedly elevated levels of hepatic copper and liver pathology. Previous studies have demonstrated that replacement of zinc in the DNA-binding domain of the estrogen receptor (ER) with copper disrupts specific binding to DNA response elements. Here, we found decreased binding of the nuclear receptors FXR, RXR, HNF4α, and LRH-1 to promoter response elements and decreased mRNA expression of nuclear receptor target genes in Atp7b⁻/⁻ mice, as well as in adult and pediatric WD patients. Excessive hepatic copper has been described in progressive familial cholestasis (PFIC), and we found that similar to individuals with WD, patients with PFIC2 or PFIC3 who have clinically elevated hepatic copper levels exhibit impaired nuclear receptor activity. Together, these data demonstrate that copper-mediated nuclear receptor dysfunction disrupts liver function in WD and potentially in other disorders associated with increased hepatic copper levels.

  14. Local motifs involved in the canonical structure of the ligand-binding domain in the nuclear receptor superfamily.

    PubMed

    Tsuji, Motonori

    2014-03-01

    Structural and sequence alignment analyses have revealed the existence of class-dependent and -independent local motifs involved in the overall fold of the ligand-binding domain (LBD) in the nuclear receptor (NR) superfamily. Of these local motifs, three local motifs, i.e., AF-2 fixed motifs, were involved in the agonist conformation of the activation function-2 (AF-2) region of the LBD. Receptor-agonist interactions increased the stability of these AF-2 fixed motifs in the agonist conformation. In contrast, perturbation of the AF-2 fixed motifs by a ligand or another protein molecule led the AF-2 architecture to adopt an antagonist conformation. Knowledge of this process should provide us with novel insights into the 'agonism' and 'antagonism' of NRs.

  15. Local field distribution near corrugated interfaces: Green's function formulation

    NASA Astrophysics Data System (ADS)

    Yu, K. W.; Wan, Jones T. K.

    2001-12-01

    We have developed a Green's function formalism to compute the local field distribution near an interface separating two media of different dielectric constants. The Maxwell's equations are converted into a surface integral equation; thus it greatly simplifies the solutions and yields accurate results for interfaces of arbitrary shape. The integral equation is solved and the local field distribution is obtained for a periodic interface.

  16. The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress.

    PubMed

    Kuan, Man I; O'Dowd, John M; Fortunato, Elizabeth A

    2016-10-01

    Our electron microscopy study (Kuan et al., 2016) found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introduction of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion.

  17. fMRI alignment based on local functional connectivity patterns

    NASA Astrophysics Data System (ADS)

    Jiang, Di; Du, Yuhui; Cheng, Hewei; Jiang, Tianzi; Fan, Yong

    2012-02-01

    In functional neuroimaging studies, the inter-subject alignment of functional magnetic resonance imaging (fMRI) data is a necessary precursor to improve functional consistency across subjects. Traditional structural MRI based registration methods cannot achieve accurate inter-subject functional consistency in that functional units are not necessarily consistently located relative to anatomical structures due to functional variability across subjects. Although spatial smoothing commonly used in fMRI data preprocessing can reduce the inter-subject functional variability, it may blur the functional signals and thus lose the fine-grained information. In this paper we propose a novel functional signal based fMRI image registration method which aligns local functional connectivity patterns of different subjects to improve the inter-subject functional consistency. Particularly, the functional connectivity is measured using Pearson correlation. For each voxel of an fMRI image, its functional connectivity to every voxel in its local spatial neighborhood, referred to as its local functional connectivity pattern, is characterized by a rotation and shift invariant representation. Based on this representation, the spatial registration of two fMRI images is achieved by minimizing the difference between their corresponding voxels' local functional connectivity patterns using a deformable image registration model. Experiment results based on simulated fMRI data have demonstrated that the proposed method is more robust and reliable than the existing fMRI image registration methods, including maximizing functional correlations and minimizing difference of global connectivity matrices across different subjects. Experiment results based on real resting-state fMRI data have further demonstrated that the proposed fMRI registration method can statistically significantly improve functional consistency across subjects.

  18. Surface localization of the nuclear receptor CAR in influenza A virus-infected cells

    SciTech Connect

    Takahashi, Tadanobu; Moriyama, Yusuke; Ikari, Akira; Sugatani, Junko; Suzuki, Takashi; Miwa, Masao

    2008-04-11

    Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism.

  19. Do nuclear collisions create a locally equilibrated quark-gluon plasma?

    NASA Astrophysics Data System (ADS)

    Romatschke, P.

    2017-01-01

    Experimental results on azimuthal correlations in high energy nuclear collisions (nucleus-nucleus, proton-nucleus, and proton-proton) seem to be well described by viscous hydrodynamics. It is often argued that this agreement implies either local thermal equilibrium or at least local isotropy. In this note, I present arguments why this is not the case. Neither local near-equilibrium nor near-isotropy are required in order for hydrodynamics to offer a successful and accurate description of experimental results. However, I predict the breakdown of hydrodynamics at momenta of order seven times the temperature, corresponding to a smallest possible QCD liquid drop size of 0.15 fm.

  20. Nuclear localization of Rad51B is independent of BRCA2

    SciTech Connect

    Miller, K A; Hinz, J M; Yamada, A; Thompson, L H; Albala, J S

    2005-06-28

    Human Rad51 is critical for the maintenance of genome stability through its role in the repair of DNA double-strand breaks. Rad51B (Rad51L1/hRec2) is one of the five known paralogs of human Rad51 found in a multi-protein complex with three other Rad51 paralogs, Rad51C, Rad51D and Xrcc2. Examination of EGFP-Rad51B fusion protein in HeLa S3 cells and immunofluorescence in several human cell lines confirms the nuclear localization of Rad51B. This is the first report to detail putative interactions of a Rad51 paralog protein with BRCA2. Utilization of a BRCA2 mutant cell line, CAPAN-1 suggests that Rad51B localizes to the nucleus independent of BRCA2. Although both Rad51B and BRCA2 are clearly involved in the homologous recombinational repair pathway, Rad51B and BRCA2 do not appear to associate directly. Furthermore, mutations in the KKLK motif of Rad51B, amino acid residues 4-7, mislocalizes Rad51B to the cytoplasm suggesting that this is the nuclear localization signal for the Rad51B protein. Examination of wild-type EGFP-Rad51B fusion protein in mammalian cells deficient in Rad51C showed that Rad51B localizes to the nucleus independent of Rad51C; further suggesting that Rad51B, like Rad51C, contains its own nuclear localization signal.

  1. Local Function Conservation in Sequence and Structure Space

    PubMed Central

    Weinhold, Nils; Sander, Oliver; Domingues, Francisco S.; Lengauer, Thomas; Sommer, Ingolf

    2008-01-01

    We assess the variability of protein function in protein sequence and structure space. Various regions in this space exhibit considerable difference in the local conservation of molecular function. We analyze and capture local function conservation by means of logistic curves. Based on this analysis, we propose a method for predicting molecular function of a query protein with known structure but unknown function. The prediction method is rigorously assessed and compared with a previously published function predictor. Furthermore, we apply the method to 500 functionally unannotated PDB structures and discuss selected examples. The proposed approach provides a simple yet consistent statistical model for the complex relations between protein sequence, structure, and function. The GOdot method is available online (http://godot.bioinf.mpi-inf.mpg.de). PMID:18604264

  2. Local function conservation in sequence and structure space.

    PubMed

    Weinhold, Nils; Sander, Oliver; Domingues, Francisco S; Lengauer, Thomas; Sommer, Ingolf

    2008-07-04

    We assess the variability of protein function in protein sequence and structure space. Various regions in this space exhibit considerable difference in the local conservation of molecular function. We analyze and capture local function conservation by means of logistic curves. Based on this analysis, we propose a method for predicting molecular function of a query protein with known structure but unknown function. The prediction method is rigorously assessed and compared with a previously published function predictor. Furthermore, we apply the method to 500 functionally unannotated PDB structures and discuss selected examples. The proposed approach provides a simple yet consistent statistical model for the complex relations between protein sequence, structure, and function. The GOdot method is available online (http://godot.bioinf.mpi-inf.mpg.de).

  3. A novel bipartite nuclear localization signal with an atypically long linker in DOF transcription factors.

    PubMed

    Krebs, Jonas; Mueller-Roeber, Bernd; Ruzicic, Slobodan

    2010-05-01

    Large molecules require a nuclear localization signal (NLS) for translocation into the nucleus. Classical NLSs are rich in basic amino acids and they represent three groups, based on their structural features: SV40 T-antigen-type, yeast mating factor Matalpha-2-type, and bipartite NLSs. DNA-binding-with-one-finger (DOF) transcription factors play important roles in plants, and although their nuclear localization has been demonstrated in several cases, public protein localization prediction tools fail to detect NLS motifs in these proteins. Here, we demonstrate that an atypical bipartite NLS with a 17 amino acid long linker between its flanking basic regions directs Arabidopsis thaliana DOF proteins to the cell nucleus. The novel bipartite NLS is highly conserved in plant DOF transcription factors, including the single DOF protein in the green alga Chlamydomonas reinhardtii.

  4. Nuclear charge and neutron radii and nuclear matter: Trend analysis in Skyrme density-functional-theory approach

    NASA Astrophysics Data System (ADS)

    Reinhard, P.-G.; Nazarewicz, W.

    2016-05-01

    Background: Radii of charge and neutron distributions are fundamental nuclear properties. They depend on both nuclear interaction parameters related to the equation of state of infinite nuclear matter and on quantal shell effects, which are strongly impacted by the presence of nuclear surface. Purpose: In this work, by studying the correlation of charge and neutron radii, and neutron skin, with nuclear matter parameters, we assess different mechanisms that drive nuclear sizes. Method: We apply nuclear density functional theory using a family of Skyrme functionals obtained by means of optimization protocols, which do not include any radius information. By performing the Monte Carlo sampling of reasonable functionals around the optimal parametrization, we scan all correlations between nuclear matter properties and observables characterizing charge and neutron distributions of spherical closed-shell nuclei 48Ca,208Pb, and 298Fl. Results: By considering the influence of various nuclear matter properties on charge and neutron radii in a multidimensional parameter space of Skyrme functionals, we demonstrate the existence of two strong relationships: (i) between the nuclear charge radii and the saturation density of symmetric nuclear matter ρ0, and (ii) between the neutron skins and the slope of the symmetry energy L . The impact of other nuclear matter properties on nuclear radii is weak or nonexistent. For functionals optimized to experimental binding energies only, proton and neutron radii are found to be weakly correlated due to canceling trends from different nuclear matter characteristics. Conclusion: The existence of only two strong relations connecting nuclear radii with nuclear matter properties has important consequences. First, by requiring that the nuclear functional reproduces the empirical saturation point of symmetric nuclear matter practically fixes the charge (or proton) radii, and vice versa. This explains the recent results of ab initio calculations

  5. Functional coupling between the extracellular matrix and nuclear lamina by Wnt signaling in progeria.

    PubMed

    Hernandez, Lidia; Roux, Kyle J; Wong, Esther Sook Miin; Mounkes, Leslie C; Mutalif, Rafidah; Navasankari, Raju; Rai, Bina; Cool, Simon; Jeong, Jae-Wook; Wang, Honghe; Lee, Hyun-Shik; Kozlov, Serguei; Grunert, Martin; Keeble, Thomas; Jones, C Michael; Meta, Margarita D; Young, Stephen G; Daar, Ira O; Burke, Brian; Perantoni, Alan O; Stewart, Colin L

    2010-09-14

    The segmental premature aging disease Hutchinson-Gilford Progeria (HGPS) is caused by a truncated and farnesylated form of Lamin A. In a mouse model for HGPS, a similar Lamin A variant causes the proliferative arrest and death of postnatal, but not embryonic, fibroblasts. Arrest is due to an inability to produce a functional extracellular matrix (ECM), because growth on normal ECM rescues proliferation. The defects are associated with inhibition of canonical Wnt signaling, due to reduced nuclear localization and transcriptional activity of Lef1, but not Tcf4, in both mouse and human progeric cells. Defective Wnt signaling, affecting ECM synthesis, may be critical to the etiology of HGPS because mice exhibit skeletal defects and apoptosis in major blood vessels proximal to the heart. These results establish a functional link between the nuclear envelope/lamina and the cell surface/ECM and may provide insights into the role of Wnt signaling and the ECM in aging.

  6. Visualization of nuclear localization of transcription factors with cyan and green fluorescent proteins in the red alga Porphyra yezoensis.

    PubMed

    Uji, Toshiki; Takahashi, Megumu; Saga, Naotsune; Mikami, Koji

    2010-04-01

    Transcription factors play a central role in expression of genomic information in all organisms. The objective of our study is to analyze the function of transcription factors in red algae. One way to analyze transcription factors in eukaryotic cells is to study their nuclear localization, as reported for land plants and green algae using fluorescent proteins. There is, however, no report documenting subcellular localization of transcription factors from red algae. In the present study, using the marine red alga Porphyra yezoensis, we confirmed for the first time successful expression of humanized fluorescent proteins (ZsGFP and ZsYFP) from a reef coral Zoanthus sp. and land plant-adapted sGFP(S65T) in gametophytic cells comparable to expression of AmCFP. Following molecular cloning and characterization of transcription factors DP-E2F-like 1 (PyDEL1), transcription elongation factor 1 (PyElf1) and multiprotein bridging factor 1 (PyMBF1), we then demonstrated that ZsGFP and AmCFP can be used to visualize nuclear localization of PyElf1 and PyMBF1. This is the first report to perform visualization of subcellular localization of transcription factors as genome-encoded proteins in red algae.

  7. Localization of human T-cell lymphotropic virus type II Tax protein is dependent upon a nuclear localization determinant in the N-terminal region.

    PubMed

    Turci, Marco; Romanelli, Maria Grazia; Lorenzi, Pamela; Righi, Paola; Bertazzoni, Umberto

    2006-01-03

    Human T-cell lymphotropic viruses (HTLV) types I and II are closely related oncogenic retroviruses that have been associated with lymphoproliferative and neurological disorders. The proviral genome encodes a trans-regulatory Tax protein that activates viral genes and upregulates various cellular genes involved in both cell growth and transformation. Tax proteins of HTLV-I (Tax-I) and HTLV-II (Tax-II) exhibit more than 77% aa homology and expression of either Tax-I or Tax-II is sufficient for immortalization of cultured T lymphocytes. Tax-I shuttles from the nucleus to the cytoplasm and accumulates within the nucleus, whereas Tax-II is found mainly in the cytoplasm. In the present study we have used recombinant vectors to analyze the size and structure of the nuclear localization domain within the Tax-II protein sequence. The Tax-II protein was expressed in HeLa cells either as the complete protein, or regions thereof, that were individually fused to the green fluorescent protein (GFP). Immunoblot analysis of the fused Tax-II products confirmed their expression and size. Fluorescence microscopy studies indicated that the complete Tax-II as well as N-truncated forms presented a punctuate cytoplasmic distribution and that a nuclear localization determinant is confined to within the first 60 aa of Tax-II. Accordingly, site directed mutagenesis and deletion of specific sequences within the first 60 aa showed that the nuclear determinant lies within the first 41 residues of Tax-II. These results point to a direct involvement of the amino-terminal residues of Tax-II protein in determining its nuclear functionality.

  8. Characterization of a nuclear localization signal of canine parvovirus capsid proteins.

    PubMed

    Vihinen-Ranta, M; Kakkola, L; Kalela, A; Vilja, P; Vuento, M

    1997-12-01

    We investigated the abilities of synthetic peptides mimicking the potential nuclear localization signal of canine parvovirus (CPV) capsid proteins to translocate a carrier protein to the nucleus following microinjection into the cytoplasm of A72 cells. Possible nuclear localization sequences were chosen for synthesis from CPV capsid protein sequences (VP1, VP2) on the basis of the presence of clustered basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear targeting activity was found within the N-terminal residues 4-13 (PAKRARRGYK) of the VP1 capsid protein. While replacement of Arg10 with glycine did not affect the activity, replacement of Lys6, Arg7, or Arg9 with glycine abolished it. The targeting activity was found to residue in a cluster of basic residues, Lys5, Arg7, and Arg9. Nuclear import was saturated by excess of unlabelled peptide conjugates (showing that it was a receptor-mediated process). Transport into the nucleus was an energy-dependent and temperature-dependent process actively mediated by the nuclear pores and inhibited by wheat germ agglutinin.

  9. Nuclear chiral and magnetic rotation in covariant density functional theory

    NASA Astrophysics Data System (ADS)

    Meng, Jie; Zhao, Pengwei

    2016-05-01

    Excitations of chiral rotation observed in triaxial nuclei and magnetic and/or antimagnetic rotations (AMR) seen in near-spherical nuclei have attracted a lot of attention. Unlike conventional rotation in well-deformed or superdeformed nuclei, here the rotational axis is not necessary coinciding with any principal axis of the nuclear density distribution. Thus, tilted axis cranking (TAC) is mandatory to describe these excitations self-consistently in the framework of covariant density functional theory (CDFT). We will briefly introduce the formalism of TAC-CDFT and its application for magnetic and AMR phenomena. Configuration-fixed CDFT and its predictions for nuclear chiral configurations and for favorable triaxial deformation parameters are also presented, and the discoveries of the multiple chiral doublets in 133Ce and 103Rh are discussed.

  10. Emerging functional roles of nuclear receptors in breast cancer.

    PubMed

    Doan, Tram B; Graham, J Dinny; Clarke, Christine L

    2017-04-01

    Nuclear receptors (NRs) have been targets of intensive drug development for decades due to their roles as key regulators of multiple developmental, physiological and disease processes. In breast cancer, expression of the estrogen and progesterone receptor remains clinically important in predicting prognosis and determining therapeutic strategies. More recently, there is growing evidence supporting the involvement of multiple nuclear receptors other than the estrogen and progesterone receptors, in the regulation of various processes important to the initiation and progression of breast cancer. We review new insights into the mechanisms of action of NRs made possible by recent advances in genomic technologies and focus on the emerging functional roles of NRs in breast cancer biology, including their involvement in circadian regulation, metabolic reprogramming and breast cancer migration and metastasis.

  11. Characterization of the nuclear localization signal of high risk HPV16 E2 protein

    SciTech Connect

    Klucevsek, Kristin; Wertz, Mary; Lucchi, John; Leszczynski, Anna; Moroianu, Junona . E-mail: moroianu@bc.edu

    2007-03-30

    The E2 protein of high risk human papillomavirus type 16 (HPV16) contains an amino-terminal (N) domain, a hinge (H) region and a carboxyl-terminal (C) DNA-binding domain. Using enhanced green fluorescent protein (EGFP) fusions with full length E2 and E2 domains in transfection assays in HeLa cells, we found that the C domain is responsible for the nuclear localization of E2 in vivo, whereas the N and H domains do not contain additional nuclear localization signals (NLSs). Deletion analysis of EGFP-E2 and EGFP-cE2 determined that the C domain contains an {alpha} helix cNLS that overlaps with the DNA-binding region. Mutational analysis revealed that the arginine and lysine residues in this cNLS are essential for nuclear localization of HPV16 E2. Interestingly, these basic amino acid residues are well conserved among the E2 proteins of BPV-1 and some high risk HPV types but not in the low risk HPV types, suggesting that there are differences between the NLSs and corresponding nuclear import pathways between these E2 proteins.

  12. How do electron localization functions describe π-electron delocalization?

    PubMed

    Steinmann, Stephan N; Mo, Yirong; Corminboeuf, Clemence

    2011-12-14

    Scalar fields provide an intuitive picture of chemical bonding. In particular, the electron localization function (ELF) has proven to be highly valuable in interpreting a broad range of bonding patterns. The discrimination between enhanced or reduced electron (de)localization within cyclic π-conjugated systems remains, however, challenging for ELF. In order to clearly distinguish between the local properties of ten highly and weakly π-(de)localized prototype systems, we compare the ELFs of both the canonical wave functions and electron-localized states (diabatic) with those of two closely related scalar fields: the electron localizability indicator (ELI-D) and the localized orbital locator (LOL). The simplest LOL function distinguishes enhanced from weak π-(de)localization in an insightful and reliable manner. LOL offers the finest contrast between annulenes with 4n/4n + 2 π electrons and their inorganic analogues as well as between hyperconjugated cyclopentadiene derivatives. LOL(π) also gives an appealing and intuitive picture of the π-bond. In contrast, the most popular ELF fails to capture subtle contrasting local electronic properties and suffers from the arbitrariness of the σ/π dissection. The orbital separation of the most recent ELI-D is clear-cut but the interpretations sometime less straightforward in the present context.

  13. EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    PubMed Central

    Reina, Jeffrey; Morais Freitas, Vanessa

    2016-01-01

    Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally

  14. Production of ABA responses requires both the nuclear and cytoplasmic functional involvement of PYR1.

    PubMed

    Park, EunJoo; Kim, Tae-Houn

    2017-02-26

    Abscisic acid (ABA) enhances stress tolerant responses in plants against unfavorable environmental conditions. In Arabidopsis, ABA promotes interactions between PYR/PYL/RCARs and PP2C, thereby allowing SnRK2s to phosphorylate downstream components required for the regulation of gene expression or for gating ion channels. Because PYR1 is known to localize to nucleus and cytoplasm it is a question whether nuclear or cytoplasmic PYR1 confer different functions to the ABA signaling pathway, as has been previously shown for regulatory proteins. In order to answer this question, transgenic lines expressing nuclear PYR1 were generated in an ABA insensitive mutant background. Enforced nuclear expression of PYR1 was examined by confocal microscopy and western blot analysis. Physiological analyses of the transgenic lines demonstrated that nuclear PYR1 is sufficient to generate ABA responses, such as, the inhibition of seed germination, root growth inhibition, the induction of gene expression, and stomatal closing movement. However, for the full recovery of ABA responses in the mutant background cytoplasmic PYR1 was required. The study suggests both nuclear and cytoplasmic PYR1 participate in the control of ABA signal transduction.

  15. Characterization of the subcellular localization and nuclear import molecular mechanisms of herpes simplex virus 1 UL2.

    PubMed

    Cai, Mingsheng; Huang, Zebin; Liao, Zongmin; Chen, Tao; Wang, Ping; Jiang, Si; Chen, Daixiong; Peng, Tao; Bian, Yun; Hong, Gengde; Yang, Hang; Zeng, Zhancheng; Li, Xiaowei; Li, Meili

    2017-04-01

    As a crucial protein, the herpes simplex virus 1 (HSV-1) UL2 protein has been shown to take part in various stages of viral infection, nonetheless, its exact subcellular localization and transport molecular determinants are not well known thus far. In the present study, by using live cells fluorescent microscopy assay, UL2 tagged with enhanced yellow fluorescent protein was transiently expressed in live cells and showed a completely nuclear accumulation without the presence of other HSV-1 proteins. Moreover, the nuclear transport of UL2 was characterized to be assisted by multiple transport pathways through Ran-, importin α1-, α5-, α7-, β1- and transportin-1 cellular transport receptors. Consequently, these results will improve understanding of UL2-mediated biological functions in HSV-1 infection cycles.

  16. Structure of importin-α bound to a non-classical nuclear localization signal of the influenza A virus nucleoprotein.

    PubMed

    Nakada, Ryohei; Hirano, Hidemi; Matsuura, Yoshiyuki

    2015-10-12

    A non-classical nuclear localization signal (ncNLS) of influenza A virus nucleoprotein (NP) is critical for nuclear import of viral genomic RNAs that transcribe and replicate in the nucleus of infected cells. Here we report a 2.3 Å resolution crystal structure of mouse importin-α1 in complex with NP ncNLS. The structure reveals that NP ncNLS binds specifically and exclusively to the minor NLS-binding site of importin-α. Structural and functional analyses identify key binding pockets on importin-α as potential targets for antiviral drug development. Unlike many other NLSs, NP ncNLS binds to the NLS-binding domain of importin-α weakly with micromolar affinity. These results suggest that a modest inhibitor with low affinity to importin-α could have anti-influenza activity with minimal cytotoxicity.

  17. Oscillator strengths, first-order properties, and nuclear gradients for local ADC(2)

    SciTech Connect

    Schütz, Martin

    2015-06-07

    We describe theory and implementation of oscillator strengths, orbital-relaxed first-order properties, and nuclear gradients for the local algebraic diagrammatic construction scheme through second order. The formalism is derived via time-dependent linear response theory based on a second-order unitary coupled cluster model. The implementation presented here is a modification of our previously developed algorithms for Laplace transform based local time-dependent coupled cluster linear response (CC2LR); the local approximations thus are state specific and adaptive. The symmetry of the Jacobian leads to considerable simplifications relative to the local CC2LR method; as a result, a gradient evaluation is about four times less expensive. Test calculations show that in geometry optimizations, usually very similar geometries are obtained as with the local CC2LR method (provided that a second-order method is applicable). As an exemplary application, we performed geometry optimizations on the low-lying singlet states of chlorophyllide a.

  18. Oscillator strengths, first-order properties, and nuclear gradients for local ADC(2)

    NASA Astrophysics Data System (ADS)

    Schütz, Martin

    2015-06-01

    We describe theory and implementation of oscillator strengths, orbital-relaxed first-order properties, and nuclear gradients for the local algebraic diagrammatic construction scheme through second order. The formalism is derived via time-dependent linear response theory based on a second-order unitary coupled cluster model. The implementation presented here is a modification of our previously developed algorithms for Laplace transform based local time-dependent coupled cluster linear response (CC2LR); the local approximations thus are state specific and adaptive. The symmetry of the Jacobian leads to considerable simplifications relative to the local CC2LR method; as a result, a gradient evaluation is about four times less expensive. Test calculations show that in geometry optimizations, usually very similar geometries are obtained as with the local CC2LR method (provided that a second-order method is applicable). As an exemplary application, we performed geometry optimizations on the low-lying singlet states of chlorophyllide a.

  19. A Local and Global Function Model of the Liver

    PubMed Central

    Wang, Hesheng; Feng, Mary; Jackson, Andrew; Ten Haken, Randall K.; Lawrence, Theodore S.; Cao, Yue

    2015-01-01

    Purposes To develop a local and global function model in the liver based upon regional and organ function measurements to support individualized adaptive radiation therapy (RT). Methods and Materials A local and global model for liver function was developed to include both functional volume and the effect of functional variation of subunits. Adopting the assumption of parallel architecture in the liver, the global function was composed of a sum of local function probabilities of subunits, varying between 0 and 1. The model was fit to 59 datasets of liver regional and organ function measures from 23 patients obtained prior to, during and 1 month after RT. The local function probabilities of subunits were modeled by a sigmoid function in relating to MRI-derived portal venous perfusion values. The global function was fitted to a logarithm of an indocyanine green retention rate at 15 min (an overall liver function measure). Cross-validation was performed by leave-m-out tests. The model was further evaluated by fitting to the data divided based upon whether the patients had hepatocellular carcinoma (HCC) or not. Results The liver function model showed that 1) a perfusion value of 68.6 ml/(100g·min) yielded a local function probability of 0.5; 2) the probability reached 0.9 at a perfusion value of 98 ml/(100g·min), and 3) at a probability of 0.03 (corresponding perfusion of 38 ml/(100g·min)) or lower, the contribution to global function was lost. Cross-validations showed that the model parameters were stable. The model fitted to the data from the patients with HCC indicated that the same amount of portal venous perfusion was translated into less local function probability than the patients with non-HCC tumors. Conclusions The developed liver function model could provide a means to better assess individual and regional dose responses of hepatic functions, and provide guidance for individualized treatment planning of RT. PMID:26700712

  20. Nuclear localization of the tight junction protein ZO-2 in epithelial cells.

    PubMed

    Islas, Socorro; Vega, Jesús; Ponce, Lissette; González-Mariscal, Lorenza

    2002-03-10

    The tight junction constitutes the major barrier to solute and water flow through the paracellular space of epithelia and endothelia. It is formed by transmembrane proteins and submembranous molecules such as the MAGUKs ZOs. We have previously found that several MAGUKs, including those of the tight (ZO-1, ZO-2, and ZO-3) and septate junction (tamou and Dlg), contain one or two nuclear sorting signals located at their first PDZ and GK domains. Now we show that these proteins also contain a nuclear export signal and focus our study on the nuclear membrane shuttling of ZO-2. In sparse cultures this molecule concentrates at the nucleus in clusters, where it partially colocalizes with splicing factor SC35. Nuclear staining diminishes as the monolayer acquires confluence through a process sensitive to the nuclear export inhibitor leptomycin B. Nuclear localization can be induced by impairing cell-cell contacts, by mechanical injury. ZO-2 that shuttles from the cell periphery into the nucleus is not newly synthesized but originates from a preexistent pool. The movement of this protein is mediated by the actin cytoskeleton.

  1. Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly

    SciTech Connect

    Tannukit, Sissada; Crabb, Tara L.; Hertel, Klemens J.; Wen, Xin; Jans, David A.; Paine, Michael L.

    2009-12-18

    Tuftelin-interacting protein 11 (TFIP11) is a protein component of the spliceosome complex that promotes the release of the lariat-intron during late-stage splicing through a direct recruitment and interaction with DHX15/PRP43. Expression of TFIP11 is essential for cell and organismal survival. TFIP11 contains a G-patch domain, a signature motif of RNA-processing proteins that is responsible for TFIP11-DHX15 interactions. No other functional domains within TFIP11 have been described. TFIP11 is localized to distinct speckled regions within the cell nucleus, although excluded from the nucleolus. In this study sequential C-terminal deletions and mutational analyses have identified two novel protein elements in mouse TFIP11. The first domain covers amino acids 701-706 (VKDKFN) and is an atypical nuclear localization signal (NLS). The second domain is contained within amino acids 711-735 and defines TFIP11's distinct speckled nuclear localization. The identification of a novel TFIP11 nuclear speckle-targeting sequence (TFIP11-STS) suggests that this domain directly interacts with additional spliceosomal components. These data help define the mechanism of nuclear/nuclear speckle localization of the splicing factor TFIP11, with implications for it's function.

  2. Novel Nuclear Localization of Fatty Acid Synthase Correlates with Prostate Cancer Aggressiveness

    PubMed Central

    Madigan, Allison A.; Rycyna, Kevin J.; Parwani, Anil V.; Datiri, Yeipyeng J.; Basudan, Ahmed M.; Sobek, Kathryn M.; Cummings, Jessica L.; Basse, Per H.; Bacich, Dean J.; O'Keefe, Denise S.

    2015-01-01

    Fatty acid synthase is up-regulated in a variety of cancers, including prostate cancer. Up-regulation of fatty acid synthase not only increases production of fatty acids in tumors but also contributes to the transformed phenotype by conferring growth and survival advantages. In addition, increased fatty acid synthase expression in prostate cancer correlates with poor prognosis, although the mechanism(s) by which this occurs are not completely understood. Because fatty acid synthase is expressed at low levels in normal cells, it is currently a major target for anticancer drug design. Fatty acid synthase is normally found in the cytosol; however, we have discovered that it also localizes to the nucleus in a subset of prostate cancer cells. Analysis of the fatty acid synthase protein sequence indicated the presence of a nuclear localization signal, and subcellular fractionation of LNCaP prostate cancer cells, as well as immunofluorescent confocal microscopy of patient prostate tumor tissue and LNCaPs confirmed nuclear localization of this protein. Finally, immunohistochemical analysis of prostate cancer tissue indicated that nuclear localization of fatty acid synthase correlates with Gleason grade, implicating a potentially novel role in prostate cancer progression. Possible clinical implications include improving the accuracy of prostate biopsies in the diagnosis of low- versus intermediate-risk prostate cancer and the uncovering of novel metabolic pathways for the therapeutic targeting of androgen-independent prostate cancer. PMID:24907642

  3. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    PubMed

    Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

    2011-01-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

  4. Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization

    SciTech Connect

    Kim, Chung Kwon; Lee, Sang Bae; Nguyen, Truong L.X.; Lee, Kyung-Hoon; Um, Sung Hee; Kim, Jihoe; Ahn, Jee-Yin

    2012-01-15

    p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.

  5. Nuclear pore proteins are involved in the biogenesis of functional tRNA.

    PubMed Central

    Simos, G; Tekotte, H; Grosjean, H; Segref, A; Sharma, K; Tollervey, D; Hurt, E C

    1996-01-01

    Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA. Images PMID:8641292

  6. Functional renal imaging: new trends in radiology and nuclear medicine.

    PubMed

    Durand, Emmanuel; Chaumet-Riffaud, Philippe; Grenier, Nicolas

    2011-01-01

    The objective of this work is to compare the characteristics of various techniques for functional renal imaging, with a focus on nuclear medicine and magnetic resonance imaging. Even with low spatial resolution and rather poor signal-to-noise ratio, classical nuclear medicine has the advantage of linearity and good sensitivity. It remains the gold standard technique for renal relative functional assessment. Technetium-99m ((99m)Tc)-labeled diethylenetriamine penta-acetate remains the reference glomerular tracer. Tubular tracers have been improved: (123)I- or (131)I-hippuran, (99m)Tc-MAG3 and, recently, (99m)Tc-nitrilotriacetic acid. However, advancement in molecular imaging has not produced a groundbreaking tracer. Renal magnetic resonance imaging with classical gadolinated tracers probably has potential in this domain but has a lack of linearity and, therefore, its value still needs evaluation. Moreover, the advent of nephrogenic systemic fibrosis has delayed its expansion. Other developments, such as diffusion or blood oxygen level-dependent imaging, may have a role in the future. The other modalities have a limited role in clinical practice for functional renal imaging.

  7. Augmented Lagrangian method for constrained nuclear density functional theory

    NASA Astrophysics Data System (ADS)

    Staszczak, A.; Stoitsov, M.; Baran, A.; Nazarewicz, W.

    2010-10-01

    The augmented Lagrangiam method (ALM), widely used in quantum chemistry constrained optimization problems, is applied in the context of the nuclear Density Functional Theory (DFT) in the self-consistent constrained Skyrme Hartree-Fock-Bogoliubov (CHFB) variant. The ALM allows precise calculations of multi-dimensional energy surfaces in the space of collective coordinates that are needed to, e.g., determine fission pathways and saddle points; it improves the accuracy of computed derivatives with respect to collective variables that are used to determine collective inertia; and is well adapted to supercomputer applications.

  8. Nuclear clustering in the energy density functional approach

    SciTech Connect

    Ebran, J.-P.; Khan, E.; Nikšić, T.; Vretenar, D.

    2015-10-15

    Nuclear Energy Density Functionals (EDFs) are a microscopic tool of choice extensively used over the whole chart to successfully describe the properties of atomic nuclei ensuing from their quantum liquid nature. In the last decade, they also have proved their ability to deal with the cluster phenomenon, shedding a new light on its fundamental understanding by treating on an equal footing both quantum liquid and cluster aspects of nuclei. Such a unified microscopic description based on nucleonic degrees of freedom enables to tackle the question pertaining to the origin of the cluster phenomenon and emphasizes intrinsic mechanisms leading to the emergence of clusters in nuclei.

  9. Impaired protein stability and nuclear localization of NOBOX variants associated with premature ovarian insufficiency.

    PubMed

    Ferrari, Ilaria; Bouilly, Justine; Beau, Isabelle; Guizzardi, Fabiana; Ferlin, Alberto; Pollazzon, Marzia; Salerno, Mariacarolina; Binart, Nadine; Persani, Luca; Rossetti, Raffaella

    2016-10-23

    Premature ovarian insufficiency (POI) is a clinical syndrome defined by a loss of ovarian activity before the age of 40. Its pathogenesis is still largely unknown, but increasing evidences support a genetic basis in most cases. Among these, heterozygous mutations in NOBOX, a homeobox gene encoding a transcription factor expressed specifically by oocyte and granulosa cells within the ovary, have been reported in ∼6% of women with sporadic POI. The pivotal role of NOBOX in early folliculogenesis is supported by findings in knock-out mice. Here, we report the genetic screening of 107 European women with idiopathic POI, recruited in various settings, and the molecular and functional characterization of the identified variants to evaluate their involvement in POI onset. Specifically, we report the identification of two novel and two recurrent heterozygous NOBOX variants in 7 out of 107 patients, with a prevalence of 6.5% (upper 95% confidence limit of 11.17%). Furthermore, immunolocalization, Western Blot and transcriptional assays conducted in either HEK293T or CHO cells revealed that all the studied variants (p.R44L, p.G91W, p.G111R, p.G152R, p.K273*, p.R449* and p.D452N) display variable degrees of functional impairment, including defects in transcriptional activity, autophagosomal degradation, nuclear localization or protein instability. Several variants conserve the ability to interact with FOXL2 in intracellular aggregates. Their inability to sustain gene expression, together with their likely aberrant effects on protein stability and degradation, make the identified NOBOX mutations a plausible cause of POI onset.

  10. Characterization of the nuclear localization signal of the hepatitis delta virus antigen

    SciTech Connect

    Alves, Carolina; Freitas, Natalia; Cunha, Celso

    2008-01-05

    The delta antigen (HDAg) is the only protein encoded by the hepatitis delta virus (HDV) RNA genome. The HDAg contains an RNA binding domain, a dimerization domain, and a nuclear localization signal (NLS). The nuclear import of HDV RNPs is thought to be one of the first tasks of the HDAg during the HDV replication cycle. Using c-myc-PK fusions with several regions of the HDAg in transfection assays in Huh7 cells, we found that the HDAg NLS consists of a single stretch of 10 amino acids, EGAPPAKRAR, located in positions 66-75. Deletion and mutation analysis of this region showed that both the acidic glutamic acid residue at position 66 and the basic arginine residue at position 75 are essential for promoting nuclear import.

  11. Bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells

    SciTech Connect

    Hisada-Ishii, Shoji; Ebihara, Mizuki; Kobayashi, Nao; Kitagawa, Yasuo . E-mail: yasuok@agr.nagoya-u.ac.jp

    2007-03-02

    Matrin 3, a nuclear matrix protein has potential (1) to withhold promiscuously edited RNAs within the nucleus in cooperation with p54{sup nrb} and PSF (2) to mediate NMDA-induced neuronal death, and (3) to modulate promoter activity of genes proximal to matrix/scaffold attachment region (MAR/SAR). We identified a bipartite nuclear localization signal (NLS) of chicken matrin 3 (cmatr3) at residues 583-602. By expressing green fluorescent protein (GFP) fused to the NLS mutant in chicken DT40 cells, we showed an essential role of the NLS for cell proliferation. Furthermore, we showed that both clusters of basic amino acids and a linker of the bipartite NLS were essential and sufficient for the nuclear import of GFP. Exogenous cmatr3 rescued the HeLa cells where human matrin 3 was suppressed by RNA interference, but cmatr3 containing deletions at either of the basic amino acid clusters or the linker could not.

  12. Nuclear localization and secretion competence is conserved amongst henipavirus matrix proteins.

    PubMed

    McLinton, Elisabeth C; Wagstaff, Kylie M; Lee, Alexander; Moseley, Gregory W; Marsh, Glenn A; Wang, Lin-Fa; Jans, David A; Lieu, Kim G; Netter, Hans

    2017-01-05

    Viruses of the genus Henipavirus of the family Paramyxoviridae are zoonotic pathogens, which have emerged in South East Asia, Australia and Africa. Nipah virus (NiV) and Hendra virus (HeV) are highly virulent pathogens transmitted from bats to animals and humans, whilst the henipavirus Cedar virus (CedV) seems to be non-pathogenic in infection studies. The full replication cycle of the Paramyxoviridae occurs in the host cell's cytoplasm where viral assembly is orchestrated by the matrix (M) protein. Unexpectedly, the NiV-M protein traffics through the nucleus as an essential step to engage the plasma membrane in preparation for viral budding/release. Comparative studies were performed to assess whether M protein nuclear localization is a common feature of the henipaviruses including the recently sequenced (although not yet isolated) Ghanaian bat henipavirus (Kumasi virus, GH-M74a virus, KV) and Mojiang virus (MojV). Live-cell confocal microscopy revealed that nuclear translocation of GFP-fused M protein is conserved between henipaviruses in both human and bat-derived cell lines. However, the efficiency of M protein nuclear localization and virus-like particle budding competency varied. Additionally, CedV-, KV- and MojV-M proteins were mutated in a bipartite nuclear localization signal indicating that a key lysine residue is essential for nuclear import, export and for the induction of budding events as previously reported for NiV-M. The results of this study suggest that the M proteins of henipaviruses may utilize a similar nucleocytoplasmic trafficking pathway as an essential step during viral replication in both humans and bats.

  13. Nuclear matter properties from local chiral interactions with Δ isobar intermediate states

    NASA Astrophysics Data System (ADS)

    Logoteta, Domenico; Bombaci, Ignazio; Kievsky, Alejandro

    2016-12-01

    Using two-nucleon and three-nucleon interactions derived in the framework of chiral perturbation theory (ChPT) with and without the explicit Δ isobar contributions, we calculate the energy per particle of symmetric nuclear matter and pure neutron matter in the framework of the microscopic Brueckner-Hartree-Fock approach. In particular, we present for the first time nuclear matter calculations using the new fully local in coordinate-space two-nucleon interaction at the next-to-next-to-next-to-leading-order (N3LO) of ChPT with Δ isobar intermediate states (N 3 LO Δ ) recently developed by Piarulli et al. [arXiv:1606.06335]. We find that using this N 3 LO Δ potential, supplemented with a local N2LO three-nucleon interaction with explicit Δ isobar degrees of freedom, it is possible to obtain a satisfactory saturation point of symmetric nuclear matter. For this combination of two- and three-nucleon interactions we also calculate the nuclear symmetry energy and we compare our results with the empirical constraints on this quantity obtained using the excitation energies to isobaric analog states in nuclei and using experimental data on the neutron skin thickness of heavy nuclei, finding a very good agreement in all the considered nucleonic density range. In addition, we find that the explicit inclusion of Δ isobars diminishes the strength of the three-nucleon interactions needed to get a good saturation point of symmetric nuclear matter. We also compare the results of our calculations with those obtained by other research groups using chiral nuclear interactions with different many-body methods, finding in many cases a very satisfactory agreement.

  14. Nuclear localization signal sequence is required for VACM-1/CUL5-dependent regulation of cellular growth.

    PubMed

    Willis, Angelica N; Dean, Shirley E Bradley; Habbouche, Joe A; Kempers, Brian T; Ludwig, Megan L; Sayfie, Aaron D; Lewis, Steven P; Harrier, Stephanie; DeBruine, Zachary J; Garrett, Richard; Burnatowska-Hledin, Maria A

    2017-04-01

    VACM-1/CUL5 is a member of the cullin family of proteins involved in the E3 ligase-dependent degradation of diverse proteins that regulate cellular proliferation. The ability of VACM-1/CUL5 to inhibit cellular growth is affected by its posttranslational modifications and its localization to the nucleus. Since the mechanism of VACM-1/CUL5 translocation to the nucleus is not clear, the goal of this project was to determine the role that the putative nuclear localization signal (NLS) we identified in the VACM-1/CUL5 ((640)PKLKRQ(646)) plays in the cellular localization of VACM-1/CUL5 and its effect on cellular growth. We used site-directed mutagenesis to change Lys642 and Lys644 to Gly and the mutated cDNA constructs were transfected into COS-1 cells. Mutation of the NLS in VACM-1/CUL5 significantly reduced its localization to the nucleus and compromised its effect on cellular growth. We have shown previously that the antiproliferative effect of VACM-1/CUL5 could be reversed by mutation of PKA-specific phosphorylation sequence ((S730A)VACM-1/CUL5), which was associated with its increased nuclear localization and modification by NEDD8. Thus, we examined whether these properties can be controlled by the NLS. The mutation of NLS in (S730A)VACM-1/CUL5 cDNA compromised its proliferative effect and reduced its localization to the nucleus. The immunocytochemistry results showed that, in cells transfected with the mutant cDNAs, the nuclear NEDD8 signal was decreased. Western blot analysis of total cell lysates, however, showed that VACM-1/CUL5 neddylation was not affected. Together, these results suggest that the presence of the NLS, both in VACM-1/CUL5 and in (S730A)VACM-1/CUL5 sequences, is critical for their control of cell proliferation.

  15. Nuclear localization of Rad52 is pre-requisite for its sumoylation

    SciTech Connect

    Ohuchi, Takashi; Seki, Masayuki Enomoto, Takemi

    2008-07-18

    In Saccharomyces cerevisiae, Rad52 plays major roles in several types of homologous recombination. Here, we found that rad52-K200R mutation greatly reduced sumoylation of Rad52. The rad52-K200R mutant exhibited defects in various types of recombination, such as intrachromosomal recombination and mating-type switching. The K200 residue of Rad52 is part of the nuclear localization signal (NLS), which is important for transport into the nucleus. Indeed, the addition of a SV40 NLS to Rad52-K200R suppressed the sumoylation defect of Rad52-K200R. These findings indicate that nuclear localization of Rad52 is pre-requisite for its sumoylation.

  16. Characterization of the nuclear localization signal of the mouse TET3 protein

    SciTech Connect

    Xiao, Peng; Zhou, Xiao-long; Zhang, Hong-xiao; Xiong, Kai; Teng, Yun; Huang, Xian-ju; Cao, Rui; Wang, Yi; Liu, Hong-lin

    2013-09-27

    Highlights: •Amino acid sequence KKRK is responsible for nuclear localization of TET3. •Amino acid sequence KKRK are capable of targeting the cytoplasmic proteins to the nucleus. •Amino acid sequence KKRK are conserved in TET3 orthologs. -- Abstract: DNA demethylation is associated with gene activation and is mediated by a family of ten-eleven translocation (TET) dioxygenase. The TET3 protein is a 1668-amino-acid DNA demethylase that is predicted to possess five nuclear localization signals (NLSs). In this paper, we used a series of green fluorescent protein-tagged and mutation constructs to identify a conserved NLS (KKRK) embedded between amino acid 1615 and 1618 of mouse TET3. The KKRK sequence facilitates the cytoplasmic protein’s translocation into the nucleus. Additionally TET3 may be imported into the nucleus by importin-α and importin-β.

  17. Local negotiation on compensation siting of the spent nuclear fuel repository in Finland

    SciTech Connect

    Kojo, Matti

    2007-07-01

    The aim of the paper is to analyse the local negotiation process between the Municipality of Eurajoki and the nuclear power company Teollisuuden Voima (TVO) and the nuclear waste management company Posiva Oy. The aim of the negotiations was to find an acceptable form of compensation for siting a spent nuclear fuel repository in Olkiluoto, Finland. The paper includes background information on the siting process in Finland, the local political setting in the Municipality of Eurajoki and a description of the negotiation process. The analysis of the negotiations on compensation is important for better understanding the progress of the Finnish siting process. The paper describes the picture of the contest to host the spent nuclear fuel repository. It also provides more information on the relationship between the Municipality of Eurajoki and the power company TVO. The negotiations on compensation and the roles of various players in the negotiations have not been studied in detail because the minutes of the Vuojoki liaison group were not available before the decision of the Supreme Administrative Court in May 2006. (author)

  18. Nuclear materials transportation workshops: USDOE outreach to local governments. Final report

    SciTech Connect

    Not Available

    1987-09-28

    To provide direct outreach to local governments, the Transportation Management Division of the United States Department of Energy asked the Urban Consortium and its Energy Task Force to assemble representatives for two workshops focusing on the transport of nuclear materials. The first session, for jurisdictions east of the Mississippi River, was held in New Orleans on May 5--6, 1988; the second was conducted on June 6--7, 1988 in Denver for jurisdictions to the west. Twenty local government professionals with management or operational responsibility for hazardous materials transportation within their jurisdictions were selected to attend each workshop. The discussions identified five major areas of concern to local government professionals; coordination; training; information resources; marking and placarding; and responder resources. Integrated federal, state, and local levels of government emerged as a priority coordination issue along with the need for expanded availability of training and training resources for first-reponders.

  19. Nuclear localization of nucleoside diphosphate kinase type B (nm23-H2) in cultured cells.

    PubMed

    Kraeft, S K; Traincart, F; Mesnildrey, S; Bourdais, J; Véron, M; Chen, L B

    1996-08-25

    Nucleoside diphosphate (NDP) kinases are metabolic enzymes found ubiquitously in cells. Recently, two known human isoforms of NDP kinase (A and B), identical to the protein products of the genes nm23-H1 and nm23-H2, respectively, have been implicated in cancer metastasis and transcriptional regulation. To date, NDP kinase has been studied extensively in tissue sections and its cellular localization was described as being cytoplasmic. However, the recently discovered role of the nm23-H2 gene product in transcriptional activation of the c-myc proto-oncogene also suggests a nuclear localization of the protein. In this study, we used isoform-specific antibodies against NDPK-B to examine the subcellular localization of the nm23-H2 gene product. The cytoplasmic fluorescence is intense and homogeneous with pronounced labeling in the centromere region. The distribution of NDPK-B in interphase nuclei exhibits a pattern of numerous uniformly dispersed fine dots with reduced staining of the nucleoli. To further characterize the nuclear localization of NDPK-B, in situ sequential extraction of nuclear components was performed. Brief exposure to Triton X-100 and subsequent treatment with RNase A do not change the nuclear staining pattern of NDPK-B. In contrast, treatment of Triton X-100-permeabilized nuclei with DNase I results in a significant loss of fluorescence. In mitotic prophase cells, the protein segregates from forming chromosomes and reappears in newly formed daughter nuclei after cell division. Taken together, the results indicate an association of NDPK-B with chromatin in interphase nuclei, supporting its proposed role in transcription.

  20. Characterizing dynamic local functional connectivity in the human brain

    PubMed Central

    Deng, Lifu; Sun, Junfeng; Cheng, Lin; Tong, Shanbao

    2016-01-01

    Functional connectivity (FC), obtained from functional magnetic resonance imaging (fMRI), brings insights into the functional organization of the brain. Recently, rich and complex behaviour of brain has been revealed by the dynamic fluctuation of FC, which had previously been regarded as confounding ‘noise’. While the dynamics of long-distance, inter-regional FC has been extensively studied, the dynamics of local FC within a few millimetres in space remains largely unexplored. In this study, the local FC was depicted by regional homogeneity (ReHo), and the dynamics of local FC was obtained using sliding windows method. We observed a robust positive correlation between ReHo and its temporal variability, which was shown to be an intrinsic feature of the brain rather than a pure stochastic effect. Furthermore, fluctuation of ReHo was associated with global functional organization: (i) brain regions with higher centrality of inter-regional FC tended to possess higher ReHo variability; (ii) coherence of ReHo fluctuation was higher within brain’s functional modules. Finally, we observed alteration of ReHo variability during a motor task compared with resting-state. Our findings associated the temporal fluctuation of ReHo with brain function, opening up the possibility of dynamic local FC study in the future. PMID:27231194

  1. Transcription-dependent nuclear localization of DAZAP1 requires an N-terminal signal

    SciTech Connect

    Lin, Yi-Tzu; Wen, Wan-Ching; Yen, Pauline H.

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer DAZAP1 shuttles between the nucleus and the cytoplasm. Black-Right-Pointing-Pointer DAZAP1 accumulates in the cytoplasm when the nuclear transcription is inhibited. Black-Right-Pointing-Pointer DAZAP1's transcription-dependent nuclear localization requires N-terminal N42. Black-Right-Pointing-Pointer SLIRP binds to N42 and may be involved in the process. -- Abstract: Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous hnRNP protein required for normal development and spermatogenesis. It resides predominantly in the nucleus and moves between the nucleus and the cytoplasm via a ZNS shuttling signal at its C-terminus. DAZAP1 accumulates in the cytoplasm when RNA polymerase II activity is inhibited by actinomycin D. Here we report the mapping of a 42-amino acid segment (N42) at the N-terminus of DAZAP1 that is both necessary and sufficient for its transcription-dependent nuclear localization. In addition, using a yeast two-hybrid system, we have identified SLIRP as a N42-binding protein which may regulate DAZAP1 subcellular localization.

  2. Vinculin promotes nuclear localization of TAZ to inhibit ECM stiffness-dependent differentiation into adipocytes.

    PubMed

    Kuroda, Mito; Wada, Hiroki; Kimura, Yasuhisa; Ueda, Kazumitsu; Kioka, Noriyuki

    2017-03-01

    Extracellular matrix (ECM) stiffness regulates the lineage commitment of mesenchymal stem cells (MSCs). Although cells sense ECM stiffness through focal adhesions, how cells sense ECM stiffness and regulate ECM stiffness-dependent differentiation remains largely unclear. In this study, we show that the cytoskeletal focal adhesion protein vinculin plays a critical role in the ECM stiffness-dependent adipocyte differentiation of MSCs. ST2 mouse MSCs differentiate into adipocytes and osteoblasts in an ECM stiffness-dependent manner. We find that a rigid ECM increases the amount of cytoskeleton-associated vinculin and promotes the nuclear localization and activity of the transcriptional coactivator paralogs Yes-associated protein (YAP, also known as YAP1) and transcriptional coactivator with a PDZ-binding motif (TAZ, also known as WWTR1) (hereafter YAP/TAZ). Vinculin is necessary for enhanced nuclear localization and activity of YAP/TAZ on the rigid ECM but it does not affect the phosphorylation of the YAP/TAZ kinase LATS1. Furthermore, vinculin depletion promotes differentiation into adipocytes on rigid ECM, while it inhibits differentiation into osteoblasts. Finally, TAZ knockdown was less effective at promoting adipocyte differentiation in vinculin-depleted cells than in control cells. These results suggest that vinculin promotes the nuclear localization of transcription factor TAZ to inhibit the adipocyte differentiation on rigid ECM.

  3. Symmetry-adapted Wannier functions in the maximal localization procedure

    NASA Astrophysics Data System (ADS)

    Sakuma, R.

    2013-06-01

    A procedure to construct symmetry-adapted Wannier functions in the framework of the maximally localized Wannier function approach [Marzari and Vanderbilt, Phys. Rev. BPRBMDO1098-012110.1103/PhysRevB.56.12847 56, 12847 (1997); Souza, Marzari, and Vanderbilt, Phys. Rev. BPRBMDO1098-012110.1103/PhysRevB.65.035109 65, 035109 (2001)] is presented. In this scheme, the minimization of the spread functional of the Wannier functions is performed with constraints that are derived from symmetry properties of the specified set of the Wannier functions and the Bloch functions used to construct them, therefore one can obtain a solution that does not necessarily yield the global minimum of the spread functional. As a test of this approach, results of atom-centered Wannier functions for GaAs and Cu are presented.

  4. Global NLO Analysis of Nuclear Parton Distribution Functions

    SciTech Connect

    Hirai, M.; Kumano, S.; Nagai, T.-H.

    2008-02-21

    Nuclear parton distribution functions (NPDFs) are determined by a global analysis of experimental measurements on structure-function ratios F{sub 2}{sup A}/F{sub 2}{sup A{sup '}} and Drell-Yan cross section ratios {sigma}{sub DY}{sup A}/{sigma}{sub DY}{sup A{sup '}}, and their uncertainties are estimated by the Hessian method. The NPDFs are obtained in both leading order (LO) and next-to-leading order (NLO) of {alpha}{sub s}. As a result, valence-quark distributions are relatively well determined, whereas antiquark distributions at x>0.2 and gluon distributions in the whole x region have large uncertainties. The NLO uncertainties are slightly smaller than the LO ones; however, such a NLO improvement is not as significant as the nucleonic case.

  5. Next Generation Nuclear Plant Resilient Control System Functional Analysis

    SciTech Connect

    Lynne M. Stevens

    2010-07-01

    Control Systems and their associated instrumentation must meet reliability, availability, maintainability, and resiliency criteria in order for high temperature gas-cooled reactors (HTGRs) to be economically competitive. Research, perhaps requiring several years, may be needed to develop control systems to support plant availability and resiliency. This report functionally analyzes the gaps between traditional and resilient control systems as applicable to HTGRs, which includes the Next Generation Nuclear Plant; defines resilient controls; assesses the current state of both traditional and resilient control systems; and documents the functional gaps existing between these two controls approaches as applicable to HTGRs. This report supports the development of an overall strategy for applying resilient controls to HTGRs by showing that control systems with adequate levels of resilience perform at higher levels, respond more quickly to disturbances, increase operational efficiency, and increase public protection.

  6. Interaction of nucleosome assembly proteins abolishes nuclear localization of DGK{zeta} by attenuating its association with importins

    SciTech Connect

    Okada, Masashi; Hozumi, Yasukazu; Ichimura, Tohru; Tanaka, Toshiaki; Hasegawa, Hiroshi; Yamamoto, Masakazu; Takahashi, Nobuya; Iseki, Ken; Yagisawa, Hitoshi; Shinkawa, Takashi; Isobe, Toshiaki; Goto, Kaoru

    2011-12-10

    Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGK{zeta}, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGK{zeta}. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGK{zeta} binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGK{zeta} and NAP1Ls prohibits nuclear import of DGK{zeta} because binding of NAP1Ls to DGK{zeta} blocks import carrier proteins, Qip1 and NPI1, to interact with DGK{zeta}, leading to cytoplasmic tethering of DGK{zeta}. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGK{zeta} and provide a clue to examine functional significance of its translocation under pathological conditions.

  7. Computational identification of post-translational modification-based nuclear import regulations by characterizing nuclear localization signal-import receptor interaction.

    PubMed

    Lin, Jhih-Rong; Liu, Zhonghao; Hu, Jianjun

    2014-10-01

    The binding affinity between a nuclear localization signal (NLS) and its import receptor is closely related to corresponding nuclear import activity. PTM-based modulation of the NLS binding affinity to the import receptor is one of the most understood mechanisms to regulate nuclear import of proteins. However, identification of such regulation mechanisms is challenging due to the difficulty of assessing the impact of PTM on corresponding nuclear import activities. In this study we proposed NIpredict, an effective algorithm to predict nuclear import activity given its NLS, in which molecular interaction energy components (MIECs) were used to characterize the NLS-import receptor interaction, and the support vector regression machine (SVR) was used to learn the relationship between the characterized NLS-import receptor interaction and the corresponding nuclear import activity. Our experiments showed that nuclear import activity change due to NLS change could be accurately predicted by the NIpredict algorithm. Based on NIpredict, we developed a systematic framework to identify potential PTM-based nuclear import regulations for human and yeast nuclear proteins. Application of this approach has identified the potential nuclear import regulation mechanisms by phosphorylation of two nuclear proteins including SF1 and ORC6.

  8. Steroidogenesis in the skin: implications for local immune functions

    PubMed Central

    Slominski, Andrzej; Zbytek, Bazej; Nikolakis, Georgios; Manna, Pulak R.; Skobowiat, Cezary; Zmijewski, Michal; Li, Wei; Janjetovic, Zorica; Postlethwaite, Arnold; Zouboulis, Christos C.; Tuckey, Robert C.

    2013-01-01

    The skin has developed a hierarchy of systems that encompasses the skin immune and local steroidogenic activities in order to protect the body against the external environment and biological factors and to maintain local homeostasis. Most recently it has been established that skin cells contain the entire biochemical apparatus necessary for production of glucocorticoids, androgens and estrogens either from precursors of systemic origin or, alternatively, through the conversion of cholesterol to pregnenolone and its subsequent transformation to biologically active steroids. Examples of these products are corticosterone, cortisol, testosterone, dihydrotesterone and estradiol. Their local production can be regulated by locally produced corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH) or cytokines. Furthermore the production of glucocorticoids is affected by ultraviolet B radiation. The level of production and nature of the final steroid products are dependent on the cell type or cutaneous compartment, e.g., epidermis, dermis, adnexal structures or adipose tissue. Locally produced glucocorticoids, androgens and estrogens affect functions of the epidermis and adnexal structures as well as local immune activity. Malfunction of these steroidogenic activities can lead to inflammatory disorders or autoimmune diseases. The cutaneous steroidogenic system can also have systemic effects, which are emphasized by significant skin contribution to circulating androgens and/or estrogens. Furthermore, local activity of CYP11A1 can produce novel 7 -steroids and secosteroids that are biologically active. Therefore, modulation of local steroidogenic activity may serve as a new therapeutic approach for treatment of inflammatory disorders, autoimmune processes or other skin disorders. In conclusion, the skin can be defined as an independent steroidogenic organ, whose activity can affect its functions and the development of local or systemic inflammatory or

  9. Steroidogenesis in the skin: implications for local immune functions.

    PubMed

    Slominski, Andrzej; Zbytek, Blazej; Nikolakis, Georgios; Manna, Pulak R; Skobowiat, Cezary; Zmijewski, Michal; Li, Wei; Janjetovic, Zorica; Postlethwaite, Arnold; Zouboulis, Christos C; Tuckey, Robert C

    2013-09-01

    The skin has developed a hierarchy of systems that encompasses the skin immune and local steroidogenic activities in order to protect the body against the external environment and biological factors and to maintain local homeostasis. Most recently it has been established that skin cells contain the entire biochemical apparatus necessary for production of glucocorticoids, androgens and estrogens either from precursors of systemic origin or, alternatively, through the conversion of cholesterol to pregnenolone and its subsequent transformation to biologically active steroids. Examples of these products are corticosterone, cortisol, testosterone, dihydrotesterone and estradiol. Their local production can be regulated by locally produced corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH) or cytokines. Furthermore the production of glucocorticoids is affected by ultraviolet B radiation. The level of production and nature of the final steroid products are dependent on the cell type or cutaneous compartment, e.g., epidermis, dermis, adnexal structures or adipose tissue. Locally produced glucocorticoids, androgens and estrogens affect functions of the epidermis and adnexal structures as well as local immune activity. Malfunction of these steroidogenic activities can lead to inflammatory disorders or autoimmune diseases. The cutaneous steroidogenic system can also have systemic effects, which are emphasized by significant skin contribution to circulating androgens and/or estrogens. Furthermore, local activity of CYP11A1 can produce novel 7Δ-steroids and secosteroids that are biologically active. Therefore, modulation of local steroidogenic activity may serve as a new therapeutic approach for treatment of inflammatory disorders, autoimmune processes or other skin disorders. In conclusion, the skin can be defined as an independent steroidogenic organ, whose activity can affect its functions and the development of local or systemic inflammatory or

  10. Alimentary Tract Absorption (f1 Values) for Radionuclides in Local and Regional Fallout from Nuclear Tests

    PubMed Central

    Ibrahim, Shawki; Simon, Steven L; Bouville, André; Melo, Dunstana; Beck, Harold

    2009-01-01

    This paper presents gastrointestinal absorption fractions (f1 values) for estimating internal doses from local and regional fallout radionuclides due to nuclear tests. The choice of f1 values are based on specific circumstances of weapons test conditions and a review of reported f1 values for elements in different physical and chemical states. Special attention is given to fallout from nuclear tests conducted at the Marshall Islands. We make a distinction between the f1 values for intakes of radioactive materials immediately after deposition (acute intakes) and intakes that occur in the course of months and years after deposition, following incorporation into terrestrial and aquatic foodstuffs (chronic intakes). Multiple f1 values for different circumstances where persons are exposed to radioactive fallout (e.g. local vs. regional fallout and coral vs. continental tests) are presented when supportive information is available. In some cases, our selected f1 values are similar to those adopted by the ICRP (e.g. iodine and most actinides). However, f1 values for cesium and strontium derived from urine bioassay data of the Marshallese population are notably lower than the generic f1 values recommended by ICRP, particularly for acute intakes from local fallout (0.4 and 0.05 for Cs and Sr, respectively. The f1 values presented here form the first complete set of values relevant to realistic dose assessments for exposure to local or regional radioactive fallout. PMID:20622554

  11. Alimentary tract absorption (f1 values) for radionuclides in local and regional fallout from nuclear tests.

    PubMed

    Ibrahim, Shawki A; Simon, Steven L; Bouville, André; Melo, Dunstana; Beck, Harold L

    2010-08-01

    This paper presents gastrointestinal absorption fractions (f1 values) for estimating internal doses from local and regional fallout radionuclides due to nuclear tests. The choice of f1 values are based on specific circumstances of weapons test conditions and a review of reported f1 values for elements in different physical and chemical states. Special attention is given to fallout from nuclear tests conducted at the Marshall Islands. We make a distinction between the f1 values for intakes of radioactive materials immediately after deposition (acute intakes) and intakes that occur in the course of months and years after deposition, following incorporation into terrestrial and aquatic foodstuffs (chronic intakes). Multiple f1 values for different circumstances where persons are exposed to radioactive fallout (e.g., local vs. regional fallout and coral vs. continental tests) are presented when supportive information is available. In some cases, our selected f1 values are similar to those adopted by the International Commission on Radiological Protection (ICRP) (e.g., iodine and most actinides). However, f1 values for cesium and strontium derived from urine bioassay data of the Marshallese population are notably lower than the generic f1 values recommended by ICRP, particularly for acute intakes from local fallout (0.4 and 0.05 for Cs and Sr, respectively). The f1 values presented here form the first complete set of values relevant to realistic dose assessments for exposure to local or regional radioactive fallout.

  12. CRM1-dependent nuclear export and dimerization with hMSH5 contribute to the regulation of hMSH4 subcellular localization

    SciTech Connect

    Neyton, Sophie; Lespinasse, Francoise; Lahaye, Francois; Staccini, Pascal; Paquis-Flucklinger, Veronique; Santucci-Darmanin, Sabine

    2007-10-15

    MSH4 and MSH5 are members of the MutS homolog family, a conserved group of proteins involved in DNA mismatch correction and homologous recombination. Although several studies have provided compelling evidences suggesting that MSH4 and MSH5 could act together in early and late stages of meiotic recombination, their precise roles are poorly understood and recent findings suggest that the human MSH4 protein may also exert a cytoplasmic function. Here we show that MSH4 is present in the cytoplasm and the nucleus of both testicular cells and transfected somatic cells. Confocal studies on transfected cells provide the first evidence that the subcellular localization of MSH4 is regulated, at least in part, by an active nuclear export pathway dependent on the exportin CRM1. We used deletion mapping and mutagenesis to define two functional nuclear export sequences within the C-terminal part of hMSH4 that mediate nuclear export through the CRM1 pathway. Our results suggest that CRM1 is also involved in MSH5 nuclear export. In addition, we demonstrate that dimerization of MSH4 and MSH5 facilitates their nuclear localization suggesting that dimerization may regulate the intracellular trafficking of these proteins. Our findings suggest that nucleocytoplasmic traffic may constitute a regulatory mechanism for MSH4 and MSH5 functions.

  13. Charge regulation and local dielectric function in planar polyelectrolyte brushes.

    PubMed

    Kumar, Rajeev; Sumpter, Bobby G; Kilbey, S Michael

    2012-06-21

    Understanding the effect of inhomogeneity on the charge regulation and dielectric properties, and how it depends on the conformational characteristics of the macromolecules is a long-standing problem. In order to address this problem, we have developed a field-theory to study charge regulation and local dielectric function in planar polyelectrolyte brushes. The theory is used to study a polyacid brush, which is comprised of chains end-grafted at the solid-fluid interface, in equilibrium with a bulk solution containing monovalent salt ions, solvent molecules, and pH controlling acid. In particular, we focus on the effects of the concentration of added salt and pH of the bulk in determining the local charge and dielectric function. Our theoretical investigations reveal that the dipole moment of the ion-pairs formed as a result of counterion adsorption on the chain backbones play a key role in affecting the local dielectric function. For polyelectrolytes made of monomers having dipole moments lower than the solvent molecules, dielectric decrement is predicted inside the brush region. However, the formation of ion-pairs (due to adsorption of counterions coming from the dissociation of added salt) more polar than the solvent molecules is shown to increase the magnitude of the dielectric function with respect to its bulk value. Furthermore, an increase in the bulk salt concentration is shown to increase the local charge inside the brush region.

  14. The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

    SciTech Connect

    Lalime, Erin N.; Pekosz, Andrew

    2014-06-15

    The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 in addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.

  15. Structure and Function of Latency-Associated Nuclear Antigen

    PubMed Central

    Verma, S. C.; Lan, K.

    2011-01-01

    Latency-associated nuclear antigen (LANA) encoded by open reading frame 73 (ORF73) is the major latent protein expressed in all forms of KSHV-associated malignancies. LANA is a large (222–234 kDa) nuclear protein that interacts with various cellular as well as viral proteins. LANA has been classified as an oncogenic protein as it dysregulates various cellular pathways including tumor suppressor pathways associated with pRb and p53 and can transform primary rat embryo fibroblasts in cooperation with the cellular oncogene Hras. It associates with GSK-3β, an important modulator of Wnt signaling pathway leading to the accumulation of cytoplasmic β-catenin, which upregulates Tcf/Lef regulated genes after entering into the nucleus. LANA also blocks the expression of RTA, the reactivation transcriptional activator, which is critical for the latency to lytic switch, and thus helps in maintaining viral latency. LANA tethers the viral episomal DNA to the host chromosomes by directly binding to its cognate binding sequence within the TR region of the genome through its C terminus and to the nucleosomes through the N terminus of the molecule. Tethering to the host chromosomes helps in efficient partitioning of the viral episomes in the dividing cells. Disruptions of LANA expression led to reduction in the episomal copies of the viral DNA, supporting its role in persistence of the viral DNA. The functions known so far suggest that LANA is a key player in KSHV-mediated pathogenesis. PMID:17089795

  16. Using the electron localization function to correct for confinement physics in semi-local density functional theory

    SciTech Connect

    Hao, Feng Mattsson, Ann E.; Armiento, Rickard

    2014-05-14

    We have previously proposed that further improved functionals for density functional theory can be constructed based on the Armiento-Mattsson subsystem functional scheme if, in addition to the uniform electron gas and surface models used in the Armiento-Mattsson 2005 functional, a model for the strongly confined electron gas is also added. However, of central importance for this scheme is an index that identifies regions in space where the correction provided by the confined electron gas should be applied. The electron localization function (ELF) is a well-known indicator of strongly localized electrons. We use a model of a confined electron gas based on the harmonic oscillator to show that regions with high ELF directly coincide with regions where common exchange energy functionals have large errors. This suggests that the harmonic oscillator model together with an index based on the ELF provides the crucial ingredients for future improved semi-local functionals. For a practical illustration of how the proposed scheme is intended to work for a physical system we discuss monoclinic cupric oxide, CuO. A thorough discussion of this system leads us to promote the cell geometry of CuO as a useful benchmark for future semi-local functionals. Very high ELF values are found in a shell around the O ions, and take its maximum value along the Cu–O directions. An estimate of the exchange functional error from the effect of electron confinement in these regions suggests a magnitude and sign that could account for the error in cell geometry.

  17. Using the electron localization function to correct for confinement physics in semi-local density functional theory.

    PubMed

    Hao, Feng; Armiento, Rickard; Mattsson, Ann E

    2014-05-14

    We have previously proposed that further improved functionals for density functional theory can be constructed based on the Armiento-Mattsson subsystem functional scheme if, in addition to the uniform electron gas and surface models used in the Armiento-Mattsson 2005 functional, a model for the strongly confined electron gas is also added. However, of central importance for this scheme is an index that identifies regions in space where the correction provided by the confined electron gas should be applied. The electron localization function (ELF) is a well-known indicator of strongly localized electrons. We use a model of a confined electron gas based on the harmonic oscillator to show that regions with high ELF directly coincide with regions where common exchange energy functionals have large errors. This suggests that the harmonic oscillator model together with an index based on the ELF provides the crucial ingredients for future improved semi-local functionals. For a practical illustration of how the proposed scheme is intended to work for a physical system we discuss monoclinic cupric oxide, CuO. A thorough discussion of this system leads us to promote the cell geometry of CuO as a useful benchmark for future semi-local functionals. Very high ELF values are found in a shell around the O ions, and take its maximum value along the Cu-O directions. An estimate of the exchange functional error from the effect of electron confinement in these regions suggests a magnitude and sign that could account for the error in cell geometry.

  18. Divergent Evolution of Nuclear Localization Signal Sequences in Herpesvirus Terminase Subunits.

    PubMed

    Sankhala, Rajeshwer S; Lokareddy, Ravi K; Cingolani, Gino

    2016-05-20

    The tripartite terminase complex of herpesviruses assembles in the cytoplasm of infected cells and exploits the host nuclear import machinery to gain access to the nucleus, where capsid assembly and genome-packaging occur. Here we analyzed the structure and conservation of nuclear localization signal (NLS) sequences previously identified in herpes simplex virus 1 (HSV-1) large terminase and human cytomegalovirus (HCMV) small terminase. We found a monopartite NLS at the N terminus of large terminase, flanking the ATPase domain, that is conserved only in α-herpesviruses. In contrast, small terminase exposes a classical NLS at the far C terminus of its helical structure that is conserved only in two genera of the β-subfamily and absent in α- and γ-herpesviruses. In addition, we predicted a classical NLS in the third terminase subunit that is partially conserved among herpesviruses. Bioinformatic analysis revealed that both location and potency of NLSs in terminase subunits evolved more rapidly than the rest of the amino acid sequence despite the selective pressure to keep terminase gene products active and localized in the nucleus. We propose that swapping NLSs among terminase subunits is a regulatory mechanism that allows different herpesviruses to regulate the kinetics of terminase nuclear import, reflecting a mechanism of virus:host adaptation.

  19. REV-ERBα influences the stability and nuclear localization of the glucocorticoid receptor.

    PubMed

    Okabe, Takashi; Chavan, Rohit; Fonseca Costa, Sara S; Brenna, Andrea; Ripperger, Jürgen A; Albrecht, Urs

    2016-11-01

    REV-ERBα (encoded by Nr1d1) is a nuclear receptor that is part of the circadian clock mechanism and regulates metabolism and inflammatory processes. The glucocorticoid receptor (GR, encoded by Nr3c1) influences similar processes, but is not part of the circadian clock, although glucocorticoid signaling affects resetting of the circadian clock in peripheral tissues. Because of their similar impact on physiological processes, we studied the interplay between these two nuclear receptors. We found that REV-ERBα binds to the C-terminal portion and GR to the N-terminal portion of HSP90α and HSP90β, a chaperone responsible for the activation of proteins to ensure survival of a cell. The presence of REV-ERBα influences the stability and nuclear localization of GR by an unknown mechanism, thereby affecting expression of GR target genes, such as IκBα (Nfkbia) and alcohol dehydrogenase 1 (Adh1). Our findings highlight an important interplay between two nuclear receptors that influence the transcriptional potential of each other. This indicates that the transcriptional landscape is strongly dependent on dynamic processes at the protein level.

  20. REV-ERBα influences the stability and nuclear localization of the glucocorticoid receptor

    PubMed Central

    Okabe, Takashi; Chavan, Rohit; Fonseca Costa, Sara S.; Brenna, Andrea; Ripperger, Jürgen A.

    2016-01-01

    ABSTRACT REV-ERBα (encoded by Nr1d1) is a nuclear receptor that is part of the circadian clock mechanism and regulates metabolism and inflammatory processes. The glucocorticoid receptor (GR, encoded by Nr3c1) influences similar processes, but is not part of the circadian clock, although glucocorticoid signaling affects resetting of the circadian clock in peripheral tissues. Because of their similar impact on physiological processes, we studied the interplay between these two nuclear receptors. We found that REV-ERBα binds to the C-terminal portion and GR to the N-terminal portion of HSP90α and HSP90β, a chaperone responsible for the activation of proteins to ensure survival of a cell. The presence of REV-ERBα influences the stability and nuclear localization of GR by an unknown mechanism, thereby affecting expression of GR target genes, such as IκBα (Nfkbia) and alcohol dehydrogenase 1 (Adh1). Our findings highlight an important interplay between two nuclear receptors that influence the transcriptional potential of each other. This indicates that the transcriptional landscape is strongly dependent on dynamic processes at the protein level. PMID:27686098

  1. Molecular mechanism by which acyclic retinoid induces nuclear localization of transglutaminase 2 in human hepatocellular carcinoma cells

    PubMed Central

    Shrestha, R; Tatsukawa, H; Shrestha, R; Ishibashi, N; Matsuura, T; Kagechika, H; Kose, S; Hitomi, K; Imamoto, N; Kojima, S

    2015-01-01

    Nuclear accumulation of transglutaminase 2 (TG2) is an important step in TG2-dependent cell death. However, the underlying molecular mechanisms for nuclear translocation of TG2 are still poorly understood. In this study, we demonstrated that acyclic retinoid (ACR) induced nuclear accumulation of TG2 in JHH-7 cells, a hepatocellular carcinoma (HCC) leading to their apoptosis. We further demonstrated molecular mechanism in nuclear-cytoplasmic trafficking of TG2 and an effect of ACR on it. We identified a novel 14-amino acid nuclear localization signal (NLS) 466AEKEETGMAMRIRV479 in the ‘C' domain and a leucine-rich nuclear export signal (NES) 657LHMGLHKL664 in the ‘D' domain that allowed TG2 to shuttle between the nuclear and cytosolic milieu. Increased nuclear import of GAPDH myc-HIS fused with the identified NLS was observed, confirming its nuclear import ability. Leptomycin B, an inhibitor of exportin-1 as well as point mutation of all leucine residues to glutamine residues in the NES of TG2 demolished its nuclear export. TG2 formed a trimeric complex with importin-α and importin-β independently from transamidase activity which strongly suggested the involvement of a NLS-based translocation of TG2 to the nucleus. ACR accelerated the formation of the trimeric complex and that may be at least in part responsible for enhanced nuclear localization of TG2 in HCC cells treated with ACR. PMID:26633708

  2. HuR and GRSF1 modulate the nuclear export and mitochondrial localization of the lncRNA RMRP

    PubMed Central

    Noh, Ji Heon; Kim, Kyoung Mi; Abdelmohsen, Kotb; Yoon, Je-Hyun; Panda, Amaresh C.; Munk, Rachel; Kim, Jiyoung; Curtis, Jessica; Moad, Christopher A.; Wohler, Christina M.; Indig, Fred E.; de Paula, Wilson; Dudekula, Dawood B.; De, Supriyo; Piao, Yulan; Yang, Xiaoling; Martindale, Jennifer L.; de Cabo, Rafael; Gorospe, Myriam

    2016-01-01

    Some mitochondrial long noncoding RNAs (lncRNAs) are encoded by nuclear DNA, but the mechanisms that mediate their transport to mitochondria are poorly characterized. Using affinity RNA pull-down followed by mass spectrometry analysis, we found two RNA-binding proteins (RBPs), HuR (human antigen R) and GRSF1 (G-rich RNA sequence-binding factor 1), that associated with the nuclear DNA-encoded lncRNA RMRP and mobilized it to mitochondria. In cultured human cells, HuR bound RMRP in the nucleus and mediated its CRM1 (chromosome region maintenance 1)-dependent export to the cytosol. After RMRP was imported into mitochondria, GRSF1 bound RMRP and increased its abundance in the matrix. Loss of GRSF1 lowered the mitochondrial levels of RMRP, in turn suppressing oxygen consumption rates and modestly reducing mitochondrial DNA replication priming. Our findings indicate that RBPs HuR and GRSF1 govern the cytoplasmic and mitochondrial localization of the lncRNA RMRP, which is encoded by nuclear DNA but has key functions in mitochondria. PMID:27198227

  3. hnRNP M interacts with PSF and p54{sup nrb} and co-localizes within defined nuclear structures

    SciTech Connect

    Marko, Marija; Leichter, Michael; Patrinou-Georgoula, Meropi; Guialis, Apostolia

    2010-02-01

    The abundant heterogeneous nuclear ribonucleoprotein M (hnRNP M) is able to associate with early spliceosomes and to influence splicing patterns of specific pre-mRNAs. Here, by a combination of immunoprecipitation and pull-down assays, we have identified PSF (polypyrimidine tract-binding protein-associated splicing factor) and p54{sup nrb}, two highly related proteins involved in transcription and RNA processing, as new binding partners of hnRNP M. HnRNP M was found to co-localize with PSF within a subset of nuclear paraspeckles and to largely co-fractionate with PSF and p54{sup nrb} in biochemical nuclear matrix preparations. In cells transfected with an alternatively spliced preprotachykinin (PPT) minigene expression of hnRNP M promoted exon skipping while expression of PSF favours exon inclusion. The latter effect was reverted specifically by co-expressing the full length hnRNP M or a deletion mutant capable of interaction with PSF and p54{sup nrb}. Together our data provide new insights and some functional implications on the hnRNP M network of interactions.

  4. Crystal structure of rice importin-α and structural basis of its interaction with plant-specific nuclear localization signals.

    PubMed

    Chang, Chiung-Wen; Couñago, Rafael Lemos Miguez; Williams, Simon J; Bodén, Mikael; Kobe, Boštjan

    2012-12-01

    In the classical nucleocytoplasmic import pathway, nuclear localization signals (NLSs) in cargo proteins are recognized by the import receptor importin-α. Importin-α has two separate NLS binding sites (the major and the minor site), both of which recognize positively charged amino acid clusters in NLSs. Little is known about the molecular basis of the unique features of the classical nuclear import pathway in plants. We determined the crystal structure of rice (Oryza sativa) importin-α1a at 2-Å resolution. The structure reveals that the autoinhibitory mechanism mediated by the importin-β binding domain of importin-α operates in plants, with NLS-mimicking sequences binding to both minor and major NLS binding sites. Consistent with yeast and mammalian proteins, rice importin-α binds the prototypical NLS from simian virus 40 large T-antigen preferentially at the major NLS binding site. We show that two NLSs, previously described as plant specific, bind to and are functional with plant, mammalian, and yeast importin-α proteins but interact with rice importin-α more strongly. The crystal structures of their complexes with rice importin-α show that they bind to the minor NLS binding site. By contrast, the crystal structures of their complexes with mouse (Mus musculus) importin-α show preferential binding to the major NLS binding site. Our results reveal the molecular basis of a number of features of the classical nuclear transport pathway specific to plants.

  5. The regulation and functions of the nuclear RNA exosome complex.

    PubMed

    Kilchert, Cornelia; Wittmann, Sina; Vasiljeva, Lidia

    2016-04-01

    The RNA exosome complex is the most versatile RNA-degradation machine in eukaryotes. The exosome has a central role in several aspects of RNA biogenesis, including RNA maturation and surveillance. Moreover, it is emerging as an important player in regulating the expression levels of specific mRNAs in response to environmental cues and during cell differentiation and development. Although the mechanisms by which RNA is targeted to (or escapes from) the exosome are still not fully understood, general principles have begun to emerge, which we discuss in this Review. In addition, we introduce and discuss novel, previously unappreciated functions of the nuclear exosome, including in transcription regulation and in the maintenance of genome stability.

  6. Correlated electron-nuclear dynamics with conditional wave functions.

    PubMed

    Albareda, Guillermo; Appel, Heiko; Franco, Ignacio; Abedi, Ali; Rubio, Angel

    2014-08-22

    The molecular Schrödinger equation is rewritten in terms of nonunitary equations of motion for the nuclei (or electrons) that depend parametrically on the configuration of an ensemble of generally defined electronic (or nuclear) trajectories. This scheme is exact and does not rely on the tracing out of degrees of freedom. Hence, the use of trajectory-based statistical techniques can be exploited to circumvent the calculation of the computationally demanding Born-Oppenheimer potential-energy surfaces and nonadiabatic coupling elements. The concept of the potential-energy surface is restored by establishing a formal connection with the exact factorization of the full wave function. This connection is used to gain insight from a simplified form of the exact propagation scheme.

  7. Compositionally distinct nuclear pore complexes of functionally distinct dimorphic nuclei in ciliate Tetrahymena.

    PubMed

    Iwamoto, Masaaki; Osakada, Hiroko; Mori, Chie; Fukuda, Yasuhiro; Nagao, Koji; Obuse, Chikashi; Hiraoka, Yasushi; Haraguchi, Tokuko

    2017-04-06

    The nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of about 30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type, or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Tetrahymena thermophila, each cell contains both a transcriptionally-active macronucleus (MAC) and a germline micronucleus (MIC). By combining in silico analysis, mass spectrometry analysis for immuno-isolated proteins, and subcellular localization analysis of GFP fused proteins, we identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-160 scaffold complex were enriched in MIC NPCs. Strikingly, two paralogs of Nup214 and of Nup153 localized exclusively to either MAC or MIC NPCs. Furthermore, the transmembrane components Pom121 and Pom82 localize exclusively to MAC and MIC NPCs, respectively. Our results argue that functional nuclear dimorphism in ciliates is likely to depend on compositional and structural specificity of NPCs.

  8. Nuclear localization of amyloid-β precursor protein-binding protein Fe65 is dependent on regulated intramembrane proteolysis

    PubMed Central

    Koistinen, Niina A.; Edlund, Anna K.; Menon, Preeti K.; Ivanova, Elena V.; Bacanu, Smaranda

    2017-01-01

    Fe65 is an adaptor protein involved in both processing and signaling of the Alzheimer-associated amyloid-β precursor protein, APP. Here, the subcellular localization was further investigated using TAP-tagged Fe65 constructs expressed in human neuroblastoma cells. Our results indicate that PTB2 rather than the WW domain is important for the nuclear localization of Fe65. Electrophoretic mobility shift of Fe65 caused by phosphorylation was not detected in the nuclear fraction, suggesting that phosphorylation could restrict nuclear localization of Fe65. Furthermore, both ADAM10 and γ-secretase inhibitors decreased nuclear Fe65 in a similar way indicating an important role also of α-secretase in regulating nuclear translocation. PMID:28323844

  9. Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins.

    PubMed

    Michishita, Eriko; Park, Jean Y; Burneskis, Jenna M; Barrett, J Carl; Horikawa, Izumi

    2005-10-01

    Sir2 is a NAD+-dependent protein deacetylase that extends lifespan in yeast and worms. This study examines seven human proteins homologous to Sir2 (SIRT1 through SIRT7) for cellular localization, expression profiles, protein deacetylation activity, and effects on human cell lifespan. We found that: 1) three nuclear SIRT proteins (SIRT1, SIRT6, and SIRT7) show different subnuclear localizations: SIRT6 and SIRT7 are associated with heterochromatic regions and nucleoli, respectively, where yeast Sir2 functions; 2) SIRT3, SIRT4, and SIRT5 are localized in mitochondria, an organelle that links aging and energy metabolism; 3) cellular p53 is a major in vivo substrate of SIRT1 deacetylase, but not the other six SIRT proteins; 4) SIRT1, but not the other two nuclear SIRT proteins, shows an in vitro deacetylase activity on histone H4 and p53 peptides; and 5) overexpression of any one of the seven SIRT proteins does not extend cellular replicative lifespan in normal human fibroblasts or prostate epithelial cells. This study supports the notion that multiple human SIRT proteins have evolutionarily conserved and nonconserved functions at different cellular locations and reveals that the lifespan of normal human cells, in contrast to that of lower eukaryotes, cannot be manipulated by increased expression of a single SIRT protein.

  10. The nuclear localization of low risk HPV11 E7 protein mediated by its zinc binding domain is independent of nuclear import receptors

    SciTech Connect

    Piccioli, Zachary; McKee, Courtney H.; Leszczynski, Anna; Onder, Zeynep; Hannah, Erin C.; Mamoor, Shahan; Crosby, Lauren; Moroianu, Junona

    2010-11-10

    We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7{sub 39-98} localized mostly to the nucleus. The GST-11E7 and GST-11cE7{sub 39-98} were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.

  11. Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. Jr; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

  12. Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

  13. An impact source localization technique for a nuclear power plant by using sensors of different types.

    PubMed

    Choi, Young-Chul; Park, Jin-Ho; Choi, Kyoung-Sik

    2011-01-01

    In a nuclear power plant, a loose part monitoring system (LPMS) provides information on the location and the mass of a loosened or detached metal impacted onto the inner surface of the primary pressure boundary. Typically, accelerometers are mounted on the surface of a reactor vessel to localize the impact location caused by the impact of metallic substances on the reactor system. However, in some cases, the number of accelerometers is not sufficient to estimate the impact location precisely. In such a case, one of useful methods is to utilize other types of sensor that can measure the vibration of the reactor structure. For example, acoustic emission (AE) sensors are installed on the reactor structure to detect leakage or cracks on the primary pressure boundary. However, accelerometers and AE sensors have a different frequency range. The frequency of interest of AE sensors is higher than that of accelerometers. In this paper, we propose a method of impact source localization by using both accelerometer signals and AE signals, simultaneously. The main concept of impact location estimation is based on the arrival time difference of the impact stress wave between different sensor locations. However, it is difficult to find the arrival time difference between sensors, because the primary frequency ranges of accelerometers and AE sensors are different. To overcome the problem, we used phase delays of an envelope of impact signals. This is because the impact signals from the accelerometer and the AE sensor are similar in the whole shape (envelope). To verify the proposed method, we have performed experiments for a reactor mock-up model and a real nuclear power plant. The experimental results demonstrate that we can enhance the reliability and precision of the impact source localization. Therefore, if the proposed method is applied to a nuclear power plant, we can obtain the effect of additional installed sensors.

  14. Real-time Kadanoff-Baym approach to nuclear response functions

    NASA Astrophysics Data System (ADS)

    Köhler, H. S.; Kwong, N. H.

    2016-03-01

    Linear density response functions are calculated for symmetric nuclear matter of normal density by time-evolving two-time Green's functions in real time. Of particular interest is the effect of correlations. The system is therefore initially time-evolved with a collision term calculated in a direct Born approximation until fully correlated. An external time-dependent potential is then applied. The ensuing density fluctuations are recorded to calculate the density response. This method was previously used by Kwong and Bonitz for studying plasma oscillations in a correlated electron gas. The energy-weighted sum-rule for the response function is guaranteed by using conserving self-energy insertions as the method then generates the full vertex-functions. These can alternatively be calculated by solving a Bethe -Salpeter equation as done in works by Bozek et al. The (first order) mean field is derived from a momentum-dependent (non-local) interaction while 2nd order self-energies are calculated using a particle-hole two-body effective (or residual) interaction given by a gaussian local potential. We show results of calculations of the response function S(ɷ,q0 ) for q0 = 0.2, 0.4 and 0.8fm -1. Comparison is made with the nucleons being un-correlated i.e. with only the first order mean field included. We discuss the relation of our work with the Landau quasi-particle theory as applied to nuclear systems by Babu and Brown and followers.

  15. Localized nuclear and perinuclear Ca2+ signals in intact mouse skeletal muscle fibers

    PubMed Central

    Georgiev, Tihomir; Svirin, Mikhail; Jaimovich, Enrique; Fink, Rainer H. A.

    2015-01-01

    Nuclear Ca2+ is important for the regulation of several nuclear processes such as gene expression. Localized Ca2+ signals (LCSs) in skeletal muscle fibers of mice have been mainly studied as Ca2+ release events from the sarcoplasmic reticulum. Their location with regard to cell nuclei has not been investigated. Our study is based on the hypothesis that LCSs associated with nuclei are present in skeletal muscle fibers of adult mice. Therefore, we carried out experiments addressing this question and we found novel Ca2+ signals associated with nuclei of skeletal muscle fibers (with possibly attached satellite cells). We measured localized nuclear and perinuclear Ca2+ signals (NLCSs and PLCSs) alongside cytosolic localized Ca2+ signals (CLCSs) during a hypertonic treatment. We also observed NLCSs under isotonic conditions. The NLCSs and PLCSs are Ca2+ signals in the range of micrometer [FWHM (full width at half maximum): 2.75 ± 0.27 μm (NLCSs) and 2.55 ± 0.17 μm (PLCSs), S.E.M.]. Additionally, global nuclear Ca2+ signals (NGCSs) were observed. To investigate which type of Ca2+ channels contribute to the Ca2+ signals associated with nuclei in skeletal muscle fibers, we performed measurements with the RyR blocker dantrolene, the DHPR blocker nifedipine or the IP3R blocker Xestospongin C. We observed Ca2+ signals associated with nuclei in the presence of each blocker. Nifedipine and dantrolene had an inhibitory effect on the fraction of fibers with PLCSs. The situation for the fraction of fibers with NLCSs is more complex indicating that RyR is less important for the generation of NLCSs compared to the generation of PLCSs. The fraction of fibers with NLCSs and PLCSs is not reduced in the presence of Xestospongin C. The localized perinuclear and intranuclear Ca2+ signals may be a powerful tool for the cell to regulate adaptive processes as gene expression. The intranuclear Ca2+ signals may be particularly interesting in this respect. PMID:26483696

  16. Isotropic proton-detected local-field nuclear magnetic resonancein solids

    SciTech Connect

    Havlin, Robert H.; Walls, Jamie D.; Pines, Alexander

    2004-08-04

    A new nuclear magnetic resonance (NMR) method is presented which produces linear, isotropic proton-detected local-field spectra for InS spin systems in powdered samples. The method, HETeronuclear Isotropic Evolution (HETIE), refocuses the anisotropic portion of the heteronuclear dipolar coupling frequencies by evolving the system under a series of specially designed Hamiltonians and evolution pathways. The theory behind HETIE is represented along with experimental studies conducted on a powdered sample of ferrocene, demonstrating the methodology outlined in this paper. Applications of HETIE for structural determination in solid-state NMR are discussed.

  17. Nuclear localization of a putative Phytophthora sojae bZIP1 transcription factor is mediated by multiple targeting motifs.

    PubMed

    Fang, Yufeng; Tyler, Brett M

    2017-02-18

    Oomycetes are fungal-like eukaryotic microbes in the kingdom Stramenopila. We recently found that the oomycete plant pathogen Phytophthora sojae uses nuclear localization signals (NLSs) for translocation of proteins into the nucleus that differ from conventional well-characterized NLSs from mammals and yeast. Here we have characterized in depth the nuclear localization signals of a P. sojae basic leucine zipper transcription factor, PsbZIP1. Nuclear localization of PsbZIP1 was determined by a central conserved region overlapping the DNA binding domain. Mutational analysis of this region identified four distinct elements that contributed multiplicatively to nuclear localization, but the conserved DNA binding residues were not required. Three of the elements showed autonomous NLS activity and the fourth served as a nuclear localization enhancer. Sequences within two of the nuclear localization elements defined a new form of bipartite NLS consisting of a triplet of basic residues followed by a tail of scattered basic amino acids. This article is protected by copyright. All rights reserved.

  18. Range Separation and Local Hybridization in Density Functional Theory†

    PubMed Central

    Henderson, Thomas M.; Janesko, Benjamin G.; Scuseria, Gustavo E.

    2016-01-01

    Kohn–Sham density functional theory has become a standard method for modeling energetic, spectroscopic, and chemical reactivity properties of large molecules and solids. Density functional theory provides a rigorous theoretical framework for modeling the many-body exchange-correlation effects that dominate the computational cost of traditional wave function approaches. The advent of hybrid exchange-correlation functionals which incorporate a fraction of nonlocal exact exchange has solidified the prominence of density functional theory within computational chemistry. Hybrids provide accurate treatments of properties such as thermochemistry and molecular geometry. But they also exhibit some rather spectacular failures, and often contain multiple empirical parameters. This article reviews our work on developing novel exchange-correlation functionals that build upon the successes of global hybrids. We focus on more flexible functional forms, including local and range-separated hybrid functionals, constructed to obey known exact constraints and (ideally) to incorporate a minimum of empirical parametrization. The article places our work within the context of some other new approximate density functionals and discusses prospects for future work. PMID:19006280

  19. Applying microscopy to the analysis of nuclear structure and function.

    PubMed

    Iborra, Francisco; Cook, Peter R; Jackson, Dean A

    2003-02-01

    One of the ultimate goals of biological research is to understand mechanisms of cell function within living organisms. With this in mind, many sophisticated technologies that allow us to inspect macromolecular structure in exquisite detail have been developed. Although knowledge of structure derived from techniques such as X-ray crystallography and nuclear magnetic resonance is of vital importance, these approaches cannot reveal the remarkable complexity of molecular interactions that exists in vivo. With this in mind, this review focuses on the use of microscopy techniques to analyze cell structure and function. We describe the different basic microscopic methodologies and how the routine techniques are best applied to particular biological problems. We also emphasize the specific capabilities and uses of light and electron microscopy and highlight their individual advantages and disadvantages. For completion, we also comment on the alternative possibilities provided by a variety of advanced imaging technologies. We hope that this brief analysis of the undoubted power of microscopy techniques will be enough to stimulate a wider participation in this rapidly developing area of biological discovery.

  20. Nuclear receptors CAR and PXR: Molecular, functional, and biomedical aspects.

    PubMed

    di Masi, Alessandra; De Marinis, Elisabetta; Ascenzi, Paolo; Marino, Maria

    2009-10-01

    Nuclear receptors (NRs) are ligand-activated transcription factors sharing a common evolutionary history and having similar sequence features at the protein level. Selective ligand(s) for some NRs is not known, therefore these NRs have been named "orphan receptors". Whenever ligands have been recognized for any of the orphan receptor, it has been categorized and grouped as "adopted" orphan receptor. This group includes the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). They function as sensors of toxic byproducts derived from endogenous metabolites and of exogenous chemicals, in order to enhance their elimination. This unique function of CAR and PXR sets them apart from the steroid hormone receptors. The broad response profile has established that CAR and PXR are xenobiotic sensors that coordinately regulate xenobiotic clearance in the liver and intestine via induction of genes involved in drug and xenobiotic metabolism. In the past few years, research has revealed new and mostly unsuspected roles for CAR and PXR in modulating hormone, lipid, and energy homeostasis as well as cancer and liver steatosis. The purpose of this review is to highlight the structural and molecular bases of CAR and PXR impact on human health, providing information on mechanisms through which diet, chemical exposure, and environment ultimately impact health and disease.

  1. Velocity analysis with local event slopes related probability density function

    NASA Astrophysics Data System (ADS)

    Zhang, Peng; Lu, Wenkai; Zhang, Yingqiang

    2015-12-01

    Macro velocity model plays a key role in seismic imaging and inversion. The performance of traditional velocity analysis methods is degraded by multiples and amplitude-versus-offset (AVO) anomalies. Local event slopes, containing the subsurface velocity information, have been widely used to accomplish common time-domain seismic processing, imaging and velocity estimation. In this paper, we propose a method for velocity analysis with probability density function (PDF) related to local event slopes. We first estimate local event slopes with phase information in the Fourier domain. An adaptive filter is applied to improve the performance of slopes estimator in the low signal-to-noise ratio (SNR) situation. Second, the PDF is approximated with the histogram function, which is related to attributes derived from local event slopes. As a graphical representation of the data distribution, the histogram function can be computed efficiently. By locating the ray path of the first arrival on the semblance image with straight-ray segments assumption, automatic velocity picking is carried out to establish velocity model. Unlike local event slopes based velocity estimation strategies such as averaging filters and image warping, the proposed method does not make the assumption that the errors of mapped velocity values are symmetrically distributed or that the variation of amplitude along the offset is slight. Extension of the method to prestack time-domain migration velocity estimation is also given. With synthetic and field examples, we demonstrate that our method can achieve high resolution, even in the presence of multiples, strong amplitude variations and polarity reversals.

  2. Nuclear localization of the dystrophin-associated protein α-dystrobrevin through importin α2/β1 is critical for interaction with the nuclear lamina/maintenance of nuclear integrity.

    PubMed

    Aguilar, Areli; Wagstaff, Kylie M; Suárez-Sánchez, Rocío; Zinker, Samuel; Jans, David A; Cisneros, Bulmaro

    2015-05-01

    Although α-dystrobrevin (DB) is assembled into the dystrophin-associated protein complex, which is central to cytoskeletal organization, it has also been found in the nucleus. Here we delineate the nuclear import pathway responsible for nuclear targeting of α-DB for the first time, together with the importance of nuclear α-DB in determining nuclear morphology. We map key residues of the nuclear localization signal of α-DB within the zinc finger domain (ZZ) using various truncated versions of the protein, and site-directed mutagenesis. Pulldown, immunoprecipitation, and AlphaScreen assays showed that the importin (IMP) α2/β1 heterodimer interacts with high affinity with the ZZ domain of α-DB. In vitro nuclear import assays using antibodies to specific importins, as well as in vivo studies using siRNA or a dominant negative importin construct, confirmed the key role of IMPα2/β1 in α-DB nuclear translocation. Knockdown of α-DB expression perturbed cell cycle progression in C2C12 myoblasts, with decreased accumulation of cells in S phase and, significantly, altered localization of lamins A/C, B1, and B2 with accompanying gross nuclear morphology defects. Because α-DB interacts specifically with lamin B1 in vivo and in vitro, nuclear α-DB would appear to play a key role in nuclear shape maintenance through association with the nuclear lamina.

  3. Local Paley Wiener theorems for functions analytic on unit spheres

    NASA Astrophysics Data System (ADS)

    Damelin, S. B.; Devaney, A. J.

    2007-04-01

    The purpose of this paper is to provide new and simplified statements of local Paley-Wiener theorems on the (n - 1)-dimensional unit sphere realized as a subset of n = 2, 3 Euclidean space. More precisely, given a function f:{\\bb C}^n\\to {\\bb C}, n=2,3 , whose restriction to an n - 1 sphere is analytic, we establish necessary and sufficient conditions determining whether f is the Fourier transform of a compactly supported, bounded function F:{\\bb R}^n\\to{\\bb C} . The essence of this investigation is that, because of the local nature of the problem, the mapping f → F is not in general invertible and so the problem cannot be studied via a Fourier integral. Our proofs are new.

  4. Global functions in global-local finite-element analysis of localized stresses in prismatic structures

    NASA Technical Reports Server (NTRS)

    Dong, Stanley B.

    1989-01-01

    An important consideration in the global local finite-element method (GLFEM) is the availability of global functions for the given problem. The role and mathematical requirements of these global functions in a GLFEM analysis of localized stress states in prismatic structures are discussed. A method is described for determining these global functions. Underlying this method are theorems due to Toupin and Knowles on strain energy decay rates, which are related to a quantitative expression of Saint-Venant's principle. It is mentioned that a mathematically complete set of global functions can be generated, so that any arbitrary interface condition between the finite element and global subregions can be represented. Convergence to the true behavior can be achieved with increasing global functions and finite-element degrees of freedom. Specific attention is devoted to mathematically two-dimensional and three-dimensional prismatic structures. Comments are offered on the GLFEM analysis of NASA flat panel with a discontinuous stiffener. Methods for determining global functions for other effects are also indicated, such as steady-state dynamics and bodies under initial stress.

  5. Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry.

    PubMed

    Maguire, Orla; O'Loughlin, Kieran; Minderman, Hans

    2015-08-01

    Aberrant activity of Nuclear Factor-kappaB (NF-κB) is associated with many diseases and is therapeutically targeted. Post-translational modifications, particularly phosphorylation of the RELA/p65 sub-unit, are essential for cytoplasmic to nuclear localization of NF-κB/p65 and initiation of transcription of downstream target genes. Immunoblot and phospho-flow cytometry have been used to study the relationship between phosphorylation motifs and NF-κB activation and microscopic analysis of nuclear localization of p65 is also used as a parameter for activation. The labor intensive nature of these approaches commonly limits the number of sampling points or replicates. Recent insights into the relationship between p65 phosphorylation motifs and their nuclear localization indicate that these parameters have different significances and should not be used interchangeably. In this study, we demonstrate feasibility and reproducibility of studying the relationship between p65 phosphorylation and nuclear translocation using imaging flow cytometry (IFC). TNFα- or PMA/Ionomycin-induced phosphorylation of p65 at serine 529 in cell line models and healthy donor lymphocytes served as the experimental model. IFC analysis demonstrated that expression of phosphorylated serine 529 (P-p65(s529)) increased rapidly following stimulation and that nuclear localization of P-p65(s529) followed the nuclear localization pattern of total p65. However, in the presence of tacrolimus, P-p65(s529) expression was inhibited without affecting nuclear localization of total p65. The data demonstrate the application of IFC to simultaneously assess phosphorylation of p65 and its cellular localization and the results obtained by this analysis corroborate current insights regarding the specific effect of tacrolimus on serine 529 phosphorylation.

  6. Computation of Local and Global Properties of the Electron Localization Function Topology in Crystals.

    PubMed

    Contreras-García, J; Pendás, A Martín; Recio, J M; Silvi, B

    2009-01-13

    We present a novel computational procedure, general, automated, and robust, for the analysis of local and global properties of the electron localization function (ELF) in crystalline solids. Our algorithm successfully faces the two main shortcomings of the ELF analysis in crystals: (i) the automated identification and characterization of the ELF induced topology in periodic systems, which is impeded by the great number and concentration of critical points in crystalline cells, and (ii) the localization of the zero flux surfaces and subsequent integration of basins, whose difficulty is due to the diverse (in many occasions very flat or very steep) ELF profiles connecting the set of critical points. Application of the new code to representative crystals exhibiting different bonding patterns is carried out in order to show the performance of the algorithm and the conceptual possibilities offered by the complete characterization of the ELF topology in solids.

  7. Nuclear matrix and structural and functional compartmentalization of the eucaryotic cell nucleus.

    PubMed

    Razin, S V; Borunova, V V; Iarovaia, O V; Vassetzky, Y S

    2014-07-01

    Becoming popular at the end of the 20th century, the concept of the nuclear matrix implies the existence of a nuclear skeleton that organizes functional elements in the cell nucleus. This review presents a critical analysis of the results obtained in the study of nuclear matrix in the light of current views on the organization of the cell nucleus. Numerous studies of nuclear matrix have failed to provide evidence of the existence of such a structure. Moreover, the existence of a filamentous structure that supports the nuclear compartmentalization appears to be unnecessary, since this function is performed by the folded genome itself.

  8. Nuclear localization of TEF3-1 promotes cell cycle progression and angiogenesis in cancer.

    PubMed

    Teng, Kaixuan; Deng, Cuilan; Xu, Jie; Men, Qiuxu; Lei, Tao; Di, Da; Liu, Ting; Li, Wenhua; Liu, Xin

    2016-03-22

    TEF3-1 (transcriptional enhancer factor 3 isoform 1), also known as TEAD4 (TEA domain family member 4), was recently revealed as an oncogenic character in cancer development. However, the underlying molecular pathogenic mechanisms remain undefined. In this paper, we investigated nuclear TEF3-1 could promote G1/S transition in HUVECs, and the expression levels of cyclins and CDKs were upregulated. Additionally, if TEF3-1 was knocked down, the expression of cyclins and CDKs was downregulated while the expression of P21, a negative regulator of the cell cycle, was upregulated. A microarray analysis also confirmed that TEF3-1 overexpression upregulates genes that are related to cell cycle progression and the promotion of angiogenesis. Moreover, we observed that nuclear TEF3-1 was highly expressed during the formation of vascular structures in gastric cancer (GC). Finally, tumor xenograft experiments indicated that, when TEF3-1 was knocked down, tumor growth and angiogenesis were also suppressed. Taken together, these results demonstrate for the first time that TEF3-1 localization to the nucleus stimulates the cell cycle progression in HUVECs and specifically contributes to tumor angiogenesis. Nuclear TEF3-1 in HUVECs may serve as an oncogenic biomarker, and the suppression of TEF3-1 may be a potential target in anti-tumor therapy.

  9. Twist1 Enhances Hypoxia Induced Radioresistance in Cervical Cancer Cells by Promoting Nuclear EGFR Localization

    PubMed Central

    Xiong, Hua; Nie, Xin; Zou, Yanmei; Gong, Chen; Li, Yang; Wu, Hua; Qiu, Hong; Yang, Lin; Zhuang, Liang; Zhang, Peng; Zhang, Jing; Wang, Yihua; Xiong, Huihua

    2017-01-01

    Twist1 is a crucial transcription factor that regulates epithelial mesenchymal transition and involves in metastasis. Recent evidence suggests that Twist1 plays important role in hypoxia-induced radioresistance, but the underlying mechanism remains elusive. Here we investigated the change of Twist1 expression in human cervical squamous cancer cell line SiHa after hypoxia treatment. We also explored the role of Twist1 in radioresistance by manipulating the expression level of Twist1. We observed that hypoxia treatment elevated the expression of Twist1 in SiHa cells. Knockdown of Twist1 with siRNA increased the radiosensitivity of SiHa cells under hypoxia condition, accompanied by reduced levels of nuclear Epidermal Growth Factor Receptor (EGFR) and DNA-dependent protein kinase (DNA-PK). Conversely, overexpression of Twist1 led to increased radioresistance of SiHa cells, which in turn increased nuclear EGFR localization and expression levels of nuclear DNA-PK. Moreover, concomitant high expression of hypoxia-inducible factor-1α (HIF-1α) and Twist1 in primary tumors of cervical cancer patients correlated with the worse prognosis after irradiation treatment. Taken together, these data provide new insights into molecular mechanism underlying hypoxia-induced radioresistance in cervical cancer cells, and suggest that Twist1 is a promising molecular target to improve the efficacy of cancer radiotherapy. PMID:28261334

  10. Nuclear localization of Lyn tyrosine kinase mediated by inhibition of its kinase activity

    SciTech Connect

    Ikeda, Kikuko; Nakayama, Yuji; Togashi, Yuuki; Obata, Yuuki; Kuga, Takahisa; Kasahara, Kousuke; Fukumoto, Yasunori; Yamaguchi, Naoto

    2008-11-01

    Src-family kinases, cytoplasmic enzymes that participate in various signaling events, are found at not only the plasma membrane but also subcellular compartments, such as the nucleus, the Golgi apparatus and late endosomes/lysosomes. Lyn, a member of the Src-family kinases, is known to play a role in DNA damage response and cell cycle control in the nucleus. However, it is still unclear how the localization of Lyn to the nucleus is regulated. Here, we investigated the mechanism of the distribution of Lyn between the cytoplasm and the nucleus in epitheloid HeLa cells and hematopoietic THP-1 cells. Lyn was definitely detected in purified nuclei by immunofluorescence and immunoblotting analyses. Nuclear accumulation of Lyn was enhanced upon treatment of cells with leptomycin B (LMB), an inhibitor of Crm1-mediated nuclear export. Moreover, Lyn mutants lacking the sites for lipid modification were highly accumulated in the nucleus upon LMB treatment. Intriguingly, inhibition of the kinase activity of Lyn by SU6656, Csk overexpression, or point mutation in the ATP-binding site induced an increase in nuclear Lyn levels. These results suggest that Lyn being imported into and rapidly exported from the nucleus preferentially accumulates in the nucleus by inhibition of the kinase activity and lipid modification.

  11. Angstrom-Resolution Magnetic Resonance Imaging of Single Molecules via Wave-Function Fingerprints of Nuclear Spins

    NASA Astrophysics Data System (ADS)

    Ma, Wen-Long; Liu, Ren-Bao

    2016-08-01

    Single-molecule sensitivity of nuclear magnetic resonance (NMR) and angstrom resolution of magnetic resonance imaging (MRI) are the highest challenges in magnetic microscopy. Recent development in dynamical-decoupling- (DD) enhanced diamond quantum sensing has enabled single-nucleus NMR and nanoscale NMR. Similar to conventional NMR and MRI, current DD-based quantum sensing utilizes the "frequency fingerprints" of target nuclear spins. The frequency fingerprints by their nature cannot resolve different nuclear spins that have the same noise frequency or differentiate different types of correlations in nuclear-spin clusters, which limit the resolution of single-molecule MRI. Here we show that this limitation can be overcome by using "wave-function fingerprints" of target nuclear spins, which is much more sensitive than the frequency fingerprints to the weak hyperfine interaction between the targets and a sensor under resonant DD control. We demonstrate a scheme of angstrom-resolution MRI that is capable of counting and individually localizing single nuclear spins of the same frequency and characterizing the correlations in nuclear-spin clusters. A nitrogen-vacancy-center spin sensor near a diamond surface, provided that the coherence time is improved by surface engineering in the near future, may be employed to determine with angstrom resolution the positions and conformation of single molecules that are isotope labeled. The scheme in this work offers an approach to breaking the resolution limit set by the "frequency gradients" in conventional MRI and to reaching the angstrom-scale resolution.

  12. Dissecting Torsin/cofactor function at the nuclear envelope: a genetic study

    PubMed Central

    Laudermilch, Ethan; Tsai, Pei-Ling; Graham, Morven; Turner, Elizabeth; Zhao, Chenguang; Schlieker, Christian

    2016-01-01

    The human genome encodes four Torsin ATPases, the functions of which are poorly understood. In this study, we use CRISPR/Cas9 engineering to delete all four Torsin ATPases individually and in combination. Using nuclear envelope (NE) blebbing as a phenotypic measure, we establish a direct correlation between the number of inactivated Torsin alleles and the occurrence of omega-shaped herniations within the lumen of the NE. A similar, although not identical, redundancy is observed for LAP1 and LULL1, which serve as regulatory cofactors for a subset of Torsin ATPases. Unexpectedly, deletion of Tor2A in a TorA/B/3A-deficient background results in a stark increase of bleb formation, even though Tor2A does not respond to LAP1/LULL1 stimulation. The robustness of the observed phenotype in Torsin-deficient cells enables a structural analysis via electron microscopy tomography and a compositional analysis via immunogold labeling. Ubiquitin and nucleoporins were identified as distinctively localizing components of the omega-shaped bleb structure. These findings suggest a functional link between the Torsin/cofactor system and NE/nuclear pore complex biogenesis or homeostasis and establish a Torsin-deficient cell line as a valuable experimental platform with which to decipher Torsin function. PMID:27798237

  13. RAM function is dependent on Kapβ2-mediated nuclear entry.

    PubMed

    Gonatopoulos-Pournatzis, Thomas; Cowling, Victoria H

    2014-02-01

    Eukaryotic gene expression is dependent on the modification of the first transcribed nucleotide of pre-mRNA by the addition of the 7-methylguanosine cap. The cap protects transcripts from exonucleases and recruits complexes which mediate transcription elongation, processing and translation initiation. The cap is synthesized by a series of reactions which link 7-methylguanosine to the first transcribed nucleotide via a 5' to 5' triphosphate bridge. In mammals, cap synthesis is catalysed by the sequential action of RNGTT (RNA guanylyltransferase and 5'-phosphatase) and RNMT (RNA guanine-7 methyltransferase), enzymes recruited to RNA pol II (polymerase II) during the early stages of transcription. We recently discovered that the mammalian cap methyltransferase is a heterodimer consisting of RNMT and the RNMT-activating subunit RAM (RNMT-activating mini-protein). RAM activates and stabilizes RNMT and thus is critical for cellular cap methylation and cell viability. In the present study we report that RNMT interacts with the N-terminal 45 amino acids of RAM, a domain necessary and sufficient for maximal RNMT activation. In contrast, smaller components of this RAM domain are sufficient to stabilize RNMT. RAM functions in the nucleus and we report that nuclear import of RAM is dependent on PY nuclear localization signals and Kapβ2 (karyopherin β2) nuclear transport protein.

  14. Designed inhibitor for nuclear localization signal of polo-like kinase 1 induces mitotic arrest.

    PubMed

    Chen, Fangjin; Zhuo, Xiaolong; Qin, Tan; Guo, Xiao; Zhang, Chuanmao; Lai, Luhua

    2016-11-24

    Polo-like kinase 1 (Plk1), a member of polo-like kinase family, regulates multiple essential steps of the cell cycle progression. Plk1 is overexpressed in multiple cancer cell lines and considered to be a prime anticancer target. Plk1 accumulates in the nucleus during S and G2 phases by its bipartite nuclear localization signal (NLS) sequence, which is crucial for Plk1 regulation during normal cell cycle progression. Here, through combined computational and experimental studies, we identified compound D110, which inhibits Plk1 kinase activity with an IC50 of 85 nm and blocks the nuclear localization of Plk1 during S and G2 phases. D110-treated cancer cells were arrested at mitosis with monopolar spindle, indicating the inhibition of the Plk1 kinase activity in cell. As D110 interacts with both the ATP site and the NLS in Plk1, it demonstrates good selectivity toward Plk2 and Plk3. The strategy of simultaneously inhibiting kinase activity and its subcellular translocations offers a novel approach for selective kinase inhibitor design.

  15. Protein Sub-Nuclear Localization Based on Effective Fusion Representations and Dimension Reduction Algorithm LDA.

    PubMed

    Wang, Shunfang; Liu, Shuhui

    2015-12-19

    An effective representation of a protein sequence plays a crucial role in protein sub-nuclear localization. The existing representations, such as dipeptide composition (DipC), pseudo-amino acid composition (PseAAC) and position specific scoring matrix (PSSM), are insufficient to represent protein sequence due to their single perspectives. Thus, this paper proposes two fusion feature representations of DipPSSM and PseAAPSSM to integrate PSSM with DipC and PseAAC, respectively. When constructing each fusion representation, we introduce the balance factors to value the importance of its components. The optimal values of the balance factors are sought by genetic algorithm. Due to the high dimensionality of the proposed representations, linear discriminant analysis (LDA) is used to find its important low dimensional structure, which is essential for classification and location prediction. The numerical experiments on two public datasets with KNN classifier and cross-validation tests showed that in terms of the common indexes of sensitivity, specificity, accuracy and MCC, the proposed fusing representations outperform the traditional representations in protein sub-nuclear localization, and the representation treated by LDA outperforms the untreated one.

  16. Exponentially localized Wannier functions in periodic zero flux magnetic fields

    NASA Astrophysics Data System (ADS)

    De Nittis, G.; Lein, M.

    2011-11-01

    In this work, we investigate conditions which ensure the existence of an exponentially localized Wannier basis for a given periodic hamiltonian. We extend previous results [Panati, G., Ann. Henri Poincare 8, 995-1011 (2007), 10.1007/s00023-007-0326-8] to include periodic zero flux magnetic fields which is the setting also investigated by Kuchment [J. Phys. A: Math. Theor. 42, 025203 (2009), 10.1088/1751-8113/42/2/025203]. The new notion of magnetic symmetry plays a crucial rôle; to a large class of symmetries for a non-magnetic system, one can associate "magnetic" symmetries of the related magnetic system. Observing that the existence of an exponentially localized Wannier basis is equivalent to the triviality of the so-called Bloch bundle, a rank m hermitian vector bundle over the Brillouin zone, we prove that magnetic time-reversal symmetry is sufficient to ensure the triviality of the Bloch bundle in spatial dimension d = 1, 2, 3. For d = 4, an exponentially localized Wannier basis exists provided that the trace per unit volume of a suitable function of the Fermi projection vanishes. For d > 4 and d ⩽ 2m (stable rank regime) only the exponential localization of a subset of Wannier functions is shown; this improves part of the analysis of Kuchment [J. Phys. A: Math. Theor. 42, 025203 (2009), 10.1088/1751-8113/42/2/025203]. Finally, for d > 4 and d > 2m (unstable rank regime) we show that the mere analysis of Chern classes does not suffice in order to prove triviality and thus exponential localization.

  17. Global and local curvature in density functional theory

    NASA Astrophysics Data System (ADS)

    Zhao, Qing; Ioannidis, Efthymios I.; Kulik, Heather J.

    2016-08-01

    Piecewise linearity of the energy with respect to fractional electron removal or addition is a requirement of an electronic structure method that necessitates the presence of a derivative discontinuity at integer electron occupation. Semi-local exchange-correlation (xc) approximations within density functional theory (DFT) fail to reproduce this behavior, giving rise to deviations from linearity with a convex global curvature that is evidence of many-electron, self-interaction error and electron delocalization. Popular functional tuning strategies focus on reproducing piecewise linearity, especially to improve predictions of optical properties. In a divergent approach, Hubbard U-augmented DFT (i.e., DFT+U) treats self-interaction errors by reducing the local curvature of the energy with respect to electron removal or addition from one localized subshell to the surrounding system. Although it has been suggested that DFT+U should simultaneously alleviate global and local curvature in the atomic limit, no detailed study on real systems has been carried out to probe the validity of this statement. In this work, we show when DFT+U should minimize deviations from linearity and demonstrate that a "+U" correction will never worsen the deviation from linearity of the underlying xc approximation. However, we explain varying degrees of efficiency of the approach over 27 octahedral transition metal complexes with respect to transition metal (Sc-Cu) and ligand strength (CO, NH3, and H2O) and investigate select pathological cases where the delocalization error is invisible to DFT+U within an atomic projection framework. Finally, we demonstrate that the global and local curvatures represent different quantities that show opposing behavior with increasing ligand field strength, and we identify where these two may still coincide.

  18. Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

    SciTech Connect

    Rahayu, Retno; Ohsaki, Eriko; Omori, Hiroko; Ueda, Keiji

    2016-09-15

    In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres, and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. - Highlights: • This is the first report showing LANA dots on mitotic chromosomes by fluorescent microscopy followed by electron microscopy. • LANA dots localized randomly on condensed chromosomes other than centromere/pericentromere and telomere/peritelomre. • Cellular mitotic checkpoint should not be always involved in the segregation of KSHV genomes in the latency.

  19. Functional brain networks develop from a "local to distributed" organization.

    PubMed

    Fair, Damien A; Cohen, Alexander L; Power, Jonathan D; Dosenbach, Nico U F; Church, Jessica A; Miezin, Francis M; Schlaggar, Bradley L; Petersen, Steven E

    2009-05-01

    The mature human brain is organized into a collection of specialized functional networks that flexibly interact to support various cognitive functions. Studies of development often attempt to identify the organizing principles that guide the maturation of these functional networks. In this report, we combine resting state functional connectivity MRI (rs-fcMRI), graph analysis, community detection, and spring-embedding visualization techniques to analyze four separate networks defined in earlier studies. As we have previously reported, we find, across development, a trend toward 'segregation' (a general decrease in correlation strength) between regions close in anatomical space and 'integration' (an increased correlation strength) between selected regions distant in space. The generalization of these earlier trends across multiple networks suggests that this is a general developmental principle for changes in functional connectivity that would extend to large-scale graph theoretic analyses of large-scale brain networks. Communities in children are predominantly arranged by anatomical proximity, while communities in adults predominantly reflect functional relationships, as defined from adult fMRI studies. In sum, over development, the organization of multiple functional networks shifts from a local anatomical emphasis in children to a more "distributed" architecture in young adults. We argue that this "local to distributed" developmental characterization has important implications for understanding the development of neural systems underlying cognition. Further, graph metrics (e.g., clustering coefficients and average path lengths) are similar in child and adult graphs, with both showing "small-world"-like properties, while community detection by modularity optimization reveals stable communities within the graphs that are clearly different between young children and young adults. These observations suggest that early school age children and adults both have

  20. Melanocortin MC₄ receptor expression sites and local function.

    PubMed

    Siljee-Wong, Jacqueline E

    2011-06-11

    The melanocortin MC(4) receptor plays an important role in energy metabolism, but also affects blood pressure, heart rate and erectile function. Localization of the receptors that fulfill these distinct roles is only partially known. Mapping of the melanocortin MC(4) receptor has been stymied by the absence of a functional antibody. Several groups have examined mRNA expression of the melanocortin MC(4) receptor in the rodent brain and transgenic approaches have also been utilized to visualize melanocortin MC(4) receptor expression sites within the brain. Ligand expression and binding studies have provided additional information on the areas of the brain where this elusive receptor is functionally expressed. Finally, microinjection of melanocortin MC(4) receptor ligands in specific nuclei has further served to elucidate the function of melanocortin MC(4) receptors in these nuclei. These combined approaches have helped link the anatomy and function of this receptor, such as the role of paraventricular hypothalamic nucleus melanocortin MC(4) receptor in the regulation of food intake. Intriguingly, however, numerous expression-sites have been identified that have not been linked to a specific receptor function such as those along the optic tract and olfactory tubercle. Further research is needed to clarify the function of the melanocortin MC(4) receptor at these sites.

  1. Cancer-Related Functions and Subcellular Localizations of Septins

    PubMed Central

    Poüs, Christian; Klipfel, Laurence; Baillet, Anita

    2016-01-01

    Since the initial discovery of septin family GTPases, the understanding of their molecular organization and cellular roles keeps being refined. Septins have been involved in many physiological processes and the misregulation of specific septin gene expression has been implicated in diverse human pathologies, including neurological disorders and cancer. In this minireview, we focus on the importance of the subunit composition and subcellular localization of septins relevant to tumor initiation, progression, and metastasis. We especially underline the importance of septin polymer composition and of their association with the plasma membrane, actin, or microtubules in cell functions involved in cancer and in resistance to cancer therapies. Through their scaffolding role, their function in membrane compartmentalization or through their protective function against protein degradation, septins also emerge as critical organizers of membrane-associated proteins and of signaling pathways implicated in cancer-associated angiogenesis, apoptosis, polarity, migration, proliferation, and in metastasis. Also, the question as to which of the free monomers, hetero-oligomers, or filaments is the functional form of mammalian septins is raised and the control over their spatial and temporal localization is discussed. The increasing amount of crosstalks identified between septins and cellular signaling mediators reinforces the exciting possibility that septins could be new targets in anti-cancer therapies or in therapeutic strategies to limit drug resistance. PMID:27878118

  2. Effects of Local Compression on Peroneal Nerve Function in Humans

    NASA Technical Reports Server (NTRS)

    Hargens, Alan R.; Botte, Michael J.; Swenson, Michael R.; Gelberman, Richard H.; Rhoades, Charles E.; Akeson, Wayne H.

    1993-01-01

    A new apparatus was developed to compress the anterior compartment selectively and reproducibly in humans. Thirty-five normal volunteers were studied to determine short-term thresholds of local tissue pressure that produce significant neuromuscular dysfunction. Local tissue fluid pressure adjacent to the deep peroneal nerve was elevated by the compression apparatus and continuously monitored for 2-3 h by the slit catheter technique. Elevation of tissue fluid pressure to within 35-40 mm Hg of diastolic blood pressure (approx. 40 mm Hg of in situ pressure in our subjects) elicited a consistent progression of neuromuscular deterioration including, in order, (a) gradual loss of sensation, as assessed by Semmes-Weinstein monofilaments, (b) subjective complaints, (c) reduced nerve conduction velocity, (d) decreased action potential amplitude of the extensor digitorum brevis muscle, and (e) motor weakness of muscles within the anterior compartment. Generally, higher intracompartment at pressures caused more rapid deterioration of neuromuscular function. In two subjects, when in situ compression levels were 0 and 30 mm Hg, normal neuromuscular function was maintained for 3 h. Threshold pressures for significant dysfunction were not always the same for each functional parameter studied, and the magnitudes of each functional deficit did not always correlate with compression level. This variable tolerance to elevated pressure emphasizes the need to monitor clinical signs and symptoms carefully in the diagnosis of compartment syndromes. The nature of the present studies was short term; longer term compression of myoneural tissues may result in dysfunction at lower pressure thresholds.

  3. Isovector response function of hot nuclear matter with Skyrme interactions

    SciTech Connect

    Braghin, F.L.; Vautherin, D.; Abada, A.

    1995-11-01

    We investigate the role of the effective nucleon-nucleon interaction in the description of giant dipole resonances in hot nuclei. For this purpose we calculate the response function of hot nuclear matter to a small isovector external perturbation using various effective Skyrme interactions. We find that for Skyrme forces with an effective mass close to unity an undamped zero sound mode occurs at zero temperature. This mode gives rise in finite nuclei (calculated via the Steinwedel-Jenssen model) to a resonance whose energy agrees with the observed value. We find that zero sound disappears at a temperature of a few MeV, leaving only a broad peak in the dipole strength. For Skyrme forces with a small value of the effective mass (0.4), there is no zero sound at zero temperature but only a weak peak located too high in energy. The strength distribution in this case is nearly independent of temperature and shows small collective effects. The relevance of these results for the saturation of photon multiplicities observed in recent experiments is pointed out.

  4. Understanding Nuclear Receptor Form and Function Using Structural Biology

    PubMed Central

    Rastinejad, Fraydoon; Huang, Pengxiang; Chandra, Vikas; Khorasanizadeh, Sepideh

    2013-01-01

    Nuclear receptors (NR) are a major transcription factor family whose members selectively bind small molecule lipophilic ligands and transduce those signals into specific changes in gene programs. For over two decades, structural biology efforts were directed exclusively on the individual ligand binding domains (LBDs) or DNA binding domains (DBDs) of NRs. These analyses revealed the basis for both ligand and DNA binding, and also revealed receptor conformations representing both the activated and repressed states. Additionally, crystallographic studies explained how NR LBD surfaces recognize discrete portions of transcriptional coregulators. The many structural snapshots of LBDs have also guided the development of synthetic ligands with therapeutic potential. Yet, the exclusive structural focus on isolated NR domains has made it difficult to conceptualize how all the NR polypeptide segments are coordinated physically and functionally in the context of receptor quaternary architectures. Newly emerged crystal structures of the PPARγ-RXRα heterodimer and HNF-4α homodimer have recently revealed the higher order organizations of these receptor complexes on DNA, as well as the complexity and uniqueness of their domain-domain interfaces. These emerging structural advances promise to better explain how signals in one domain can be allosterically transmitted to distal receptor domains, also providing much better frameworks for guiding future drug discovery efforts. PMID:24103914

  5. E4orf6 variants with separate abilities to augment adenovirus replication and direct nuclear localization of the E1B 55-kilodalton protein.

    PubMed

    Orlando, Joseph S; Ornelles, David A

    2002-02-01

    The E4orf6 protein of group C adenovirus is an oncoprotein that, in association with the E1B 55-kDa protein and by E1B-independent means, promotes virus replication. An arginine-faced amphipathic alpha-helix in the E4orf6 protein is required for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein and to enhance replication of an E4 deletion virus. In this study, E4orf6 protein variants containing arginine substitutions in the amphipathic alpha-helix were analyzed. Two of the six arginine residues within the alpha-helix, arginine-241 and arginine-243, were critical for directing nuclear localization of the E1B 55-kDa protein. The four remaining arginine residues appear to provide a net positive charge for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein. The molecular determinants of the arginine-faced amphipathic alpha-helix that were required for the functional interaction between the E4orf6 and E1B 55-kDa proteins seen in the transfected cell differed from those required to support a productive infection. Several E4orf6 protein variants with arginine-to-glutamic acid substitutions that failed to direct nuclear localization of the E1B 55-kDa protein restored replication of an E4 deletion virus. Additionally, a variant containing an arginine-to-alanine substitution at position 243 that directed nuclear localization of the E1B 55-kDa protein failed to enhance virus replication. These results indicate that the ability of the E4orf6 protein to relocalize the E1B 55-kDa protein to the nucleus can be separated from the ability of the E4orf6 protein to support a productive infection.

  6. The 91-205 amino acid region of AcMNPV ORF34 (Ac34), which comprises a potential C3H zinc finger, is required for its nuclear localization and optimal virus multiplication.

    PubMed

    Qiu, Jianxiang; Tang, Zhimin; Yuan, Meijin; Wu, Wenbi; Yang, Kai

    2017-01-15

    During baculovirus infection, most viral proteins must be imported to the nucleus to support virus multiplication. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf34 (ac34) is an alphabaculovirus unique gene that is required for optimal virus production. Ac34 distributes in both the cytoplasm and the nuclei of virus-infected Sf9 cells, but contains no conventional nuclear localization signal (NLS). In this study, we investigated the nuclear targeting domains in Ac34. Transient expression assays showed that Ac34 localized in both the cytoplasm and the nuclei of Sf9 cells, indicating that no viral protein is required for Ac34 nuclear localization. Subcellular localization analysis of Ac34 truncations and internal deletions fused with green fluorescent protein in plasmid-transfected Sf9 cells identified that the 91-205 amino acid (aa) region is required for Ac34 nuclear localization. Mutations in a potential C3H zinc finger (aa 116-131) in Ac34 resulted in exclusive cytoplasmic distribution of GFP:Ac34, suggesting that the zinc finger is required for Ac34 nuclear localization. To assess the functional importance of Ac34 in the nucleus during virus replication, recombinant AcMNPV bacmids containing a series of Ac34 truncations, internal deletions, or site mutations fused with HA tags were constructed. Subcellular localization analysis showed that Ac34 with internal deletions in aa 91-205 or site mutations in the potential zinc finger was predominantly distributed in the cytoplasm. Viral plaque assays and virus growth curves indicated that disruption of Ac34 nuclear localization significantly impaired virus replication. Taken together, our findings demonstrated that the nuclear localization of Ac34 requires the 91-205 aa region and its nuclear localization is essential for optimal virus replication.

  7. Effect of local anesthetics on serotonin1A receptor function.

    PubMed

    Rao, Bhagyashree D; Shrivastava, Sandeep; Chattopadhyay, Amitabha

    2016-12-01

    The fundamental mechanism behind the action of local anesthetics is still not clearly understood. Phenylethanol (PEtOH) is a constituent of essential oils with a pleasant odor and can act as a local anesthetic. In this work, we have explored the effect of PEtOH on the function of the hippocampal serotonin1A receptor, a representative neurotransmitter receptor belonging to the G protein-coupled receptor (GPCR) family. Our results show that PEtOH induces reduction in ligand binding to the serotonin1A receptor due to lowering of binding affinity, along with a concomitant decrease in the degree of G-protein coupling. Analysis of membrane order using the environment-sensitive fluorescent probe DPH revealed decrease in membrane order with increasing PEtOH concentration, as evident from reduction in rotational correlation time of the probe. Analysis of results obtained shows that the action of local anesthetics could be attributed to the combined effects of specific interaction of the receptor with anesthetics and alteration of membrane properties (such as membrane order). These results assume relevance in the perspective of anesthetic action and could be helpful to achieve a better understanding of the possible role of anesthetics in the function of membrane receptors.

  8. Avoiding coral reef functional collapse requires local and global action.

    PubMed

    Kennedy, Emma V; Perry, Chris T; Halloran, Paul R; Iglesias-Prieto, Roberto; Schönberg, Christine H L; Wisshak, Max; Form, Armin U; Carricart-Ganivet, Juan P; Fine, Maoz; Eakin, C Mark; Mumby, Peter J

    2013-05-20

    Coral reefs face multiple anthropogenic threats, from pollution and overfishing to the dual effects of greenhouse gas emissions: rising sea temperature and ocean acidification. While the abundance of coral has declined in recent decades, the implications for humanity are difficult to quantify because they depend on ecosystem function rather than the corals themselves. Most reef functions and ecosystem services are founded on the ability of reefs to maintain their three-dimensional structure through net carbonate accumulation. Coral growth only constitutes part of a reef's carbonate budget; bioerosion processes are influential in determining the balance between net structural growth and disintegration. Here, we combine ecological models with carbonate budgets and drive the dynamics of Caribbean reefs with the latest generation of climate models. Budget reconstructions using documented ecological perturbations drive shallow (6-10 m) Caribbean forereefs toward an increasingly fragile carbonate balance. We then projected carbonate budgets toward 2080 and contrasted the benefits of local conservation and global action on climate change. Local management of fisheries (specifically, no-take marine reserves) and the watershed can delay reef loss by at least a decade under "business-as-usual" rises in greenhouse gas emissions. However, local action must be combined with a low-carbon economy to prevent degradation of reef structures and associated ecosystem services.

  9. Local and linear chemical reactivity response functions at finite temperature in density functional theory.

    PubMed

    Franco-Pérez, Marco; Ayers, Paul W; Gázquez, José L; Vela, Alberto

    2015-12-28

    We explore the local and nonlocal response functions of the grand canonical potential density functional at nonzero temperature. In analogy to the zero-temperature treatment, local (e.g., the average electron density and the local softness) and nonlocal (e.g., the softness kernel) intrinsic response functions are defined as partial derivatives of the grand canonical potential with respect to its thermodynamic variables (i.e., the chemical potential of the electron reservoir and the external potential generated by the atomic nuclei). To define the local and nonlocal response functions of the electron density (e.g., the Fukui function, the linear density response function, and the dual descriptor), we differentiate with respect to the average electron number and the external potential. The well-known mathematical relationships between the intrinsic response functions and the electron-density responses are generalized to nonzero temperature, and we prove that in the zero-temperature limit, our results recover well-known identities from the density functional theory of chemical reactivity. Specific working equations and numerical results are provided for the 3-state ensemble model.

  10. Local and linear chemical reactivity response functions at finite temperature in density functional theory

    SciTech Connect

    Franco-Pérez, Marco E-mail: ayers@mcmaster.ca E-mail: avela@cinvestav.mx; Ayers, Paul W. E-mail: ayers@mcmaster.ca E-mail: avela@cinvestav.mx; Gázquez, José L. E-mail: ayers@mcmaster.ca E-mail: avela@cinvestav.mx; Vela, Alberto E-mail: ayers@mcmaster.ca E-mail: avela@cinvestav.mx

    2015-12-28

    We explore the local and nonlocal response functions of the grand canonical potential density functional at nonzero temperature. In analogy to the zero-temperature treatment, local (e.g., the average electron density and the local softness) and nonlocal (e.g., the softness kernel) intrinsic response functions are defined as partial derivatives of the grand canonical potential with respect to its thermodynamic variables (i.e., the chemical potential of the electron reservoir and the external potential generated by the atomic nuclei). To define the local and nonlocal response functions of the electron density (e.g., the Fukui function, the linear density response function, and the dual descriptor), we differentiate with respect to the average electron number and the external potential. The well-known mathematical relationships between the intrinsic response functions and the electron-density responses are generalized to nonzero temperature, and we prove that in the zero-temperature limit, our results recover well-known identities from the density functional theory of chemical reactivity. Specific working equations and numerical results are provided for the 3-state ensemble model.

  11. Imaging local brain function with emission computed tomography

    SciTech Connect

    Kuhl, D.E.

    1984-03-01

    Positron emission tomography (PET) using /sup 18/F-fluorodeoxyglucose (FDG) was used to map local cerebral glucose utilization in the study of local cerebral function. This information differs fundamentally from structural assessment by means of computed tomography (CT). In normal human volunteers, the FDG scan was used to determine the cerebral metabolic response to conrolled sensory stimulation and the effects of aging. Cerebral metabolic patterns are distinctive among depressed and demented elderly patients. The FDG scan appears normal in the depressed patient, studded with multiple metabolic defects in patients with multiple infarct dementia, and in the patients with Alzheimer disease, metabolism is particularly reduced in the parietal cortex, but only slightly reduced in the caudate and thalamus. The interictal FDG scan effectively detects hypometabolic brain zones that are sites of onset for seizures in patients with partial epilepsy, even though these zones usually appear normal on CT scans. The future prospects of PET are discussed.

  12. Localization using nonindividualized head-related transfer functions

    NASA Astrophysics Data System (ADS)

    Wenzel, Elizabeth M.; Arruda, Marianne; Kistler, Doris J.; Wightman, Frederic L.

    1993-07-01

    The paper investigates the accuracy of localization by inexperienced listeners of the direction (azimuth and elevation) of wideband noisebursts presented in the free-field or over headphones, with headphone stimuli being synthesized using head-related transfer functions (HRTFs) from a representative subject of Wightman and Kistler (1989). Many subjects showed high rates of front-back and up-down confusions that increased significantly for virtual sources compared to the free-field stimuli. When confusions were resolved, localization of virtual sources was quite accurate and comparable to the free-field sources for 12 out of 16 subjects. The results of this study suggest that, while the interaural cues to horizontal location are robust, the spectral cues considered important for resolving location along a particular cone-of-confusion are distorted by a synthesis process that uses nonindividualized HRTFs.

  13. Library of sophisticated functions for analysis of nuclear spectra

    NASA Astrophysics Data System (ADS)

    Morháč, Miroslav; Matoušek, Vladislav

    2009-10-01

    In the paper we present compact library for analysis of nuclear spectra. The library consists of sophisticated functions for background elimination, smoothing, peak searching, deconvolution, and peak fitting. The functions can process one- and two-dimensional spectra. The software described in the paper comprises a number of conventional as well as newly developed methods needed to analyze experimental data. Program summaryProgram title: SpecAnalysLib 1.1 Catalogue identifier: AEDZ_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEDZ_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 42 154 No. of bytes in distributed program, including test data, etc.: 2 379 437 Distribution format: tar.gz Programming language: C++ Computer: Pentium 3 PC 2.4 GHz or higher, Borland C++ Builder v. 6. A precompiled Windows version is included in the distribution package Operating system: Windows 32 bit versions RAM: 10 MB Word size: 32 bits Classification: 17.6 Nature of problem: The demand for advanced highly effective experimental data analysis functions is enormous. The library package represents one approach to give the physicists the possibility to use the advanced routines simply by calling them from their own programs. SpecAnalysLib is a collection of functions for analysis of one- and two-parameter γ-ray spectra, but they can be used for other types of data as well. The library consists of sophisticated functions for background elimination, smoothing, peak searching, deconvolution, and peak fitting. Solution method: The algorithms of background estimation are based on Sensitive Non-linear Iterative Peak (SNIP) clipping algorithm. The smoothing algorithms are based on the convolution of the original data with several types of filters and algorithms based on discrete

  14. Nuclear medical determination of left ventricular diastolic function in coronary heart disease

    SciTech Connect

    Brugger, P.; Laesser, W.K.; Kullich, W.; Stoiberer, I.; Klein, G.

    1985-06-01

    In 64 patients with coronary heart disease, the left ventricular diastolic function was determined by means of a new nuclear medical method (nuclear stethoscope). The investigations revealed an abnormal diastolic filling in 85.9% of the cases on the basis of the parameters peak filling rate and time to peak filling rate as manifestation of a disturbed ventricular function.

  15. The star formation rate distribution function of the local Universe

    NASA Astrophysics Data System (ADS)

    Bothwell, M. S.; Kennicutt, R. C.; Johnson, B. D.; Wu, Y.; Lee, J. C.; Dale, D.; Engelbracht, C.; Calzetti, D.; Skillman, E.

    2011-08-01

    We present total infrared (IR) and ultraviolet (UV) luminosity functions derived from large representative samples of galaxies at z˜ 0, selected at IR and UV wavelengths from the Imperial IRAS Faint Source Catalogue redshift data base (IIFSCz) catalogue, and the GALEX All-Sky Imaging Survey (AIS), respectively. We augment these with deep Spitzer and GALEX imaging of galaxies in the 11 Mpc Local Volume Legacy (LVL) Survey, allowing us to extend these luminosity functions to lower luminosities (˜106 L⊙), and providing good constraints on the slope of the luminosity function at the extreme faint end for the first time. Using conventional star formation prescriptions, we generate from our data the star formation rate (SFR) distribution function for the local Universe. We find that it has a Schechter form, the faint-end slope has a constant value (to the limits of our data) of α=-1.51 ± 0.08 and the ‘characteristic’ SFR ψ* is 9.2 M⊙ yr-1. We also show the distribution function of the SFR volume density; we then use this to calculate a value for the total SFR volume density at z˜ 0 of 0.025 ± 0.0016 M⊙ yr-1 Mpc-3, of which ˜20 per cent is occurring in starbursts. Decomposing the total star formation by infrared luminosity, it can be seen that 9 ± 1 per cent is due to LIRGs, and 0.7 ± 0.2 per cent is occurring in ULIRGs. By comparing UV and IR emission for galaxies in our sample, we also calculate the fraction of star formation occurring in dust-obscured environments, and examine the distribution of dusty star formation: we find a very shallow slope at the highly extincted end, which may be attributable to line-of-sight orientation effects as well as conventional internal extinction.

  16. A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function.

    PubMed

    Okray, Zeynep; de Esch, Celine E F; Van Esch, Hilde; Devriendt, Koen; Claeys, Annelies; Yan, Jiekun; Verbeeck, Jelle; Froyen, Guy; Willemsen, Rob; de Vrij, Femke M S; Hassan, Bassem A

    2015-04-01

    Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5' untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

  17. Nuclear translocation of glutathione S-transferase {pi} is mediated by a non-classical localization signal

    SciTech Connect

    Kawakatsu, Miho; Goto, Shinji; Yoshida, Takako; Urata, Yoshishige; Li, Tao-Sheng

    2011-08-12

    Highlights: {yields} Nuclear translocation of GST{pi} is abrogated by the deletion of the last 16 amino acid residues in the carboxy-terminal region, indicating that residues 195-208 of GST{pi} are required for nuclear translocation. {yields} The lack of a contiguous stretch of positively charged amino acid residues within the carboxy-terminal region of GST{pi}, suggests that the nuclear translocation of GST{pi} is mediated by a non-classical nuclear localization signal. {yields} An in vitro transport assay shows that the nuclear translocation of GST{pi} is dependent on cytosolic factors and ATP. -- Abstract: Glutathione S-transferase {pi} (GST{pi}), a member of the GST family of multifunctional enzymes, is highly expressed in human placenta and involved in the protection of cellular components against electrophilic compounds or oxidative stress. We have recently found that GST{pi} is expressed in the cytoplasm, mitochondria, and nucleus in some cancer cells, and that the nuclear expression of GST{pi} appears to correlate with resistance to anti-cancer drugs. Although the mitochondrial targeting signal of GST{pi} was previously identified in the amino-terminal region, the mechanism of nuclear translocation remains completely unknown. In this study, we find that the region of GST{pi}195-208 is critical for nuclear translocation, which is mediated by a novel and non-classical nuclear localization signal. In addition, using an in vitro transport assay, we demonstrate that the nuclear translocation of GST{pi} depends on the cytosolic extract and ATP. Although further experiments are needed to understand in depth the precise mechanism of nuclear translocation of GST{pi}, our results may help to establish more efficient anti-cancer therapy, especially with respect to resistance to anti-cancer drugs.

  18. Structure of Importin-α from a Filamentous Fungus in Complex with a Classical Nuclear Localization Signal.

    PubMed

    Bernardes, Natalia E; Takeda, Agnes A S; Dreyer, Thiago R; Freitas, Fernanda Z; Bertolini, Maria Célia; Fontes, Marcos R M

    2015-01-01

    Neurospora crassa is a filamentous fungus that has been extensively studied as a model organism for eukaryotic biology, providing fundamental insights into cellular processes such as cell signaling, growth and differentiation. To advance in the study of this multicellular organism, an understanding of the specific mechanisms for protein transport into the cell nucleus is essential. Importin-α (Imp-α) is the receptor for cargo proteins that contain specific nuclear localization signals (NLSs) that play a key role in the classical nuclear import pathway. Structures of Imp-α from different organisms (yeast, rice, mouse, and human) have been determined, revealing that this receptor possesses a conserved structural scaffold. However, recent studies have demonstrated that the Impα mechanism of action may vary significantly for different organisms or for different isoforms from the same organism. Therefore, structural, functional, and biophysical characterization of different Impα proteins is necessary to understand the selectivity of nuclear transport. Here, we determined the first crystal structure of an Impα from a filamentous fungus which is also the highest resolution Impα structure already solved to date (1.75 Å). In addition, we performed calorimetric analysis to determine the affinity and thermodynamic parameters of the interaction between Imp-α and the classical SV40 NLS peptide. The comparison of these data with previous studies on Impα proteins led us to demonstrate that N. crassa Imp-α possess specific features that are distinct from mammalian Imp-α but exhibit important similarities to rice Imp-α, particularly at the minor NLS binding site.

  19. Functional analysis of the nuclear LIM domain interactor NLI.

    PubMed Central

    Jurata, L W; Gill, G N

    1997-01-01

    LIM homeodomain and LIM-only (LMO) transcription factors contain two tandemly arranged Zn2+-binding LIM domains capable of mediating protein-protein interactions. These factors have restricted patterns of expression, are found in invertebrates as well as vertebrates, and are required for cell type specification in a variety of developing tissues. A recently identified, widely expressed protein, NLI, binds with high affinity to the LIM domains of LIM homeodomain and LMO proteins in vitro and in vivo. In this study, a 38-amino-acid fragment of NLI was found to be sufficient for the association of NLI with nuclear LIM domains. In addition, NLI was shown to form high affinity homodimers through the amino-terminal 200 amino acids, but dimerization of NLI was not required for association with the LIM homeodomain protein Lmxl. Chemical cross-linking analysis revealed higher-order complexes containing multiple NLI molecules bound to Lmx1, indicating that dimerization of NLI does not interfere with LIM domain interactions. Additionally, NLI formed complexes with Lmx1 on the rat insulin I promoter and inhibited the LIM domain-dependent synergistic transcriptional activation by Lmx1 and the basic helix-loop-helix protein E47 from the rat insulin I minienhancer. These studies indicate that NLI contains at least two functionally independent domains and may serve as a negative regulator of synergistic transcriptional responses which require direct interaction via LIM domains. Thus, NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining specific partner interactions. PMID:9315627

  20. Insulin in the brain: sources, localization and functions.

    PubMed

    Ghasemi, Rasoul; Haeri, Ali; Dargahi, Leila; Mohamed, Zahurin; Ahmadiani, Abolhassan

    2013-02-01

    Historically, insulin is best known for its role in peripheral glucose homeostasis, and insulin signaling in the brain has received less attention. Insulin-independent brain glucose uptake has been the main reason for considering the brain as an insulin-insensitive organ. However, recent findings showing a high concentration of insulin in brain extracts, and expression of insulin receptors (IRs) in central nervous system tissues have gathered considerable attention over the sources, localization, and functions of insulin in the brain. This review summarizes the current status of knowledge of the peripheral and central sources of insulin in the brain, site-specific expression of IRs, and also neurophysiological functions of insulin including the regulation of food intake, weight control, reproduction, and cognition and memory formation. This review also considers the neuromodulatory and neurotrophic effects of insulin, resulting in proliferation, differentiation, and neurite outgrowth, introducing insulin as an attractive tool for neuroprotection against apoptosis, oxidative stress, beta amyloid toxicity, and brain ischemia.

  1. Semiparametric Bayesian local functional models for diffusion tensor tract statistics☆

    PubMed Central

    Hua, Zhaowei; Dunson, David B.; Gilmore, John H.; Styner, Martin A.; Zhu, Hongtu

    2012-01-01

    We propose a semiparametric Bayesian local functional model (BFM) for the analysis of multiple diffusion properties (e.g., fractional anisotropy) along white matter fiber bundles with a set of covariates of interest, such as age and gender. BFM accounts for heterogeneity in the shape of the fiber bundle diffusion properties among subjects, while allowing the impact of the covariates to vary across subjects. A nonparametric Bayesian LPP2 prior facilitates global and local borrowings of information among subjects, while an infinite factor model flexibly represents low-dimensional structure. Local hypothesis testing and credible bands are developed to identify fiber segments, along which multiple diffusion properties are significantly associated with covariates of interest, while controlling for multiple comparisons. Moreover, BFM naturally group subjects into more homogeneous clusters. Posterior computation proceeds via an efficient Markov chain Monte Carlo algorithm. A simulation study is performed to evaluate the finite sample performance of BFM. We apply BFM to investigate the development of white matter diffusivities along the splenium of the corpus callosum tract and the right internal capsule tract in a clinical study of neurodevelopment in new born infants. PMID:22732565

  2. Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen☆

    PubMed Central

    Ko, Rinkei; Bennett, Samuel E.

    2011-01-01

    Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl2. The functional significance of the UNG2·PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30–810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-3H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl2. The stimulatory effect of PCNA was increased by the addition of MgCl2; however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl2 and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA. PMID:16216562

  3. The unlikelihood of localized corrosion of nuclear waste packages arising from deliquescent brine formation

    NASA Astrophysics Data System (ADS)

    Apted, M.; Arthur, R.; King, F.; Langmuir, D.; Kessler, J.

    2005-01-01

    The Nuclear Waste Technical Review Board (NWTRB) recently postulated a scenario for the formation of a deliquescent, divalent-cation (Ca, Mg) chloride brine from wind-blown dust on the surface of an alloy 22 container designed to hold radioactive waste. The NWTRB suggested that such brines could lead to localized penetrations of waste packages at a proposed repository in Yucca Mountain, Nevada. In response, the Electric Power Research Institute (EPRI) sponsored an independent analysis, specifically examining and responding to a series of decision points in the NWTRB’s scenario. Only if all of these questions could be answered affirmatively would NWTRB’s scenario result in potential noncompliance with regulatory criteria. The EPRI analysis found that none of the questions has an affirmative answer.

  4. Localization of interchromatin granule cluster and Cajal body components in oocyte nuclear bodies of the hemipterans.

    PubMed

    Bogolyubov, D S; Batalova, F M; Ogorzałek, A

    2007-10-01

    An oocyte nucleus contains different extrachromosomal nuclear domains collectively called nuclear bodies (NBs). In the present work we revealed, using immunogold labeling electron microscopy, some marker components of interchromatin granule clusters (IGCs) and Cajal bodies (CBs) in morphologically heterogeneous oocyte NBs studied in three hemipteran species: Notostira elongata, Capsodes gothicus (Miridae) and Velia caprai (Veliidae). Both IGC and CB counterparts were revealed in oocyte nuclei of the studied species but morphological and biochemical criteria were found to be not sufficient to determine carefully the define type of oocyte NBs. We found that the molecular markers of the CBs (coilin and non-phosphorylated RNA polymerase II) and IGCs (SC35 protein) may be localized in the same NB. Anti-SC35 antibody may decorate not only a granular material representing "true" interchromatin granules but also masks some fibrillar parts of complex NBs. Our first observations on the hemipteran oocyte NBs confirm the high complexity and heterogeneity of insect oocyte IGCs and CBs in comparison with those in mammalian somatic cells and amphibian oocytes.

  5. A mid-infrared look at the dusty nuclear environments of local active galaxies

    NASA Astrophysics Data System (ADS)

    Alonso Herrero, Almudena

    2016-08-01

    Active galactic nuclei are largely explained in the context of a unified theory, by which a geometrically and optically thick torus of gas and dust obscures the AGN central engine. The torus intercepts a substantial amount of flux from the central engine and and reradiates it in the infrared. There are still many open questions about the nature of the torus material and the role of nuclear (< 100 pc) starbursts in feeding and/or obscuring AGNs. Ground-based mid-infrared imaging and spectroscopy on 8-10m class telescopes allow us to study the dusty environments of nearby active galactic nuclei on physical scales of less than 100pc. In this talk I will present results from a mid-infrared sub-arcsecond resolution imaging and spectroscopy survey of a sample of local AGN. The observations were mostly taken with CanariCam on the 10.4m Gran Telescopio Canarias (GTC) through an ESO/GTC large programme and the CanariCam AGN guaranteed time program. I will discuss results on the torus properties of different types of AGN from the modelling of the unresolved infrared emission with the CLUMPY torus models. I will also show that the molecules responsible for the 11.3micron PAH feature survive in the vicinity of the active nucleus and thus this PAH feature can be used to study the nuclear star formation activity in AGN.

  6. Nuclear localization is required for induction of apoptotic cell death by the Rb-associated p84N5 death domain protein.

    PubMed

    Evans, Randall L; Poe, Bryan S; Goodrich, David W

    2002-07-11

    The mechanisms utilized to transduce apoptotic signals that originate from within the nucleus, in response to DNA damage for example, are not well understood. Identifying these mechanisms is important for predicting how tumor cells will respond to genotoxic radiation or chemotherapy. The Rb tumor suppressor protein can inhibit apoptosis triggered by DNA damage, but how it does so is unclear. We have previously characterized a death domain protein, p84N5, that specifically associates with an amino-terminal domain of Rb protein. The p84N5 death domain is required for its ability to trigger apoptotic cell death. Association with Rb protein inhibits p84N5-induced apoptosis suggesting that it may be a mediator of Rb's effects on apoptosis. Unlike other death domain-containing apoptotic signaling proteins, however, p84N5 is localized predominantly within the nucleus of interphase cells. Here we test whether p84N5 requires nuclear localization in order to trigger apoptosis. We identify the p84N5 nuclear localization signal and demonstrate that nuclear localization is required for p84N5-induced apoptosis. To our knowledge, this identifies p84N5 as the first death-domain containing apoptotic signaling protein that functions within the nucleus. By analogy to other death domain containing proteins, p84N5 may play some role in apoptotic signaling within the nucleus. Further, p84N5 is a potential mediator of Rb protein's effects on DNA damage induced apoptosis.

  7. Cloning, localization, and axonemal function of Tetrahymena centrin.

    PubMed

    Guerra, Charles; Wada, Yuuko; Leick, Vagn; Bell, Aaron; Satir, Peter

    2003-01-01

    Centrin, an EF hand Ca(2+) binding protein, has been cloned in Tetrahymena thermophila. It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron. It has > 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25. Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin. Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme. The function of centrin in Ca(2+) control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca(2+) or the Ca(2+)-mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca(2+), increased MT sliding velocity. Antibodies to centrin abrogated this increase. This is the first demonstration of a specific centrin function associated with axonemal dynein. It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca(2+) responses, including ciliary reversal or chemotaxis.

  8. Local functional descriptors for surface comparison based binding prediction

    PubMed Central

    2012-01-01

    Background Molecular recognition in proteins occurs due to appropriate arrangements of physical, chemical, and geometric properties of an atomic surface. Similar surface regions should create similar binding interfaces. Effective methods for comparing surface regions can be used in identifying similar regions, and to predict interactions without regard to the underlying structural scaffold that creates the surface. Results We present a new descriptor for protein functional surfaces and algorithms for using these descriptors to compare protein surface regions to identify ligand binding interfaces. Our approach uses descriptors of local regions of the surface, and assembles collections of matches to compare larger regions. Our approach uses a variety of physical, chemical, and geometric properties, adaptively weighting these properties as appropriate for different regions of the interface. Our approach builds a classifier based on a training corpus of examples of binding sites of the target ligand. The constructed classifiers can be applied to a query protein providing a probability for each position on the protein that the position is part of a binding interface. We demonstrate the effectiveness of the approach on a number of benchmarks, demonstrating performance that is comparable to the state-of-the-art, with an approach with more generality than these prior methods. Conclusions Local functional descriptors offer a new method for protein surface comparison that is sufficiently flexible to serve in a variety of applications. PMID:23176080

  9. Serial assessment of local peripheral vascular function after eccentric exercise.

    PubMed

    Stacy, Mitchel R; Bladon, Kallie J; Lawrence, Jennifer L; McGlinchy, Sarah A; Scheuermann, Barry W

    2013-12-01

    Muscle damage is a common response to unaccustomed eccentric exercise; however, the effects of skeletal muscle damage on local vascular function and blood flow are poorly understood. This study examined serial local vascular responses to flow-mediated (endothelial-dependent) and nitroglycerin-mediated (endothelial-independent) dilation in the brachial artery after strenuous eccentric exercise and serially assessed resting blood flow. Ten healthy males performed 50 maximal eccentric unilateral arm contractions to induce muscle damage to the biceps brachii. Changes in maximal isometric strength and vascular responses were assessed 1, 24, 48, and 96 h after exercise. Mean blood velocities and arterial diameters, measured with Doppler ultrasound, were used to calculate blood flow and shear stress (expressed as area under the curve). Eccentric exercise resulted in impaired maximal isometric strength for up to 96 h (p < 0.001). Reductions in flow-mediated dilation (before exercise, 9.4% ± 2.6%; 1 h after exercise, 5.1% ± 2.2%) and nitroglycerin responses (before exercise, 26.3% ± 6.5%; 1 h after exercise, 20.7% ± 4.7%) were observed in the 1 h after exercise and remained lower for 96 h (p < 0.05). The shear stress response was attenuated immediately after exercise and remained impaired for 48 h (p < 0.05). Resting blood pressure and muscle blood flow remained similar throughout the study. Results suggest that muscle damage from eccentric exercise leads to impaired local endothelial and vascular smooth muscle function. Lower shear stress after exercise might contribute to the observed reduction in flow-mediated dilation responses, but the mechanism responsible for the attenuated endothelial-independent vasodilation remains unclear.

  10. Localization using nonindividualized head-related transfer functions.

    PubMed

    Wenzel, E M; Arruda, M; Kistler, D J; Wightman, F L

    1993-07-01

    A recent development in human-computer interfaces is the virtual acoustic display, a device that synthesizes three-dimensional, spatial auditory information over headphones using digital filters constructed from head-related transfer functions (HRTFs). The utility of such a display depends on the accuracy with which listeners can localize virtual sound sources. A previous study [F. L. Wightman and D. J. Kistler, J. Acoust. Soc. Am. 85, 868-878 (1989)] observed accurate localization by listeners for free-field sources and for virtual sources generated from the subjects' own HRTFs. In practice, measurement of the HRTFs of each potential user of a spatial auditory display may not be feasible. Thus, a critical research question is whether listeners can obtain adequate localization cues from stimuli based on nonindividualized transforms. Here, inexperienced listeners judged the apparent direction (azimuth and elevation) of wideband noisebursts presented in the free-field or over headphones; headphone stimuli were synthesized using HRTFs from a representative subject of Wightman and Kistler. When confusions were resolved, localization of virtual sources was quite accurate and comparable to the free-field sources for 12 of the 16 subjects. Of the remaining subjects, 2 showed poor elevation accuracy in both stimulus conditions, and 2 showed degraded elevation accuracy with virtual sources. Many of the listeners also showed high rates of front-back and up-down confusions that increased significantly for virtual sources compared to the free-field stimuli. These data suggest that while the interaural cues to horizontal location are robust, the spectral cues considered important for resolving location along a particular cone-of-confusion are distorted by a synthesis process that uses nonindividualized HRTFs.

  11. Anomalous organic magnetoresistance from competing carrier-spin-dependent interactions with localized electronic and nuclear spins

    NASA Astrophysics Data System (ADS)

    Flatté, Michael E.

    Transport of carriers through disordered electronic energy landscapes occurs via hopping or tunneling through various sites, and can enhance the effects of carrier spin dynamics on the transport. When incoherent hopping preserves the spin orientation of carriers, the magnetic-field-dependent correlations between pairs of spins influence the charge conductivity of the material. Examples of these phenomena have been identified in hopping transport in organic semiconductors and colloidal quantum dots, as well as tunneling through oxide barriers in complex oxide devices, among other materials. The resulting room-temperature magnetic field effects on the conductivity or electroluminescence require external fields of only a few milliTesla. These magnetic field effects can be dramatically modified by changes in the local spin environment. Recent theoretical and experimental work has identified a regime for low-field magnetoresistance in organic semiconductors in which the spin-relaxing effects of localized nuclear spins and electronic spins interfere1. The regime is studied experimentally by the controlled addition of localized electronic spins, through the addition of a stable free radical (galvinoxyl) to a material (MEH-PPV) that exhibits substantial room-temperature magnetoresistance (20 initially suppressed by the doping, as the localized electronic spin mixes one of the two spins whose correlation controls the transport. At intermediate doping, when one spin is fully decohered but the other is not, there is a regime where the magnetoresistance is insensitive to the doping level. For much greater doping concentrations the magnetoresistance is fully suppressed as both spins that control the charge conductivity of the material are mixed. The behavior is described within a theoretical model describing the effect of carrier spin dynamics on the current. Generalizations to amorphous and other disordered crystalline semiconductors will also be described. This work was

  12. The methylation of the C-terminal region of hnRNPQ (NSAP1) is important for its nuclear localization

    SciTech Connect

    Passos, Dario O.; Quaresma, Alexandre J.C.; Kobarg, Joerg . E-mail: jkobarg@lnls.br

    2006-07-28

    Protein arginine methylation is an irreversible post-translational protein modification catalyzed by a family of at least nine different enzymes entitled PRMTs (protein arginine methyl transferases). Although PRMT1 is responsible for 85% of the protein methylation in human cells, its substrate spectrum has not yet been fully characterized nor are the functional consequences of methylation for the protein substrates well understood. Therefore, we set out to employ the yeast two-hybrid system in order to identify new substrate proteins for human PRMT1. We were able to identify nine different PRMT1 interacting proteins involved in different aspects of RNA metabolism, five of which had been previously described either as substrates for PRMT1 or as functionally associated with PRMT1. Among the four new identified possible protein substrates was hnRNPQ3 (NSAP1), a protein whose function has been implicated in diverse steps of mRNA maturation, including splicing, editing, and degradation. By in vitro methylation assays we were able to show that hnRNPQ3 is a substrate for PRMT1 and that its C-terminal RGG box domain is the sole target for methylation. By further studies with the inhibitor of methylation Adox we provide evidence that hnRNPQ1-3 are methylated in vivo. Finally, we demonstrate by immunofluorescence analysis of HeLa cells that the methylation of hnRNPQ is important for its nuclear localization, since Adox treatment causes its re-distribution from the nucleus to the cytoplasm.

  13. Exact density functional and wave function embedding schemes based on orbital localization

    NASA Astrophysics Data System (ADS)

    Hégely, Bence; Nagy, Péter R.; Ferenczy, György G.; Kállay, Mihály

    2016-08-01

    Exact schemes for the embedding of density functional theory (DFT) and wave function theory (WFT) methods into lower-level DFT or WFT approaches are introduced utilizing orbital localization. First, a simple modification of the projector-based embedding scheme of Manby and co-workers [J. Chem. Phys. 140, 18A507 (2014)] is proposed. We also use localized orbitals to partition the system, but instead of augmenting the Fock operator with a somewhat arbitrary level-shift projector we solve the Huzinaga-equation, which strictly enforces the Pauli exclusion principle. Second, the embedding of WFT methods in local correlation approaches is studied. Since the latter methods split up the system into local domains, very simple embedding theories can be defined if the domains of the active subsystem and the environment are treated at a different level. The considered embedding schemes are benchmarked for reaction energies and compared to quantum mechanics (QM)/molecular mechanics (MM) and vacuum embedding. We conclude that for DFT-in-DFT embedding, the Huzinaga-equation-based scheme is more efficient than the other approaches, but QM/MM or even simple vacuum embedding is still competitive in particular cases. Concerning the embedding of wave function methods, the clear winner is the embedding of WFT into low-level local correlation approaches, and WFT-in-DFT embedding can only be more advantageous if a non-hybrid density functional is employed.

  14. An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

    PubMed

    Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

    2016-04-19

    Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins.

  15. Aurora B–mediated localized delays in nuclear envelope formation facilitate inclusion of late-segregating chromosome fragments

    PubMed Central

    Karg, Travis; Warecki, Brandt; Sullivan, William

    2015-01-01

    To determine how chromosome segregation is coordinated with nuclear envelope formation (NEF), we examined the dynamics of NEF in the presence of lagging acentric chromosomes in Drosophila neuroblasts. Acentric chromosomes often exhibit delayed but ultimately successful segregation and incorporation into daughter nuclei. However, it is unknown whether these late-segregating acentric fragments influence NEF to ensure their inclusion in daughter nuclei. Through live analysis, we show that acentric chromosomes induce highly localized delays in the reassembly of the nuclear envelope. These delays result in a gap in the nuclear envelope that facilitates the inclusion of lagging acentrics into telophase daughter nuclei. Localized delays of nuclear envelope reassembly require Aurora B kinase activity. In cells with reduced Aurora B activity, there is a decrease in the frequency of local nuclear envelope reassembly delays, resulting in an increase in the frequency of acentric-bearing, lamin-coated micronuclei. These studies reveal a novel role of Aurora B in maintaining genomic integrity by promoting the formation of a passageway in the nuclear envelope through which late-segregating acentric chromosomes enter the telophase daughter nucleus. PMID:25877868

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

    SciTech Connect

    Patterson, Edward I.; Dombrovski, Andrew K.; Swarbrick, Crystall M.D.; Raidal, Shane R.; Forwood, Jade K.

    2013-09-06

    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.

  17. Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity

    PubMed Central

    Darricarrère, Nicole; Liu, Na; Watanabe, Toshiaki; Lin, Haifan

    2013-01-01

    The Piwi protein subfamily is essential for Piwi-interacting RNA (piRNA) biogenesis, transposon silencing, and germ-line development, all of which have been proposed to require Piwi endonuclease activity, as validated for two cytoplasmic Piwi proteins in mice. However, recent evidence has led to questioning of the generality of this mechanism for the Piwi members that reside in the nucleus. Drosophila offers a distinct opportunity to study the function of nuclear Piwi proteins because, among three Drosophila Piwi proteins—called Piwi, Aubergine, and Argonaute 3—Piwi is the only member of this subfamily that is localized in the nucleus and expressed in both germ-line and somatic cells in the gonad, where it is responsible for piRNA biogenesis and regulatory functions essential for fertility. In this study, we demonstrate beyond doubt that the slicer activity of Piwi is not required for any known functions in vivo. We show that, in transgenic flies with the DDX catalytic triad of PIWI mutated, neither primary nor secondary piRNA biogenesis is detectably affected, transposons remain repressed, and fertility is normal. Our observations demonstrate that the mechanism of Piwi is independent of its in vitro endonuclease activity. Instead, it is consistent with the alternative mode of Piwi function as a molecule involved in the piRNA-directed guidance of epigenetic factors to chromatin. PMID:23297219

  18. Local, Regional and National Responses for Medical Management of a Radiological/Nuclear Incident

    PubMed Central

    Dainiak, Nicholas; Skudlarska, Beata; Albanese, Joseph

    2013-01-01

    Radiological and nuclear devices may be used by terrorists or may be the source of accidental exposure. A tiered approach has been recommended for response to a terrorist event wherein local, regional, state and federal assets become involved sequentially, as the magnitude in severity of the incident increases. State-wide hospital plans have been developed and published for Connecticut, New York and California. These plans address delineation of responsibilities of various categories of health professionals, protection of healthcare providers, identification and classification of individuals who might have been exposed to and/or contaminated by radiation and, in the case of Connecticut response plan, early management of victims. Regional response programs such as the New England Regional Health Compact (consisting of 6 member states) have been developed to manage consequences of radiation injury. The Department of Homeland Security is ultimately responsible for managing both health consequences and the crisis. Multiple US national response assets may be called upon for use in radiological incidents. These include agencies and programs that have been developed by the Department of Energy, the Environmental Protection Agency and the Department of Defense. Coordination of national, regional and state assets with local response efforts is necessary to provide a timely and efficient response. PMID:23447742

  19. Mechanisms of cellular penetration and nuclear localization of an anti-double strand DNA autoantibody.

    PubMed

    Zack, D J; Stempniak, M; Wong, A L; Taylor, C; Weisbart, R H

    1996-09-01

    An anti-dsDNA Ab, mAb 3E10, was identified that bound membranes of fixed human renal tubular cells, penetrated live murine renal tubular cells in vivo, and localized in the cell nucleus. mAb 3E10 binds both dsDNA and an extracellular matrix protein, HP8/HEVIN, expressed in high endothelial venules. Previous studies showed both shared and distinct binding determinants of mAb 3E10 VH for DNA and HP8/HEVIN. To independently assess the requirement of DNA and HP8/HEVIN in cellular penetration, site-directed mutants of mAb 3E10 VH and V kappa were studied for penetrating kidney cell lines. The results showed that residues required for binding DNA, but not HP8/HEVIN, were necessary for Ab penetration, indicating that cellular penetration required the presence of DNA or binding of Ab to a membrane determinant precisely resembling DNA. Ab Fab penetrated cells, indicating that neither the Fc nor multivalent Ab binding is necessary for cellular penetration. Ab synthesized in the cytoplasm as a result of deleting heavy and light chain signal peptides was not translocated to the nucleus, indicating a mechanism distinct from the usual protein nuclear localization signals and suggesting the need for a membrane-mediated pathway or for post-translational modification of the Ab.

  20. Sequential expression, activity and nuclear localization of prolyl oligopeptidase protein in the developing rat brain.

    PubMed

    Hannula, Mirva J; Männistö, Pekka T; Myöhänen, Timo T

    2011-01-01

    Prolyl oligopeptidase (POP) is a serine protease that hydrolyzes peptides shorter than 30-mer. Some evidence has recently been obtained that POP can generate protein-protein interactions and therefore participate in various physiological and pathological events. Several studies have reported that POP may be involved in neurogenesis since its activity increases during development and can be found in the nucleus of proliferating tissues. In cell cultures, POP has been shown to be localized in the nucleus, but only early in the development, since during maturation it is moved to the cytosol. We have now studied for the first time the expression of POP protein, its enzymatic activity and nuclear localization in vivo in the developing rat brain. We observed that enzymatic activity of POP is highest on embryonic day 18 while the protein amounts reach their peak at birth. Furthermore, POP is located in the nucleus only early in the development but is transferred to the cytosol already before parturition. Our in vivo results confirm the previous cell culture results supporting the role of POP in neurogenesis. A discordance of antenatal protein amounts and enzymatic activities is suggesting a tight regulation of POP activity and possibly even a nonhydrolytic role at that stage.

  1. AHM1, a Novel Type of Nuclear Matrix–Localized, MAR Binding Protein with a Single AT Hook and a J Domain–Homologous Region

    PubMed Central

    Morisawa, Gaku; Han-yama, Atsushi; Moda, Ichiro; Tamai, Atsushi; Iwabuchi, Masaki; Meshi, Tetsuo

    2000-01-01

    Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hook–containing MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain–homologous region and a Zn finger–like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger–like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones. PMID:11041885

  2. [Nuclear cardiology: the present functions and future perspectives].

    PubMed

    Mei, Xiaoli; Fan, Chengzhong

    2013-02-01

    For the past decade, the diagnosis and treatment of coronary artery disease (CAD) has shifted from the traditional model by evaluating coronary artery stenosis with morphological imaging methods to a novel model by focusing on the detection of ischemia for risk stratification. The myocardial perfusion imaging (MPI) using stress single photon emission computed tomography (SPECT) has become the most commonly used stress imaging technique for the diagnosis and treatment of patients with suspected or known CAD. It has got strong supports, including those of the American College of Cardiology, American Heart Association, American Society of Nuclear Cardiology (ACC/AHA/ASNC) and other numerous clinical guidelines. They all stressed that the SPECT MPI is recommended to be used as the "gate keeper" to coronary angiography in order to prevent unnecessary intervention test and save the cost. However, in China the introduction and application of nuclear cardiology was late and highly unbalanced. This leads to the lack of understanding of nuclear cardiology in some clinicians, and there often is misunderstanding on correct selection of coronary angiography, cardiac CT, CT coronary angiography and others for diagnosis and treatment of CAD which results in a trend of over-application of these traditional techniques. In this article, we will focus on the status of nuclear cardiology, including SPECT, positron emission tomography (PET) MPI in the patients with CAD for the diagnosis of ischemia, risk stratification and management decision-making, and also compare it with the traditional morphological imaging techniques. In addition, we will briefly introduce the recent advances in cardiac hybrid imaging and molecular imaging. The aim of this paper is to popularize the knowledge of nuclear cardiology, and promote the rational application of nuclear cardiology in China.

  3. Nuclear Safety Functions of ITER Gas Injection System Instrumentation and Control and the Concept Design

    NASA Astrophysics Data System (ADS)

    Yang, Yu; Maruyama, S.; Fossen, A.; Villers, F.; Kiss, G.; Zhang, Bo; Li, Bo; Jiang, Tao; Huang, Xiangmei

    2016-08-01

    The ITER Gas Injection System (GIS) plays an important role on fueling, wall conditioning and distribution for plasma operation. Besides that, to support the safety function of ITER, GIS needs to implement three nuclear safety Instrumentation and Control (I&C) functions. In this paper, these three functions are introduced with the emphasis on their latest safety classifications. The nuclear I&C design concept is briefly discussed at the end.

  4. Functional implications of local DNA structures in regulatory motifs.

    PubMed

    Xiang, Qian

    2013-01-01

    The three-dimensional structure of DNA has been proposed to be a major determinant for functional transcription factors (TFs) and DNA interaction. Here, we use hydroxyl radical cleavage pattern as a measure of local DNA structure. We compared the conservation between DNA sequence and structure in terms of information content and attempted to assess the functional implications of DNA structures in regulatory motifs. We used statistical methods to evaluate the structural divergence of substituting a single position within a binding site and applied them to a collection of putative regulatory motifs. The following are our major observations: (i) we observed more information in structural alignment than in the corresponding sequence alignment for most of the transcriptional factors; (ii) for each TF, majority of positions have more information in the structural alignment as compared to the sequence alignment; (iii) we further defined a DNA structural divergence score (SD score) for each wild-type and mutant pair that is distinguished by single-base mutation. The SD score for benign mutations is significantly lower than that of switch mutations. This indicates structural conservation is also important for TFBS to be functional and DNA structures will provide previously unappreciated information for TF to realize the binding specificity.

  5. Nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program.

    PubMed

    Matsushita, Akane; Inoue, Haruhiko; Goto, Shingo; Nakayama, Akira; Sugano, Shoji; Hayashi, Nagao; Takatsuji, Hiroshi

    2013-01-01

    The transcriptional activator WRKY45 plays a major role in the salicylic acid/benzothiadiazole-induced defense program in rice. Here, we show that the nuclear ubiquitin-proteasome system (UPS) plays a role in regulating the function of WRKY45. Proteasome inhibitors induced accumulation of polyubiquitinated WRKY45 and transient up-regulation of WRKY45 target genes in rice cells, suggesting that WRKY45 is constantly degraded by the UPS to suppress defense responses in the absence of defense signals. Mutational analysis of the nuclear localization signal indicated that UPS-dependent WRKY45 degradation occurs in the nuclei. Interestingly, the transcriptional activity of WRKY45 after salicylic acid treatment was impaired by proteasome inhibition. The same C-terminal region in WRKY45 was essential for both transcriptional activity and UPS-dependent degradation. These results suggest that UPS regulation also plays a role in the transcriptional activity of WRKY45. It has been reported that AtNPR1, the central regulator of the salicylic acid pathway in Arabidopsis, is regulated by the UPS. We found that OsNPR1/NH1, the rice counterpart of NPR1, was not stabilized by proteasome inhibition under uninfected conditions. We discuss the differences in post-translational regulation of salicylic acid pathway components between rice and Arabidopsis.

  6. Multicolour localization microscopy by point-spread-function engineering

    NASA Astrophysics Data System (ADS)

    Shechtman, Yoav; Weiss, Lucien E.; Backer, Adam S.; Lee, Maurice Y.; Moerner, W. E.

    2016-09-01

    Super-resolution microscopy has revolutionized cellular imaging in recent years. Methods that rely on sequential localization of single point emitters enable spatial tracking at a resolution of ˜10-40 nm. Moreover, tracking and imaging in three dimensions is made possible by various techniques, including point-spread-function (PSF) engineering—namely, encoding the axial (z) position of a point source in the shape that it creates in the image plane. However, efficient multicolour imaging remains a challenge for localization microscopy—a task of the utmost importance for contextualizing biological data. Normally, multicolour imaging requires sequential imaging, multiple cameras or segmented dedicated fields of view. Here, we demonstrate an alternate strategy: directly encoding the spectral information (colour), in addition to three-dimensional position, in the image. By exploiting chromatic dispersion we design a new class of optical phase masks that simultaneously yield controllably different PSFs for different wavelengths, enabling simultaneous multicolour tracking or super-resolution imaging in a single optical path.

  7. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin

    PubMed Central

    Dechat, Thomas; Pfleghaar, Katrin; Sengupta, Kaushik; Shimi, Takeshi; Shumaker, Dale K.; Solimando, Liliana; Goldman, Robert D.

    2008-01-01

    Over the past few years it has become evident that the intermediate filament proteins, the types A and B nuclear lamins, not only provide a structural framework for the nucleus, but are also essential for many aspects of normal nuclear function. Insights into lamin-related functions have been derived from studies of the remarkably large number of disease-causing mutations in the human lamin A gene. This review provides an up-to-date overview of the functions of nuclear lamins, emphasizing their roles in epigenetics, chromatin organization, DNA replication, transcription, and DNA repair. In addition, we discuss recent evidence supporting the importance of lamins in viral infections. PMID:18381888

  8. A Crowdsourced nucleus: Understanding nuclear organization in terms of dynamically networked protein function

    PubMed Central

    Wood, Ashley M.; Garza-Gongora, Arturo G.; Kosak, Steven T.

    2014-01-01

    The spatial organization of the nucleus results in a compartmentalized structure that affects all aspects of nuclear function. This compartmentalization involves genome organization as well as the formation of nuclear bodies and plays a role in many functions, including gene regulation, genome stability, replication, and RNA processing. Here we review the recent findings associated with the spatial organization of the nucleus and reveal that a common theme for nuclear proteins is their ability to participate in a variety of functions and pathways. We consider this multiplicity of function in terms of Crowdsourcing, a recent phenomenon in the world of information technology, and suggest that this model provides a novel way to synthesize the many intersections between nuclear organization and function. PMID:24412853

  9. Nucleoplasmic/nucleolar translocation and identification of a nuclear localization signal (NLS) in Dictyostelium BAF60a/SMARCD1 homologue Snf12.

    PubMed

    Catalano, Andrew; O'Day, Danton H

    2012-09-01

    Dictyostelium is a model eukaryote for the study of several cellular processes; however, comparatively little is known about its nucleolus. Identification of nucleolar proteins is key to understanding this nuclear subcompartment, but only four have been identified in Dictyostelium. As discussed in this article, a potential relationship between nucleolar NumA1 and BAF60a/SMARCD1 suggested BAF60a may also reside in the nucleolus. Here, we identify BAF60a homologue Snf12 as the fifth nucleolar protein in Dictyostelium. Immunolocalization experiments demonstrate that Snf12 is nucleoplasmic, but translocates to nucleoli upon actinomycin-D-induced transcription inhibition (0.05 mg/mL, 4 h). Translocation was accompanied by a microtubule-independent protrusion of nucleolar Snf12 regions from the nucleus followed by detection of Snf12 in cytoplasmic circles for at least 48 h. Residues (372)KRKR(375) are both necessary and sufficient for nucleoplasmic localization of Snf12 and represent a functional nuclear localization signal (NLS), similar to recently identified NLSs in other Dictyostelium proteins. Since nucleolar and nucleoplasmic proteins redistribute during mitosis, we investigated Snf12 dynamics during this time. Dictyostelium undergoes closed mitosis, meaning its nuclear envelope remains intact. Despite this, during metaphase and anaphase Snf12 redistributed throughout the cytoplasm before reaccumulating in the nucleus during telophase, unlike the previously reported nucleoplasmic redistribution of nucleolar NumA1. The nuclear exit of Snf12 was independent of its putative nuclear export signal and not inhibited by exportin inhibition, suggesting that the redistribution of nuclear proteins during mitosis in Dictyostelium is mediated by other mechanisms. Snf12 is the second Dictyostelium nucleolar protein for which its dynamics during mitosis have been investigated.

  10. Human GATA-3 trans-activation, DNA-binding, and nuclear localization activities are organized into distinct structural domains.

    PubMed Central

    Yang, Z; Gu, L; Romeo, P H; Bories, D; Motohashi, H; Yamamoto, M; Engel, J D

    1994-01-01

    GATA-3 is a zinc finger transcription factor which is expressed in a highly restricted and strongly conserved tissue distribution pattern in vertebrate organisms, specifically, in a subset of hematopoietic cells, in cells within the central and peripheral nervous systems, in the kidney, and in placental trophoblasts. Tissue-specific cellular genes regulated by GATA-3 have been identified in T lymphocytes and the placenta, while GATA-3-regulated genes in the nervous system and kidney have not yet been defined. We prepared monoclonal antibodies with which we could dissect the biochemical and functional properties of human GATA-3. The results of these experiments show some anticipated phenotypes, for example, the definition of discrete domains required for specific DNA-binding site recognition (amino acids 303 to 348) and trans activation (amino acids 30 to 74). The signaling sequence for nuclear localization of human GATA-3 is a property conferred by sequences within and surrounding the amino finger (amino acids 249 to 311) of the protein, thereby assigning a function to this domain and thus explaining the curious observation that this zinc finger is dispensable for DNA binding by the GATA family of transcription factors. Images PMID:8114750

  11. On the Usage of Locally Dense Basis Sets in the Calculation of NMR Indirect Nuclear Spin-Spin Coupling Constants

    NASA Astrophysics Data System (ADS)

    Sanchez, Marina; Provasi, Patricio F.; Aucar, Gustavo A.; Sauer, Stephan P. A.

    Locally dense basis sets (nuclear spin-spin couplings in several saturated and unsaturated fluorinated hydrocarbons. We find that the choice of the basis set for each atom belonging to our studied model compounds depends on its location with respect to the coupled fluorine atoms and on the cis/trans or synperiplanar/antiperiplanar conformation of the molecule. Carbon atoms in the bonding path connecting the coupled fluorine atoms have to be described with better basis sets than the carbon atoms outside this path. For the hydrogen atoms directly connected to the coupling pathway in molecules with trans or antiperiplanar conformations and for all hydrogen atoms not directly connected to the coupling pathway one can employ a minimal basis set with only one s-function. Employing these type of LDBSs we can reduce the number of necessary basis functions by about 30% without losing more than about 1 Hz in accuracy. The analysis of the four contributions to the vicinal fluorine-fluorine coupling constants shows that the non-contact orbital paramagnetic term is the most important contribution followed by the also non-contact spin-dipolar term. The Fermi contact term is the largest contribution only in the synperiplanar conformations of 1,2-difluoroethane and -propane.

  12. A mapping of the electron localization function for earth materials

    NASA Astrophysics Data System (ADS)

    Gibbs, G. V.; Cox, D. F.; Ross, N. L.; Crawford, T. D.; Burt, J. B.; Rosso, K. M.

    2005-06-01

    The electron localization function, ELF, generated for a number of geometry-optimized earth materials, provides a graphical representation of the spatial localization of the probability electron density distribution as embodied in domains ascribed to localized bond and lone pair electrons. The lone pair domains, displayed by the silica polymorphs quartz, coesite and cristobalite, are typically banana-shaped and oriented perpendicular to the plane of the SiOSi angle at ~0.60 Å from the O atom on the reflex side of the angle. With decreasing angle, the domains increase in magnitude, indicating an increase in the nucleophilic character of the O atom, rendering it more susceptible to potential electrophilic attack. The Laplacian isosurface maps of the experimental and theoretical electron density distribution for coesite substantiates the increase in the size of the domain with decreasing angle. Bond pair domains are displayed along each of the SiO bond vectors as discrete concave hemispherically-shaped domains at ~0.70 Å from the O atom. For more closed-shell ionic bonded interactions, the bond and lone pair domains are often coalesced, resulting in concave hemispherical toroidal-shaped domains with local maxima centered along the bond vectors. As the shared covalent character of the bonded interactions increases, the bond and lone pair domains are better developed as discrete domains. ELF isosurface maps generated for the earth materials tremolite, diopside, talc and dickite display banana-shaped lone pair domains associated with the bridging O atoms of SiOSi angles and concave hemispherical toroidal bond pair domains associated with the nonbridging ones. The lone pair domains in dickite and talc provide a basis for understanding the bonded interactions between the adjacent neutral layers. Maps were also generated for beryl, cordierite, quartz, low albite, forsterite, wadeite, åkermanite, pectolite, periclase, hurlbutite, thortveitite and vanthoffite. Strategies

  13. A Mapping of the Electron Localization Function for Earth Materials

    SciTech Connect

    Gibbs, Gerald V.; Cox, David F.; Ross, Nancy; Crawford, T Daniel; Burt, Jason; Rosso, Kevin M.

    2005-06-01

    The electron localization function, ELF, generated for a number of geometry-optimized earth materials, provides a graphical representation of the spatial localization of the probability electron density distribution as embodied in domains ascribed to localized bond and lone pair electrons. The lone pair domains, displayed by the silica polymorphs quartz, coesite and cristobalite, are typically banana-shaped and oriented perpendicular to the plane of the SiOSi angle at ~0.60 Å from the O atom on the reflex side of the angle. With decreasing angle, the domains increase in magnitude, indicating an increase in the nucleophilic character of the O atom, rendering it more susceptible to potential electrophilic attack. The Laplacian isosurface maps of the experimental and theoretical electron density distribution for coesite substantiates the increase in the size of the domain with decreasing angle. Bond pair domains are displayed along each of the SiO bond vectors as discrete concave hemispherically-shaped domains at ~0.70 Å from the O atom. For more closed-shell ionic bonded interactions, the bond and lone pair domains are often coalesced, resulting in concave hemispherical toroidal-shaped domains with local maxima centered along the bond vectors. As the shared covalent character of the bonded interactions increases, the bond and lone pair domains are better developed as discrete domains. ELF isosurface maps generated for the earth materials tremolite, diopside, talc and dickite display banana-shaped lone pair domains associated with the bridging O atoms of SiOSi angles and concave hemispherical toroidal bond pair domains associated with the nonbridging ones. The lone pair domains in dickite and talc provide a basis for understanding the bonded interactions between the adjacent neutral layers. Maps were also generated for beryl, cordierite, quartz, low albite, forsterite, wadeite, åkermanite, pectolite, periclase, hurlbutite, thortveitite and vanthoffite. Strategies

  14. Testing for parity violation in nuclei using spin density matrices for nuclear density functionals

    NASA Astrophysics Data System (ADS)

    Barrett, B. R.; Giraud, B. G.

    2015-06-01

    The spin density matrix (SDM) used in atomic and molecular physics is revisited for nuclear physics, in the context of the radial density functional theory. The vector part of the SDM defines a ‘hedgehog’ situation, which exists only if nuclear states contain some amount of parity violation. A toy model is given as an illustrative example.

  15. Nuclear localization of human spermine oxidase isoforms - possible implications in drug response and disease etiology.

    PubMed

    Murray-Stewart, Tracy; Wang, Yanlin; Goodwin, Andrew; Hacker, Amy; Meeker, Alan; Casero, Robert A

    2008-06-01

    The recent discovery of the direct oxidation of spermine via spermine oxidase (SMO) as a mechanism through which specific antitumor polyamine analogues exert their cytotoxic effects has fueled interest in the study of the polyamine catabolic pathway. A major byproduct of spermine oxidation is H2O2, a source of toxic reactive oxygen species. Recent targeted small interfering RNA studies have confirmed that SMO-produced reactive oxygen species are directly responsible for oxidative stress capable of inducing apoptosis and potentially mutagenic DNA damage. In the present study, we describe a second catalytically active splice variant protein of the human spermine oxidase gene, designated SMO5, which exhibits substrate specificities and affinities comparable to those of the originally identified human spermine oxidase-1, SMO/PAOh1, and, as such, is an additional source of H2O2. Importantly, overexpression of either of these SMO isoforms in NCI-H157 human non-small cell lung carcinoma cells resulted in significant localization of SMO protein in the nucleus, as determined by confocal microscopy. Furthermore, cell lines overexpressing either SMO/PAOh1 or SMO5 demonstrated increased spermine oxidation in the nucleus, with accompanying alterations in individual nuclear polyamine concentrations. This increased oxidation of spermine in the nucleus therefore increases the production of highly reactive H2O2 in close proximity to DNA, as well as decreases nuclear spermine levels, thus altering the protective roles of spermine in free radical scavenging and DNA shielding, and resulting in an overall increased potential for oxidative DNA damage in these cells. The results of these studies therefore have considerable significance both with respect to targeting polyamine oxidation as an antineoplastic strategy, and in regard to the potential role of spermine oxidase in inflammation-induced carcinogenesis.

  16. Nuclear localization of human spermine oxidase isoforms – possible implications in drug response and disease etiology

    PubMed Central

    Murray-Stewart, Tracy; Wang, Yanlin; Goodwin, Andrew; Hacker, Amy; Meeker, Alan; Casero, Robert A.

    2013-01-01

    The recent discovery of the direct oxidation of spermine via spermine oxidase (SMO) as a mechanism through which specific antitumor polyamine analogues exert their cytotoxic effects has fueled interest in the study of the polyamine catabolic pathway. A major byproduct of spermine oxidation is H2O2, a source of toxic reactive oxygen species. Recent targeted small interfering RNA studies have confirmed that SMO-produced reactive oxygen species are directly responsible for oxidative stress capable of inducing apoptosis and potentially mutagenic DNA damage. In the present study, we describe a second catalytically active splice variant protein of the human spermine oxidase gene, designated SMO5, which exhibits substrate specificities and affinities comparable to those of the originally identified human spermine oxidase-1, SMO/PAOh1, and, as such, is an additional source of H2O2. Importantly, overexpression of either of these SMO isoforms in NCI-H157 human non-small cell lung carcinoma cells resulted in significant localization of SMO protein in the nucleus, as determined by confocal microscopy. Furthermore, cell lines overexpressing either SMO/PAOh1 or SMO5 demonstrated increased spermine oxidation in the nucleus, with accompanying alterations in individual nuclear polyamine concentrations. This increased oxidation of spermine in the nucleus therefore increases the production of highly reactive H2O2 in close proximity to DNA, as well as decreases nuclear spermine levels, thus altering the protective roles of spermine in free radical scavenging and DNA shielding, and resulting in an overall increased potential for oxidative DNA damage in these cells. The results of these studies therefore have considerable significance both with respect to targeting polyamine oxidation as an antineoplastic strategy, and in regard to the potential role of spermine oxidase in inflammation-induced carcinogenesis. PMID:18422650

  17. Isoform-specific subcellular localization and function of protein kinase A identified by mosaic imaging of mouse brain

    PubMed Central

    Ilouz, Ronit; Lev-Ram, Varda; Bushong, Eric A; Stiles, Travis L; Friedmann-Morvinski, Dinorah; Douglas, Christopher; Goldberg, Geoffrey; Ellisman, Mark H; Taylor, Susan S

    2017-01-01

    Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIβ or the RIIβ subunit results in distinct neuronal phenotypes. Although RIβ contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIβ is enriched in dendrites and co-localizes with MAP2, whereas RIIβ is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIβ in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIβ, but not of RIIβ, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization. DOI: http://dx.doi.org/10.7554/eLife.17681.001 PMID:28079521

  18. Clustered basic amino acids of the small sendai virus C protein Y1 are critical to its RAN GTPase-mediated nuclear localization.

    PubMed

    Irie, Takashi; Yoshida, Asuka; Sakaguchi, Takemasa

    2013-01-01

    The Sendai virus (SeV) C proteins are shown to exert multiple functions during the course of infection. Perhaps reflecting their many functions, they occur at multiple sites of the cell. In this study, we focused on the nuclear-localizing ability of the smaller C protein, Y1, and found that this translocation is mediated by Ran GTPase but not by passive diffusion, and that basic residues within the 149-157 amino acid region are critical for that. The mechanism of inhibition of interferon (IFN)-signaling seemed to differ between the C and Y1 proteins, since deletion of 12 C-terminal amino acids resulted in a loss of the function for the C but not for the Y1 protein. The ability of Y1 mutants to inhibit IFN-α-induced, ISRE-driven expression of a reporter gene almost paralleled with that to localize in the nucleus. These results suggest that nuclear localization of the Y1 protein might be important for the inhibitory effect on type-I IFN-stimulated gene expression.

  19. Sequences within the C Terminus of the Metabotropic Glutamate Receptor 5 (mGluR5) Are Responsible for Inner Nuclear Membrane Localization.

    PubMed

    Sergin, Ismail; Jong, Yuh-Jiin I; Harmon, Steven K; Kumar, Vikas; O'Malley, Karen L

    2017-03-03

    Traditionally, G-protein-coupled receptors (GPCR) are thought to be located on the cell surface where they transmit extracellular signals to the cytoplasm. However, recent studies indicate that some GPCRs are also localized to various subcellular compartments such as the nucleus where they appear required for various biological functions. For example, the metabotropic glutamate receptor 5 (mGluR5) is concentrated at the inner nuclear membrane (INM) where it mediates Ca(2+) changes in the nucleoplasm by coupling with Gq/11 Here, we identified a region within the C-terminal domain (amino acids 852-876) that is necessary and sufficient for INM localization of the receptor. Because these sequences do not correspond to known nuclear localization signal motifs, they represent a new motif for INM trafficking. mGluR5 is also trafficked to the plasma membrane where it undergoes re-cycling/degradation in a separate receptor pool, one that does not interact with the nuclear mGluR5 pool. Finally, our data suggest that once at the INM, mGluR5 is stably retained via interactions with chromatin. Thus, mGluR5 is perfectly positioned to regulate nucleoplasmic Ca(2+)in situ.

  20. Regulation of mammalian microRNA processing and function by cellular signaling and subcellular localization

    PubMed Central

    Smalheiser, Neil R.

    2008-01-01

    For many microRNAs, in many normal tissues and in cancer cells, the cellular levels of mature microRNAs are not simply determined by transcription of microRNA genes. This mini-review will discuss how microRNA biogenesis and function can be regulated by various nuclear and cytoplasmic processing events, including emerging evidence that microRNA pathway components can be selectively regulated by control of their subcellular localization and by modifications that occur during dynamic cellular signaling. Finally, I will briefly summarize studies of microRNAs in synaptic fractions of adult mouse forebrain, which may serve as a model for other cell types as well. PMID:18433727

  1. Identification and punctate nuclear localization of a novel noncoding RNA, Ks-1, from the honeybee brain.

    PubMed Central

    Sawata, Miyuki; Yoshino, Daisuke; Takeuchi, Hideaki; Kamikouchi, Azusa; Ohashi, Kazuaki; Kubo, Takeo

    2002-01-01

    We identified a novel gene, Ks-1, which is expressed preferentially in the small-type Kenyon cells of the honeybee brain. This gene is also expressed in some of the large soma neurons in the brain and in the suboesophageal ganglion. Reverse transcription-polymerase chain reaction experiments indicated that Ks-1 transcripts are enriched in the honeybee brain. cDNA cloning revealed that the consensus Ks-1 cDNA is over 17 kbp and contains no significant open reading frames. Furthermore, fluorescent in situ hybridization revealed that Ks-1 transcripts are located in the nuclei of the neural cells, accumulating in some scattered spots. These findings demonstrate that Ks-1 encodes a novel class of noncoding nuclear RNA and is possibly involved in the regulation of neural functions. PMID:12088150

  2. Role of a Nuclear Localization Signal on the Minor Capsid Proteins VP2 and VP3 in BKPyV Nuclear Entry

    PubMed Central

    Bennett, Shauna M.; Zhao, Linbo; Imperiale, Michael J.

    2014-01-01

    BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin β1, we found that the importin α/β pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell. PMID:25463609

  3. p27 Nuclear localization and growth arrest caused by perlecan knockdown in human endothelial cells

    SciTech Connect

    Sakai, Katsuya; Oka, Kiyomasa; Matsumoto, Kunio; Nakamura, Toshikazu

    2010-02-12

    Perlecan, a secreted heparan sulfate proteoglycan, is a major component of the vascular basement membrane and participates in angiogenesis. Here, we used small interference RNA-mediated knockdown of perlecan expression to investigate the regulatory function of perlecan in the growth of human vascular endothelial cells. Basic fibroblast growth factor (bFGF)-induced ERK phosphorylation and cyclin D1 expression were unchanged by perlecan deficiency in endothelial cells; however, perlecan deficiency inhibited the Rb protein phosphorylation and DNA synthesis induced by bFGF. By contrast to cytoplasmic localization of the cyclin-dependent kinase inhibitor p27 in control endothelial cells, p27 was localized in the nucleus and its expression increased in perlecan-deficient cells, which suggests that p27 mediates inhibition of Rb phosphorylation. In addition to the well-characterized function of perlecan as a co-receptor for heparin-binding growth factors such as bFGF, our results suggest that perlecan plays an indispensible role in endothelial cell proliferation and acts through a mechanism that involves subcellular localization of p27.

  4. Nuclear β-catenin localization supports homology of feathers, avian scutate scales, and alligator scales in early development.

    PubMed

    Musser, Jacob M; Wagner, Günter P; Prum, Richard O

    2015-01-01

    Feathers are an evolutionary novelty found in all extant birds. Despite recent progress investigating feather development and a revolution in dinosaur paleontology, the relationship of feathers to other amniote skin appendages, particularly reptile scales, remains unclear. Disagreement arises primarily from the observation that feathers and avian scutate scales exhibit an anatomical placode-defined as an epidermal thickening-in early development, whereas alligator and other avian scales do not. To investigate the homology of feathers and archosaur scales we examined patterns of nuclear β-catenin localization during early development of feathers and different bird and alligator scales. In birds, nuclear β-catenin is first localized to the feather placode, and then exhibits a dynamic pattern of localization in both epidermis and dermis of the feather bud. We found that asymmetric avian scutate scales and alligator scales share similar patterns of nuclear β-catenin localization with feathers. This supports the hypothesis that feathers, scutate scales, and alligator scales are homologous during early developmental stages, and are derived from early developmental stages of an asymmetric scale present in the archosaur ancestor. Furthermore, given that the earliest stage of β-catenin localization in feathers and archosaur scales is also found in placodes of several mammalian skin appendages, including hair and mammary glands, we hypothesize that a common skin appendage placode originated in the common ancestor of all amniotes. We suggest a skin placode should not be defined by anatomical features, but as a local, organized molecular signaling center from which an epidermal appendage develops.

  5. Cell-specific integration of nuclear receptor function at the genome.

    PubMed

    Everett, Logan J; Lazar, Mitchell A

    2013-01-01

    Nuclear receptors (NRs) encompass a family of regulatory proteins that directly couple small-molecule signaling to transcriptional regulation. Initial studies of specific NR targets led to a model in which NRs bind highly specific DNA motifs in proximal promoter regions and strongly induce gene transcription in response to ligand binding. More recently, genome-wide studies have added to the complexity of this classic model of NR function. In particular, binding of NRs at weaker or alternate motifs is common in the context of DNA assembled into chromatin, and ligand responsiveness varies at different NR target genes. Such findings have led to proposed modifications to the classic view of NR regulation, including the 'assisted loading' model in which NRs assist in opening chromatin rather than compete for binding sites, and context-specific models in which genomic and epigenomic features influence the NR function locally at each binding site. Further elucidation of these mechanisms will be particularly important for understanding cell-specific and ligand-specific functions of each NR. Emerging genomic technologies such as ChIP-seq and GRO-seq provide insights on a larger scale leading to deeper understanding of the complexities of transcriptional regulation by NRs.

  6. Identification and characterization of a nuclear localization signal of TRIM28 that overlaps with the HP1 box.

    PubMed

    Moriyama, Tetsuji; Sangel, Percival; Yamaguchi, Hiroki; Obuse, Chikashi; Miyamoto, Yoichi; Oka, Masahiro; Yoneda, Yoshihiro

    2015-07-03

    Tripartite motif-containing 28 (TRIM28) is a transcription regulator, which forms a repressor complex containing heterochromatin protein 1 (HP1). Here, we report identification of a nuclear localization signal (NLS) within the 462-494 amino acid region of TRIM28 that overlaps with its HP1 binding site, HP1 box. GST-pulldown experiments revealed the interaction of the arginine-rich TRIM28 NLS with various importin α subtypes (α1, α2 and α4). In vitro transport assay demonstrated that nuclear localization of GFP-TRIM28 NLS is mediated by importin αs, in conjunction with importin β1 and Ran. Further, we demonstrated that HP1 and importin αs compete for binding to TRIM28. Together, our findings suggest that importin α has an essential role in the nuclear delivery and preferential HP1 interaction of TRIM28.

  7. A Study of Community Leaders in a Nuclear Host Community: Local Issues, Expectations and Support and Opposition.

    ERIC Educational Resources Information Center

    Bronfman, B. H.

    As part of a continuing effort to assess the social impacts on communities of energy facility planning, construction, operation, and decommissioning, a May 1977 survey of 37 community leaders in Hartsville, Tennessee (site of a nuclear power plant) establishes major local issues (past, present, and future) which leaders feel are important to…

  8. The Effects of Local Supralethal Irradiation on Renal Function

    DTIC Science & Technology

    1974-05-01

    Doyle W. G. Ewald ARMED FORCES RADIOBIOLOGY RESEARCH INSTITUTE Defense Nuclear Agency Bethesda, Maryland App-oved for public release...USN Director ARMED FORCES RADIOBIOLOGY RESEARCH INSTITUTE Defense Nuclear Agency Bethesda, Maryland Approved for public release; distribution...J. Physiol. 182:561-566, 1955. 3. Avioli, L. V., Lazor , M. Z., Cotlove, E., Brace, K. C. and Andrews, J. R. Early effects of radiation on

  9. PML nuclear bodies: regulation, function and therapeutic perspectives.

    PubMed

    Sahin, Umut; Lallemand-Breitenbach, Valérie; de Thé, Hugues

    2014-11-01

    PML nuclear bodies (NBs) were first described by electron microscopy and rediscovered through their treatment-reversible disruption in a rare leukaemia. They recruit multiple partner proteins and now emerge as interferon- and oxidative stress-responsive sumoylation factories. NBs mediate interferon-induced viral restriction, enhance proteolysis, finely tune metabolism and enforce stress-induced senescence. Apart from being markers of cellular stress, PML NBs could be harnessed pharmacologically in a number of conditions, including cancer, viral infection or neurodegenerative diseases.

  10. Role of a nuclear localization signal on the minor capsid Proteins VP2 and VP3 in BKPyV nuclear entry

    SciTech Connect

    Bennett, Shauna M.; Zhao, Linbo; Bosard, Catherine; Imperiale, Michael J.

    2015-01-01

    BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin β1, we found that the importin α/β pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell. - Highlights: • Polyomaviruses must deliver their genome to the nucleus to replicate. • The minor capsid proteins have a well-conserved nuclear localization signal. • Mutation of this NLS diminishes, but does not completely inhibit, infection.

  11. LINC'ing form and function at the nuclear envelope.

    PubMed

    Meinke, Peter; Schirmer, Eric C

    2015-09-14

    The nuclear envelope is an amazing piece of engineering. On one hand it is built like a mediaeval fortress with filament systems reinforcing its membrane walls and its double membrane structure forming a lumen like a castle moat. On the other hand its structure can adapt while maintaining its integrity like a reed bending in a river. Like a fortress it has guarded drawbridges in the nuclear pore complexes, but also has other mechanical means of communication. All this is enabled largely because of the LINC complex, a multi-protein structure that connects the intermediate filament nucleoskeleton across the lumen of the double membrane nuclear envelope to multiple cytoplasmic filament systems that themselves could act simultaneously both like mediaeval buttresses and like lines on a suspension bridge. Although many details of the greater LINC structure remain to be discerned, a number of recent findings are giving clues as to how its structural organization can yield such striking dynamic yet stable properties. Combining double- and triple-helical coiled-coils, intrinsic disorder and order, tissue-specific components, and intermediate filaments enables these unique properties.

  12. Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution

    PubMed Central

    1978-01-01

    This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex- lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis. PMID:102651

  13. Systematic Analysis of the Functional Relevance of Nuclear Structure and Mechanics in Breast Cancer Progression

    DTIC Science & Technology

    2013-07-01

    ANSI Std. Z39.18 Systematic Analysis of the Functional Relevance of Nuclear Structure and Mechanics in Breast Cancer Progression Jan Lammerding... analysis of the functional consequences of changes in the expression of lamins (A, B1, B2, and C) and lamin B receptor on nuclear morphology and...enhanced passage), proliferation, and epithelial-to- mesenchymal transition (EMT). In addition, we proposed to conduct an analysis of samples

  14. The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex Function

    PubMed Central

    Ho, Albert K.; Raczniak, Gregory A.; Ives, Eric B.; Wente, Susan R.

    1998-01-01

    Integral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered the nup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23°C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethal nup116-C phenotype was conducted. One gene (designated SNL1 for suppressor of nup116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity of gle2–1 and nic96-G3 mutant cells. The nic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporin nup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function

  15. Agrobacterium rhizogenes GALLS Protein Contains Domains for ATP Binding, Nuclear Localization, and Type IV Secretion▿

    PubMed Central

    Hodges, Larry D.; Vergunst, Annette C.; Neal-McKinney, Jason; den Dulk-Ras, Amke; Moyer, Deborah M.; Hooykaas, Paul J. J.; Ream, Walt

    2006-01-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2. PMID:17012398

  16. Thr308 determines Akt1 nuclear localization in insulin-stimulated keratinocytes

    SciTech Connect

    Goren, Itamar; Mueller, Elke; Pfeilschifter, Josef

    2008-07-18

    Here, we determined the localization and activation of protein kinase B (Akt) in acute cutaneous wound tissue in mice. Akt1 represented the major Akt isoform that was expressed and activated in wound margin keratinocytes and also in the cultured human keratinocyte line HaCaT. Mutation of Akt1 protein, exchanging the activation-essential Ser473 and Thr308 residues for inactive Ala or phosphorylation-mimicking Asp and Glu residues, revealed that phosphorylation of Ser473 represented an essential prerequisite for auto-phosphorylation of Thr308 within the Akt1 protein in keratinocytes. Moreover, cell culture experiments and transfection studies using Thr308 mutated Akt1 proteins demonstrated that phosphorylation of Akt1 at Thr308 appeared to selectively exclude the active kinase from the nucleus and direct the kinase to the cytoplasmic compartment in keratinocytes upon insulin stimulation. In summary, our data show that phosphorylation of Thr308 during insulin-mediated Akt1 activation is an essential prerequisite to exclude Akt1 from the nuclear compartment.

  17. The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen.

    PubMed Central

    Rihs, H P; Jans, D A; Fan, H; Peters, R

    1991-01-01

    We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport. Images PMID:1848177

  18. Local seismic network for monitoring of a potential nuclear power plant area

    NASA Astrophysics Data System (ADS)

    Tiira, Timo; Uski, Marja; Kortström, Jari; Kaisko, Outi; Korja, Annakaisa

    2016-04-01

    This study presents a plan for seismic monitoring of a region around a potential nuclear power plant. Seismic monitoring is needed to evaluate seismic risk. The International Atomic Energy Agency has set guidelines on seismic hazard evaluation and monitoring of such areas. According to these guidelines, we have made a plan for a local network of seismic stations to collect data for seismic source characterization and seismotectonic interpretations, as well as to monitor seismic activity and natural hazards. The detection and location capability of the network were simulated using different station configurations by computing spatial azimuthal coverages and detection threshold magnitudes. Background noise conditions around Pyhäjoki were analyzed by comparing data from different stations. The annual number of microearthquakes that should be detected with a dense local network centered around Pyhäjoki was estimated. The network should be dense enough to fulfill the requirements of azimuthal coverage better than 180° and automatic event location capability down to ML ˜ 0 within a distance of 25 km from the site. A network of 10 stations should be enough to reach these goals. With this setup, the detection threshold magnitudes are estimated to be ML = -0.1 and ML = 0.1 within a radius of 25 and 50 km from Pyhäjoki, respectively. The annual number of earthquakes detected by the network is estimated to be 2 (ML ≥ ˜ -0.1) within 25 km radius and 5 (ML ≥ ˜-0.1 to ˜0.1) within 50 km radius. The location accuracy within 25 km radius is estimated to be 1-2 and 4 km for horizontal coordinates and depth, respectively. Thus, the network is dense enough to map out capable faults with horizontal accuracy of 1-2 km within 25 km radius of the site. The estimation is based on the location accuracies of five existing networks in northern Europe. Local factors, such as seismic noise sources, geology and infrastructure might limit the station configuration and detection and

  19. Local seismic network for monitoring of a potential nuclear power plant area.

    PubMed

    Tiira, Timo; Uski, Marja; Kortström, Jari; Kaisko, Outi; Korja, Annakaisa

    2016-01-01

    This study presents a plan for seismic monitoring of a region around a potential nuclear power plant. Seismic monitoring is needed to evaluate seismic risk. The International Atomic Energy Agency has set guidelines on seismic hazard evaluation and monitoring of such areas. According to these guidelines, we have made a plan for a local network of seismic stations to collect data for seismic source characterization and seismotectonic interpretations, as well as to monitor seismic activity and natural hazards. The detection and location capability of the network were simulated using different station configurations by computing spatial azimuthal coverages and detection threshold magnitudes. Background noise conditions around Pyhäjoki were analyzed by comparing data from different stations. The annual number of microearthquakes that should be detected with a dense local network centered around Pyhäjoki was estimated. The network should be dense enough to fulfill the requirements of azimuthal coverage better than 180° and automatic event location capability down to ML ∼ 0 within a distance of 25 km from the site. A network of 10 stations should be enough to reach these goals. With this setup, the detection threshold magnitudes are estimated to be ML = -0.1 and ML = 0.1 within a radius of 25 and 50 km from Pyhäjoki, respectively. The annual number of earthquakes detected by the network is estimated to be 2 (ML ≥ ∼ -0.1) within 25 km radius and 5 (ML ≥ ∼-0.1 to ∼0.1) within 50 km radius. The location accuracy within 25 km radius is estimated to be 1-2 and 4 km for horizontal coordinates and depth, respectively. Thus, the network is dense enough to map out capable faults with horizontal accuracy of 1-2 km within 25 km radius of the site. The estimation is based on the location accuracies of five existing networks in northern Europe. Local factors, such as seismic noise sources, geology and infrastructure might limit the station

  20. What Is the Actual Local Crystalline Structure of Uranium Dioxide, UO2? A New Perspective for the Most Used Nuclear Fuel.

    PubMed

    Desgranges, L; Ma, Y; Garcia, Ph; Baldinozzi, G; Siméone, D; Fischer, H E

    2017-01-03

    Up to now, uranium dioxide, the most used nuclear fuel, was said to have a Fm3̅m crystalline structure from 30 to 3000 K, and its behavior was modeled under this assumption. However, recently X-ray diffraction experiments provided atomic pair-distribution functions of UO2, in which UO distance was shorter than the expected value for the Fm3̅m space group. Here we show neutron diffraction results that confirm this shorter UO bond, and we also modeled the corresponding pair-distribution function showing that UO2 has a local Pa3̅ symmetry. The existence of a local lower symmetry in UO2 could explain some unexpected properties of UO2 that would justify UO2 modeling to be reassessed. It also deserves more study from an academic point of view because of its good thermoelectric properties that may originate from its particular crystalline structure.

  1. Test of nuclear wave functions for pseudospin symmetry.

    PubMed

    Ginocchio, J N; Leviatan, A

    2001-08-13

    Using the fact that pseudospin is an approximate symmetry of the Dirac Hamiltonian with realistic scalar and vector mean fields, we derive the wave functions of the pseudospin partners of eigenstates of a realistic Dirac Hamiltonian and compare these wave functions with the wave functions of the Dirac eigenstates.

  2. Neuropeptide Y system in the retina: From localization to function.

    PubMed

    Santos-Carvalho, Ana; Ambrósio, António Francisco; Cavadas, Cláudia

    2015-07-01

    The retina is a highly complex structure where several types of cells communicate through countless different molecules to codify visual information. Each type of cells plays unique roles in the retina, presenting a singular expression of neurotransmitters. Some neurotransmitter systems in the retina are well understood, while others need to be better explored to unravel the intricate signaling system involved. Neuropeptide Y (NPY), a 36 amino acid peptide, is one of the most common peptide neurotransmitter in the CNS and a highly conserved peptide among species. We review the localization of NPY and NPY receptors (mainly NPY Y1, Y2, Y4 and Y5) in retinal cells. Common features of the expression of NPY and NPY receptors in mammalian and non-mammalian species indicate universal roles of this system in the retina. In the present review, we highlight the putative roles of NPY receptor activation in the retina, discussing, in particular, their involvement in retinal development, neurotransmitter release modulation, neuroprotection, microglia and Muller cells function, retinal pigmented epithelium changes, retinal endothelial physiology and proliferation of retinal progenitor cells. Further studies are needed to confirm that targeting the NPY system might be a potential therapeutic strategy for retinal degenerative diseases.

  3. Non-locality, adiabaticity, thermodynamics and electron energy probability functions

    NASA Astrophysics Data System (ADS)

    Boswell, Roderick; Zhang, Yunchao; Charles, Christine; Takahashi, Kazunori

    2016-09-01

    Thermodynamic properties are revisited for electrons that are governed by nonlocal electron energy probability functions in a plasma of low collisionality. Measurements in a laboratory helicon double layer experiment have shown that the effective electron temperature and density show a polytropic correlation with an index of γe = 1 . 17 +/- 0 . 02 along the divergent magnetic field, implying a nearly isothermal plasma (γe = 1) with heat being brought into the system. However, the evolution of electrons along the divergent magnetic field is essentially an adiabatic process, which should have a γe = 5 / 3 . The reason for this apparent contradiction is that the nearly collisionless plasma is very far from local thermodynamic equilibrium and the electrons behave nonlocally. The corresponding effective electron enthalpy has a conservation relation with the potential energy, which verifies that there is no heat transferred into the system during the electron evolution. The electrons are shown in nonlocal momentum equilibrium under the electric field and the gradient of the effective electron pressure. The convective momentum of ions, which can be assumed as a cold species, is determined by the effective electron pressure and the effective electron enthalpy is shown to be the source for ion acceleration. For these nearly collisionless plasmas, the use of traditional thermodynamic concepts can lead to very erroneous conclusions regarding the thermal conductivity.

  4. Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells

    PubMed Central

    Lundberg, Lindsay; Pinkham, Chelsea; de la Fuente, Cynthia; Brahms, Ashwini; Shafagati, Nazly; Wagstaff, Kylie M.; Jans, David A.; Tamir, Sharon; Kehn-Hall, Kylene

    2016-01-01

    The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host’s primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus. PMID:27902702

  5. Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells.

    PubMed

    Lundberg, Lindsay; Pinkham, Chelsea; de la Fuente, Cynthia; Brahms, Ashwini; Shafagati, Nazly; Wagstaff, Kylie M; Jans, David A; Tamir, Sharon; Kehn-Hall, Kylene

    2016-11-01

    The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host's primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.

  6. Function of the nuclear receptor FTZ-F1 during the pupal stage in Drosophila melanogaster.

    PubMed

    Sultan, Abdel-Rahman S; Oish, Yasuhiro; Ueda, Hitoshi

    2014-04-01

    The nuclear receptor βFTZ-F1 is expressed in most cells in a temporally specific manner, and its expression is induced immediately after decline in ecdysteroid levels. This factor plays important roles during embryogenesis, larval ecdysis, and early metamorphic stages. However, little is known about the expression pattern, regulation and function of this receptor during the pupal stage. We analyzed the expression pattern and regulation of ftz-f1 during the pupal period, as well as the phenotypes of RNAi knockdown or mutant animals, to elucidate its function during this stage. Western blotting revealed that βFTZ-F1 is expressed at a high level during the late pupal stage, and this expression is dependent on decreasing ecdysteroid levels. By immunohistological analysis of the late pupal stage, FTZ-F1 was detected in the nuclei of most cells, but cytoplasmic localization was observed only in the oogonia and follicle cells of the ovary. Both the ftz-f1 genetic mutant and temporally specific ftz-f1 knockdown using RNAi during the pupal stage showed defects in eclosion and in the eye, the antennal segment, the wing and the leg, including bristle color and sclerosis. These results suggest that βFTZ-F1 is expressed in most cells at the late pupal stage, under the control of ecdysteroids and plays important roles during pupal development.

  7. Processivity factor of KSHV contains a nuclear localization signal and binding domains for transporting viral DNA polymerase into the nucleus

    SciTech Connect

    Chen Yali; Ciustea, Mihai; Ricciardi, Robert P. . E-mail: ricciardi@biochem.dental.upenn.edu

    2005-09-30

    Kaposi's sarcoma-associated human herpesvirus (KSHV) encodes a processivity factor (PF-8, ORF59) that forms homodimers and binds to viral DNA polymerase (Pol-8, ORF9). PF-8 is essential for stabilizing Pol-8 on template DNA so that Pol-8 can incorporate nucleotides continuously. Here, the intracellular interaction of these two viral proteins was examined by confocal immunofluorescence microscopy. When individually expressed, PF-8 was observed exclusively in the nucleus, whereas Pol-8 was found only in the cytoplasm. However, when co-expressed, Pol-8 was co-translocated with PF-8 into the nucleus. Mutational analysis revealed that PF-8 contains a nuclear localization signal (NLS) as well as domains located at the N-terminus and the C-proximal regions that are required for Pol-8 binding. This study suggests that the mechanism that enables PF-8 to transport Pol-8 into the nucleus is the first critical step required for Pol-8 and PF-8 to function processively in KSHV DNA synthesis.

  8. Local Water Dynamics in Coacervated Polyelectrolytes Monitored Through Dynamic Nuclear Polarization-Enhanced 1H NMR

    PubMed Central

    Kausik, Ravinath; Srivastava, Aasheesh; Korevaar, Peter A.; Stucky, Galen; Waite, J. Herbert

    2009-01-01

    We present the first study of quantifying the diffusion coefficient of interfacial water on polyelectrolyte surfaces of systems fully dispersed in bulk water under ambient conditions. Such measurements were made possible through the implementation of a recently introduced Dynamic Nuclear Polarization (DNP) technique to selectively amplify the nuclear magnetic resonance (NMR) signal of hydration water that is interacting with specifically located spin labels on polyelectrolyte surfaces. The merit of this novel capability is demonstrated in this report through the measurement of solvent microvisosity on the surface of two types of oppositely charged polyelectrolytes, when freely dissolved versus when complexed to form a liquid-liquid colloidal phase called complex coacervates. These complex coacervates were formed through electrostatic complexation between the imidazole-based cationic homopolymer poly(N-vinylimidazole) (PVIm), and anionic polypeptide polyaspartate (PAsp) in the pH range of 4.5 – 6.0, under which conditions the coacervate droplets are highly fluidic yet densely packed with polyelectrolytes. We also investigated the rotational diffusion coefficients of the spin labels covalently bound to the polyelectrolyte chains for both PVIm and PAsp, showing a 5 fold change in the rotational correlation time as well as anisotropy parameter upon coacervation, which represents a surprisingly small decrease given the high polymer concentration inside the dense microdroplets. For both DNP and ESR experiments, the polymers were covalently tagged with stable nitroxide radical spin labels (∼1 wt %) to probe the local solvent and polymer segment dynamics. We found that the surface water diffusion coefficients near uncomplexed PVIm and PAsp at pH 8 differ, and are around D∼1.3×10−9 m2 / s. In contrast, inside the complex coacervate phase, the water diffusion coefficient in the immediate vicinity of either polyelectrolyte was D∼ 0.25×10−9 m2 / s, which is about

  9. DIFFERENT REQUIREMENTS OF THE KINASE AND UHM DOMAINS OF KIS FOR ITS NUCLEAR LOCALIZATION AND BINDING TO SPLICING FACTORS

    PubMed Central

    Manceau, Valérie; Kielkopf, Clara L.; Sobel, André; Maucuer, Alexandre

    2008-01-01

    Summary The protein kinase KIS is made by the juxtaposition of a unique kinase domain and a C-terminal domain with a U2AF Homology Motif (UHM), a sequence motif for protein interaction initially identified in the heterodimeric pre-mRNA splicing factor U2AF. This domain of KIS is closely related to the C-terminal UHM domain of the U2AF large subunit, U2AF65. KIS phosphorylates the splicing factor SF1, which in turn enhances SF1 binding to U2AF65 and the 3′ splice site, an event known to take place at an early step of spliceosome assembly. Here, the analysis of the subcellular localization of mutated forms of KIS indicates that the kinase domain of KIS is the necessary domain for its nuclear localization. As in the case of U2AF65, the UHM containing C-terminal domain of KIS is required for binding to the splicing factors SF1 and SF3b155. The efficiency of KIS binding to SF1 and SF3b155 is similar to that of U2AF65 in pull-down assays. These results further support the functional link of KIS with splicing factors. Interestingly, when compared to other UHM containing proteins, KIS presents a different specificity for the UHM docking sites that are present in the N-terminal region of SF3b155, thus providing a new insight into the variety of interactions mediated by UHM domains. PMID:18588901

  10. The "genomic storm" induced by bacterial endotoxin is calmed by a nuclear transport modifier that attenuates localized and systemic inflammation.

    PubMed

    DiGiandomenico, Antonio; Veach, Ruth Ann; Zienkiewicz, Jozef; Moore, Daniel J; Wylezinski, Lukasz S; Hutchens, Martha A; Hawiger, Jacek

    2014-01-01

    Lipopolysaccharide (LPS) is a potent microbial virulence factor that can trigger production of proinflammatory mediators involved in the pathogenesis of localized and systemic inflammation. Importantly, the role of nuclear transport of stress responsive transcription factors in this LPS-generated "genomic storm" remains largely undefined. We developed a new nuclear transport modifier (NTM) peptide, cell-penetrating cSN50.1, which targets nuclear transport shuttles importin α5 and importin β1, to analyze its effect in LPS-induced localized (acute lung injury) and systemic (lethal endotoxic shock) murine inflammation models. We analyzed a human genome database to match 46 genes that encode cytokines, chemokines and their receptors with transcription factors whose nuclear transport is known to be modulated by NTM. We then tested the effect of cSN50.1 peptide on proinflammatory gene expression in murine bone marrow-derived macrophages stimulated with LPS. This NTM suppressed a proinflammatory transcriptome of 37 out of 84 genes analyzed, without altering expression of housekeeping genes or being cytotoxic. Consistent with gene expression analysis in primary macrophages, plasma levels of 23 out of 26 LPS-induced proinflammatory cytokines, chemokines, and growth factors were significantly attenuated in a murine model of LPS-induced systemic inflammation (lethal endotoxic shock) while the anti-inflammatory cytokine, interleukin 10, was enhanced. This anti-inflammatory reprogramming of the endotoxin-induced genomic response was accompanied by complete protection against lethal endotoxic shock with prophylactic NTM treatment, and 75% protection when NTM was first administered after LPS exposure. In a murine model of localized lung inflammation caused by direct airway exposure to LPS, expression of cytokines and chemokines in the bronchoalveolar space was suppressed with a concomitant reduction of neutrophil trafficking. Thus, calming the LPS-triggered "genomic storm" by

  11. Method and basis set dependence of anharmonic ground state nuclear wave functions and zero-point energies: application to SSSH.

    PubMed

    Kolmann, Stephen J; Jordan, Meredith J T

    2010-02-07

    One of the largest remaining errors in thermochemical calculations is the determination of the zero-point energy (ZPE). The fully coupled, anharmonic ZPE and ground state nuclear wave function of the SSSH radical are calculated using quantum diffusion Monte Carlo on interpolated potential energy surfaces (PESs) constructed using a variety of method and basis set combinations. The ZPE of SSSH, which is approximately 29 kJ mol(-1) at the CCSD(T)/6-31G* level of theory, has a 4 kJ mol(-1) dependence on the treatment of electron correlation. The anharmonic ZPEs are consistently 0.3 kJ mol(-1) lower in energy than the harmonic ZPEs calculated at the Hartree-Fock and MP2 levels of theory, and 0.7 kJ mol(-1) lower in energy at the CCSD(T)/6-31G* level of theory. Ideally, for sub-kJ mol(-1) thermochemical accuracy, ZPEs should be calculated using correlated methods with as big a basis set as practicable. The ground state nuclear wave function of SSSH also has significant method and basis set dependence. The analysis of the nuclear wave function indicates that SSSH is localized to a single symmetry equivalent global minimum, despite having sufficient ZPE to be delocalized over both minima. As part of this work, modifications to the interpolated PES construction scheme of Collins and co-workers are presented.

  12. E4orf6 Variants with Separate Abilities To Augment Adenovirus Replication and Direct Nuclear Localization of the E1B 55-Kilodalton Protein

    PubMed Central

    Orlando, Joseph S.; Ornelles, David A.

    2002-01-01

    The E4orf6 protein of group C adenovirus is an oncoprotein that, in association with the E1B 55-kDa protein and by E1B-independent means, promotes virus replication. An arginine-faced amphipathic α-helix in the E4orf6 protein is required for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein and to enhance replication of an E4 deletion virus. In this study, E4orf6 protein variants containing arginine substitutions in the amphipathic α-helix were analyzed. Two of the six arginine residues within the α-helix, arginine-241 and arginine-243, were critical for directing nuclear localization of the E1B 55-kDa protein. The four remaining arginine residues appear to provide a net positive charge for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein. The molecular determinants of the arginine-faced amphipathic α-helix that were required for the functional interaction between the E4orf6 and E1B 55-kDa proteins seen in the transfected cell differed from those required to support a productive infection. Several E4orf6 protein variants with arginine-to-glutamic acid substitutions that failed to direct nuclear localization of the E1B 55-kDa protein restored replication of an E4 deletion virus. Additionally, a variant containing an arginine-to-alanine substitution at position 243 that directed nuclear localization of the E1B 55-kDa protein failed to enhance virus replication. These results indicate that the ability of the E4orf6 protein to relocalize the E1B 55-kDa protein to the nucleus can be separated from the ability of the E4orf6 protein to support a productive infection. PMID:11773420

  13. Phosphoinositide 3-Kinase Beta Protects Nuclear Envelope Integrity by Controlling RCC1 Localization and Ran Activity

    PubMed Central

    Redondo-Muñoz, Javier; Pérez-García, Vicente; Rodríguez, María J.; Valpuesta, José M.

    2014-01-01

    The nuclear envelope (NE) forms a barrier between the nucleus and the cytosol that preserves genomic integrity. The nuclear lamina and nuclear pore complexes (NPCs) are NE components that regulate nuclear events through interaction with other proteins and DNA. Defects in the nuclear lamina are associated with the development of laminopathies. As cells depleted of phosphoinositide 3-kinase beta (PI3Kβ) showed an aberrant nuclear morphology, we studied the contribution of PI3Kβ to maintenance of NE integrity. pik3cb depletion reduced the nuclear membrane tension, triggered formation of areas of lipid bilayer/lamina discontinuity, and impaired NPC assembly. We show that one mechanism for PI3Kβ regulation of NE/NPC integrity is its association with RCC1 (regulator of chromosome condensation 1), the activator of nuclear Ran GTPase. PI3Kβ controls RCC1 binding to chromatin and, in turn, Ran activation. These findings suggest that PI3Kβ regulates the nuclear envelope through upstream regulation of RCC1 and Ran. PMID:25348717

  14. The human gonadotropin releasing hormone type I receptor is a functional intracellular GPCR expressed on the nuclear membrane.

    PubMed

    Re, Michelle; Pampillo, Macarena; Savard, Martin; Dubuc, Céléna; McArdle, Craig A; Millar, Robert P; Conn, P Michael; Gobeil, Fernand; Bhattacharya, Moshmi; Babwah, Andy V

    2010-07-08

    The mammalian type I gonadotropin releasing hormone receptor (GnRH-R) is a structurally unique G protein-coupled receptor (GPCR) that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME). Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER) leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS) in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.

  15. Spectroscopic properties of nuclear skyrme energy density functionals.

    PubMed

    Tarpanov, D; Dobaczewski, J; Toivanen, J; Carlsson, B G

    2014-12-19

    We address the question of how to improve the agreement between theoretical nuclear single-particle energies (SPEs) and observations. Empirically, in doubly magic nuclei, the SPEs can be deduced from spectroscopic properties of odd nuclei that have one more or one less neutron or proton. Theoretically, bare SPEs, before being confronted with observations, must be corrected for the effects of the particle vibration coupling (PVC). In the present work, we determine the PVC corrections in a fully self-consistent way. Then, we adjust the SPEs, with PVC corrections included, to empirical data. In this way, the agreement with observations, on average, improves; nevertheless, large discrepancies still remain. We conclude that the main source of disagreement is still in the underlying mean fields, and not in including or neglecting the PVC corrections.

  16. Heat shock protein 70 chaperone overexpression ameliorates phenotypes of the spinal and bulbar muscular atrophy transgenic mouse model by reducing nuclear-localized mutant androgen receptor protein.

    PubMed

    Adachi, Hiroaki; Katsuno, Masahisa; Minamiyama, Makoto; Sang, Chen; Pagoulatos, Gerassimos; Angelidis, Charalampos; Kusakabe, Moriaki; Yoshiki, Atsushi; Kobayashi, Yasushi; Doyu, Manabu; Sobue, Gen

    2003-03-15

    Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR). The nuclear inclusions consisting of the mutant AR protein are characteristic and combine with many components of ubiquitin-proteasome and molecular chaperone pathways, raising the possibility that misfolding and altered degradation of mutant AR may be involved in the pathogenesis. We have reported that the overexpression of heat shock protein (HSP) chaperones reduces mutant AR aggregation and cell death in a neuronal cell model (Kobayashi et al., 2000). To determine whether increasing the expression level of chaperone improves the phenotype in a mouse model, we cross-bred SBMA transgenic mice with mice overexpressing the inducible form of human HSP70. We demonstrated that high expression of HSP70 markedly ameliorated the motor function of the SBMA model mice. In double-transgenic mice, the nuclear-localized mutant AR protein, particularly that of the large complex form, was significantly reduced. Monomeric mutant AR was also reduced in amount by HSP70 overexpression, suggesting the enhanced degradation of mutant AR. These findings suggest that HSP70 overexpression ameliorates SBMA phenotypes in mice by reducing nuclear-localized mutant AR, probably caused by enhanced mutant AR degradation. Our study may provide the basis for the development of an HSP70-related therapy for SBMA and other polyQ diseases.

  17. The {open_quotes}Dragon of Kovachitsa{close_quotes}: Local perceptions of radioactive pollution near the Kozlodui Nuclear Power Station (Bulgaria)

    SciTech Connect

    Konstantinov, Y.

    1995-03-01

    There is continuing uncertainty surrounding the safety of nuclear power stations in Eastern Europe, among them the one at Kozlodui, Bulgaria. It is argued in this paper that attempts on the part of the state to assert the safety of nuclear power stations are expressed in terms of an official {open_quotes}nuclear-safe{close_quotes} discourse, while the local inhabitants near the Kozlodui nuclear power station employ a vernacular mode of expression to explain possible radioactive pollution.

  18. Mbh 1: a novel gelsolin/severin-related protein which binds actin in vitro and exhibits nuclear localization in vivo.

    PubMed Central

    Prendergast, G C; Ziff, E B

    1991-01-01

    We describe the characterization of a novel cDNA, mbh1 (myc basic motif homolog-1), which was found during a search for candidate factors which might interact with the c-Myc oncoprotein. Embedded within the amino acid sequence encoded by mbh1 is a region distantly related to the basic/helix-loop-helix (B/HLH) DNA-binding motif and a potential nuclear localization signal. Mbh1 encodes a polypeptide structurally similar to the actin-severing proteins gelsolin and severin. Translation of mbh1 RNA in rabbit reticulocyte extracts produces an approximately 45 kd protein capable of binding actin-coupled agarose beads in vitro in a Ca2(+)-dependent manner. Antiserum raised to a trpE/mbh1 bacterial fusion protein recognizes an approximately 45 kb protein in murine 3T3 fibroblasts, suggesting that the cDNA encodes the complete Mbh1 protein. Examination of Mbh1 localization in 3T3 fibroblasts by indirect immunofluorescence reveals a larger cell population showing diffuse staining, and a smaller population exhibiting a distinct nuclear stain. Western analysis corroborates this intracellular localization and indicates that total cellular levels and localization of Mbh1 are not affected by the cell growth state. The data suggest that Mbh1 may play a role in regulating cytoplasmic and/or nuclear architecture through potential interactions with actin. Images PMID:1849072

  19. CTNNBL1 is a novel nuclear localization sequence-binding protein that recognizes RNA-splicing factors CDC5L and Prp31.

    PubMed

    Ganesh, Karuna; Adam, Salome; Taylor, Benjamin; Simpson, Paul; Rada, Cristina; Neuberger, Michael

    2011-05-13

    Nuclear proteins typically contain short stretches of basic amino acids (nuclear localization sequences; NLSs) that bind karyopherin α family members, directing nuclear import. Here, we identify CTNNBL1 (catenin-β-like 1), an armadillo motif-containing nuclear protein that exhibits no detectable primary sequence homology to karyopherin α, as a novel, selective NLS-binding protein. CTNNBL1 (a single-copy gene conserved from fission yeast to man) was previously found associated with Prp19-containing RNA-splicing complexes as well as with the antibody-diversifying enzyme AID. We find that CTNNBL1 association with the Prp19 complex is mediated by recognition of the NLS of the CDC5L component of the complex and show that CTNNBL1 also interacts with Prp31 (another U4/U6.U5 tri-snRNP-associated splicing factor) through its NLS. As with karyopherin αs, CTNNBL1 binds NLSs via its armadillo (ARM) domain, but displays a separate, more selective NLS binding specificity. Furthermore, the CTNNBL1/AID interaction depends on amino acids forming the AID conformational NLS with CTNNBL1-deficient cells showing a partial defect in AID nuclear accumulation. However, in further contrast to karyopherin αs, the CTNNBL1 N-terminal region itself binds karyopherin αs (rather than karyopherin β), suggesting a function divergent from canonical nuclear transport. Thus, CTNNBL1 is a novel NLS-binding protein, distinct from karyopherin αs, with the results suggesting a possible role in the selective intranuclear targeting or interactions of some splicing-associated complexes.

  20. Nuclear Pore Complexes and Nucleocytoplasmic Transport: From Structure to Function to Disease.

    PubMed

    Dickmanns, Achim; Kehlenbach, Ralph H; Fahrenkrog, Birthe

    2015-01-01

    Nucleocytoplasmic transport is an essential cellular activity and occurs via nuclear pore complexes (NPCs) that reside in the double membrane of the nuclear envelope. Significant progress has been made during the past few years in unravelling the ultrastructural organization of NPCs and their constituents, the nucleoporins, by cryo-electron tomography and X-ray crystallography. Mass spectrometry and genomic approaches have provided deeper insight into the specific regulation and fine tuning of individual nuclear transport pathways. Recent research has also focused on the roles nucleoporins play in health and disease, some of which go beyond nucleocytoplasmic transport. Here we review emerging results aimed at understanding NPC architecture and nucleocytoplasmic transport at the atomic level, elucidating the specific function individual nucleoporins play in nuclear trafficking, and finally lighting up the contribution of nucleoporins and