Immunohistological Localization of Peroxisome Proliferator-Activated Receptor α and γ in Human Sebaceous Glands.

PubMed

Furue, Masutake; Takemura, Masaki; Nishio, Kiichiroet; Sato, Yuki; Nagata, Shoko; Kan, Nagisa; Suenaga, Asako; Furue, Kazuhisa; Yoshida, Maiko; Konishi, Sawako; Tsuji, Gaku

2016-11-01

The immunohistological localization of peroxisome proliferator-activated receptor a (PPARa) and PPAR g was examined in 28 pilosebaceous units in 10 paraffin-embedded normal human skin specimens. Rabbit polyclonal antibody against human PPARa and monoclonal antibody against human PPARg were used as specific primary antibodies. The nuclear and cytoplasmic expression of PPARa was detected in basal to differentiated sebocytes. In contrast, the expression of PPARg was confined to nuclei of suprabasal to early-differentiated sebocytes. The nuclear PPARg expression was present only occasionally in the basal sebocytes. These results suggest that PPARa and PPARg are integral parts of sebocyte differentiation in human sebaceous glands.

Nuclear translocation of IGF1R by intracellular amphiregulin contributes to the resistance of lung tumour cells to EGFR-TKI.

PubMed

Guerard, Marie; Robin, Thomas; Perron, Pascal; Hatat, Anne-Sophie; David-Boudet, Laurence; Vanwonterghem, Laetitia; Busser, Benoit; Coll, Jean-Luc; Lantuejoul, Sylvie; Eymin, Beatrice; Hurbin, Amandine; Gazzeri, Sylvie

2018-04-28

Many Receptor Tyrosine Kinases translocate from the cell surface to the nucleus in normal and pathological conditions, including cancer. Here we report the nuclear expression of insulin-like growth factor-1 receptor (IGF1R) in primary human lung tumours. Using lung cancer cell lines and lung tumour xenografts, we demonstrate that the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) gefitinib induces the nuclear accumulation of IGF1R in mucinous lung adenocarcinoma by a mechanism involving the intracellular re-localization of the growth factor amphiregulin. Amphiregulin allows the binding of IGF1R to importin-β1 and promotes its nuclear transport. The nuclear accumulation of IGF1R by amphiregulin induces cell cycle arrest through p21 WAF1/CIP1 upregulation, and prevents the induction of apoptosis in response to gefitinib. These results identify amphiregulin as the first nuclear localization signal-containing protein that interacts with IGF1R and allows its nuclear translocation. Furthermore they indicate that nuclear expression of IGF1R contributes to EGFR-TKI resistance in lung cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

Nuclear Accumulation of Histone Deacetylase 4 (HDAC4) Exerts Neurotoxicity in Models of Parkinson's Disease.

PubMed

Wu, Qimei; Yang, Xiaoyu; Zhang, Lei; Zhang, Yu; Feng, Linyin

2017-11-01

Histone deacetylase 4 (HDAC4) is a class II HDAC which is highly expressed in the brain. Previous reports have shown that HDAC4 is essential for normal brain physiology and its deregulation leads to several neurodegenerative disorders. However, it remains unclear whether dysregulation of HDAC4 is specifically involved in the development of Parkinson's disease. In this study, we demonstrate that intracellular trafficking of HDAC4 is important in regulating dopaminergic cell death. While HDAC4 normally localizes to the cytoplasm, nuclear accumulation of HDAC4 was observed in dopaminergic neurons overexpressing A53T mutant α-synuclein treated with MPP + /MPTP in vitro and in vivo. Nuclear-localized HDAC4 repressed cAMP response element-binding protein (CREB) and myocyte enhancer factor 2A (MEF2A), altered neuronal gene expression, and promoted neuronal apoptosis. Furthermore, cytoplasm-to-nucleus shuttling of HDAC4 was determined by its phosphorylation status, which was regulated by PP2A and PKCε. Treatment with PKCε-specific activators, DCP-LA or Bryostatin 1, provided neuroprotection against MPP + toxicity in a dose-dependent manner. In summary, our research illustrated that intracellular trafficking of HDAC4 contributes to the vulnerability of cells expressing pathogenic α-synuclein mutants in response to oxidative stress and compounds which maintain cytoplasmic localization of HDAC4 such as PKCε activators that may serve as therapeutic agents for Parkinson's disease.

Local variance of atmospheric 14C concentrations around Fukushima Dai-ichi Nuclear Power Plant from 2010 to 2012.

PubMed

Chen, Biying; Xu, Sheng; Cook, Gordon T; Freeman, Stewart P H T; Hou, Xiaolin; Liu, Cong-Qiang; Naysmith, Philip; Yamaguchi, Katsuhiko

2017-01-01

Radiocarbon ( 14 C) has been measured in single tree ring samples collected from the southwest of the Fukushima Dai-ichi Nuclear Power Plant. Our data indicate south-westwards dispersion of radiocarbon and the highest 14 C activity observed so far in the local environment during the 2011 accident. The abnormally high 14 C activity in the late wood of 2011 ring may imply an unknown source of radiocarbon nearby after the accident. The influence of 14 C shrank from 30 km during normal reactor operation to 14 km for the accident in the northwest of FDNPP, but remains unclear in the southwest.

Distinct nuclear arrangement of active and inactive c-myc genes in control and differentiated colon carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harnicarova, Andrea; Kozubek, Stanislav; Pachernik, Jiri

2006-12-10

Using sequential RNA-DNA fluorescence in situ hybridization, the nuclear arrangement of both the active and inactive c-myc gene as well as its transcription was investigated in colon cancer HT-29 cells induced to differentiate into enterocytes. Cytogenetic studies revealed the presence of two chromosomes 8 in HT-29 cells, of which the one containing c-myc gene amplicons was substantially larger and easily distinguished from the normal chromosome. This observation enabled detection of both activity and nuclear localization of c-myc genes in single cells and in individual chromosome territories. Similar transcriptional activity of the c-myc gene was observed in both the normal andmore » derivative chromosome 8 territories showing no influence of the amplification on the c-myc gene expression. Our experiments demonstrate strikingly specific nuclear and territorial arrangements of active genes as compared with inactive ones: on the periphery of their territories facing to the very central region of the cell nucleus. Nuclear arrangement of c-myc genes and transcripts was conserved during cell differentiation and, therefore, independent of the level of differentiation-specific c-myc gene expression. However, after the induction of differentiation, a more internal territorial location was found for the single copy c-myc gene of normal chromosome 8, while amplicons conserved their territorial topography.« less

SRSF1-3 contributes to diversification of the immunoglobulin variable region gene by promoting accumulation of AID in the nucleus.

PubMed

Kawaguchi, Yuka; Nariki, Hiroaki; Kawamoto, Naoko; Kanehiro, Yuichi; Miyazaki, Satoshi; Suzuki, Mari; Magari, Masaki; Tokumitsu, Hiroshi; Kanayama, Naoki

2017-04-01

Activation-induced cytidine deaminase (AID) is essential for diversification of the Ig variable region (IgV). AID is excluded from the nucleus, where it normally functions. However, the molecular mechanisms responsible for regulating AID localization remain to be elucidated. The SR-protein splicing factor SRSF1 is a nucleocytoplasmic shuttling protein, a splicing isoform of which called SRSF1-3, has previously been shown to contribute to IgV diversification in chicken DT40 cells. In this study, we examined whether SRSF1-3 functions in IgV diversification by promoting nuclear localization of AID. AID expressed alone was localized predominantly in the cytoplasm. In contrast, co-expression of AID with SRSF1-3 led to the nuclear accumulation of both AID and SRSF1-3 and the formation of a protein complex that contained them both, although SRSF1-3 was dispensable for nuclear import of AID. Expression of either SRSF1-3 or a C-terminally-truncated AID mutant increased IgV diversification in DT40 cells. However, overexpression of exogenous SRSF1-3 was unable to further enhance IgV diversification in DT40 cells expressing the truncated AID mutant, although SRSF1-3 was able to form a protein complex with the AID mutant. These results suggest that SRSF1-3 promotes nuclear localization of AID probably by forming a nuclear protein complex, which might stabilize nuclear AID and induce IgV diversification in an AID C-terminus-dependent manner. Copyright © 2017 Elsevier Inc. All rights reserved.

Cortical Recruitment and Nuclear–Cytoplasmic Shuttling of Scd5p, a Protein Phosphatase-1-targeting Protein Involved in Actin Organization and EndocytosisD⃞

PubMed Central

Chang, Ji Suk; Henry, Kenneth; Geli, María Isabel; Lemmon, Sandra K.

2006-01-01

Scd5p regulates endocytosis and cortical actin organization as a targeting subunit for the Ser/Thr protein phosphatase-1 (PP1) in yeast. To identify localization signals in Scd5p required for cell surface recruitment, visualization of GFP-tagged Scd5 truncations and deletions was performed. Scd5p contains a PP1 binding site, a 3-repeat region of 20 amino acids (3R), and a 9-repeat region of 12 amino acids (9R). We found that the 9R is critical for cortical localization of Scd5p, but cortical recruitment is not essential for Scd5p's function in actin organization and endocytosis. We propose that Scd5p can target PP1 to endocytic factors in the cytoplasm that have been disassembled and/or inactivated by phosphorylation. We also found that Scd5p undergoes nuclear-cytoplasmic shuttling in a Crm1p-dependent manner. Scd5p-ΔCT lacking the 9R region and its nuclear export signal (NES) accumulates in the nucleus, causing cortical actin and endocytic defects. Cytoplasmic localization and function of Scd5p-ΔCT is restored by NES addition. However, removal of Scd5p's nuclear localization signal prevents nuclear entry, but endocytosis and actin organization remain relatively normal. These results indicate that nuclear-cytoplasmic shuttling is not required for regulation of Scd5p's cortical function and suggest that Scd5p has an independent nuclear function. PMID:16251346

Utilization of nuclear structural proteins for targeted therapy and detection of proliferative and differentiation disorders

DOEpatents

Lelievre, Sophie; Bissell, Mina

2001-01-01

The localization of nuclear apparatus proteins (NUMA) is used to identify tumor cells and different stages in the tumor progression and differentiation processes. There is a characteristic organization of NuMA in tumor cells and in phenotypically normal cells. NuMA distribution patterns are significantly less diffuse in proliferating non-malignant cells compared to malignant cells. The technique encompasses cell immunostaining using a NuMA specific antibody, and microscopic analysis of NuMA distribution within each nucleus.

A chromatin insulator determines the nuclear localization of DNA.

PubMed

Gerasimova, T I; Byrd, K; Corces, V G

2000-11-01

Chromatin insulators might regulate gene expression by controlling the subnuclear organization of DNA. We found that a DNA sequence normally located inside of the nucleus moved to the periphery when the gypsy insulator was placed within the sequence. The presence of the gypsy insulator also caused two sequences, normally found in different regions of the nucleus, to come together at a single location. Alterations in this subnuclear organization imposed by the gypsy insulator correlated with changes in gene expression that took place during the heat-shock response. These global changes in transcription were accompanied by dramatic alterations in the distribution of insulator proteins and DNA. The results suggest that the nuclear organization imposed by the gypsy insulator on the chromatin fiber is important for gene expression.

The DNA Helicase Activity of BLM Is Necessary for the Correction of the Genomic Instability of Bloom Syndrome Cells

PubMed Central

Neff, Norma F.; Ellis, Nathan A.; Ye, Tian Zhang; Noonan, James; Huang, Kelly; Sanz, Maureen; Proytcheva, Maria

1999-01-01

Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLM encodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of a Saccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern. PMID:10069810

Cytochemistry of the functional domains of the nucleus in normal and in pathologic conditions.

PubMed

Maraldi, N M; Zini, N; Santi, S; Ognibene, A; Rizzoli, R; Mazzotti, G; Manzoli, F A

1998-01-01

By means of ultrastructural cytochemistry significant advances have been made in understanding the functional roles of many nuclear domains. This review gives schematic information about the main nuclear domains involved in replication, transcription, processing and transport of the transcripts in normal and in pathologic conditions. Particular attention is paid to a functional domain that appears to be involved in signal transduction. Data are reported on the intranuclear specific localization of key elements of the polyphosphoinositide signal transduction system in different cell types including human osteosarcoma cell lines. Compared with the compartmentalization of the cytoplasm, the nucleus has long been considered as relatively unstructured. On the other hand, fundamental nuclear functions, such as DNA replication and RNA transcription, can be molecularly characterized also in cell-free systems, suggesting that supramolecular organization is not so strictly required as for other cell functions occurring within intact cytoplasmic organelles. Nevertheless, a stringent organization is required for packing about 200 cm of DNA in the about 30 micron 3 of the nucleus. In the absence of membrane-delimited organelles, the nuclear organization is based on functional compartments, or domains, whose spatial localization involves the nuclear matrix, which shares many properties with the cytoskeleton. The nuclear domains are defined as structural compartments, not necessarily stable but dynamically variable, which perform specific metabolic functions through the partitioning of molecular complexes. Their identification has been made possible in the last few years by the development of specific nuclear probes for confocal and electron microscope immunocytochemistry. Therefore, the complex network of structures and enzymatic functions that make up the nucleus is in several cases yielding to molecular analysis, but a large part remains unknown (Strouboulis and Wolffe, 1996; Laemmli and Tjian, 1996). Rapid advances in understanding the functional role of the nuclear domains have been made recently: in particular, of the nuclear envelope, of the nucleolus, and of RNA splicing. In other cases, e.g. the precise localization of the nuclear domains involved in signal transduction, much remains to be clarified (Forbes and Johnson, 1997). It is conceivable that in the near future unexpected new nuclear domains will come to light and new nuclear functions may emerge, especially in field of post-transcriptional processing and transport of RNAs, and in the relationships between the nucleo-skeleton and enzymic fixed sites involved in replication, transcription and signal transduction. The aim of this review is to provide information about the morphological characteristics, the associated functions and the molecular composition of the main nuclear domains found to date. To simplify the exposition, the main data on each nuclear domain are reported in Tables, together with the principal references on the subject. Figures refer to original findings on some aspects of nuclear domain organization.

Vaccinia-related kinase 3 (VRK3) sets the circadian period and amplitude by affecting the subcellular localization of clock proteins in mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Nayoung; Department of Brain Science, Ajou University School of Medicine, 164 Worldcup-ro, Yeongtong-gu, Suwon, Kyunggi-do, 16499; Song, Jieun

In the eukaryotic circadian clock machinery, negative feedback repression of CLOCK (CLK) and BMAL1 transcriptional activity by PERIOD (PER) and CRYPTOCHROME (CRY) underlies the basis for 24 h rhythmic gene expression. Thus, precise regulation of the time-dependent nuclear entry of circadian repressors is crucial to generating normal circadian rhythms. Here, we sought to identify novel kinase(s) that regulate nuclear entry of mammalian CRY1 (mCRY1) with an unbiased screening using red fluorescent protein (RFP)-tagged human kinome expression plasmids in mammalian cells. Transient expression of human vaccinia-related kinase 3 (hVRK3) reduced the nuclear presence of mCRY1. hVRK3 expression also induced alterations in themore » subcellular localization of other core clock proteins, including mCRY2, mPER2, and BMAL1. In contrast, the subcellular localization of mCLK was not changed. Given that singly expressed mCLK mostly resides in the cytoplasm and that nuclear localization sequence (NLS) mutation of hVRK3 attenuated the effect of hVRK3 co-expression on subcellular localization, ectopically expressed hVRK3 presumably reduces the retention of proteins in the nucleus. Finally, downregulation of hvrk3 using siRNA reduced the amplitude and lengthened the period of the cellular bioluminescence rhythm. Taken together, these data suggest that VRK3 plays a role in setting the amplitude and period length of circadian rhythms in mammalian cells. - Highlights: • Screening was performed to identify kinases that regulate CRY1 subcellular localization. • VRK3 alters the subcellular localization of CRY1, CRY2, PER2, and BMAL1. • VRK3 knock-down alters the circadian bioluminescence rhythm in mammalian cells.« less

Nuclear glutaredoxin 3 is critical for protection against oxidative stress-induced cell death

USDA-ARS?s Scientific Manuscript database

Mammalian glutaredoxin 3 (Grx3) has been shown to be critical in maintaining redox homeostasis and regulating cell survival pathways in cancer cells. However, the regulation of Grx3 is not fully understood. In the present study, we investigate the subcellular localization of Grx3 under normal growth...

New quantitative approaches reveal the spatial preference of nuclear compartments in mammalian fibroblasts.

PubMed

Weston, David J; Russell, Richard A; Batty, Elizabeth; Jensen, Kirsten; Stephens, David A; Adams, Niall M; Freemont, Paul S

2015-03-06

The nuclei of higher eukaryotic cells display compartmentalization and certain nuclear compartments have been shown to follow a degree of spatial organization. To date, the study of nuclear organization has often involved simple quantitative procedures that struggle with both the irregularity of the nuclear boundary and the problem of handling replicate images. Such studies typically focus on inter-object distance, rather than spatial location within the nucleus. The concern of this paper is the spatial preference of nuclear compartments, for which we have developed statistical tools to quantitatively study and explore nuclear organization. These tools combine replicate images to generate 'aggregate maps' which represent the spatial preferences of nuclear compartments. We present two examples of different compartments in mammalian fibroblasts (WI-38 and MRC-5) that demonstrate new knowledge of spatial preference within the cell nucleus. Specifically, the spatial preference of RNA polymerase II is preserved across normal and immortalized cells, whereas PML nuclear bodies exhibit a change in spatial preference from avoiding the centre in normal cells to exhibiting a preference for the centre in immortalized cells. In addition, we show that SC35 splicing speckles are excluded from the nuclear boundary and localize throughout the nucleoplasm and in the interchromatin space in non-transformed WI-38 cells. This new methodology is thus able to reveal the effect of large-scale perturbation on spatial architecture and preferences that would not be obvious from single cell imaging.

Monitoring ventricular function at rest and during exercise with a nonimaging nuclear detector.

PubMed

Wagner, H N; Rigo, P; Baxter, R H; Alderson, P O; Douglass, K H; Housholder, D F

1979-05-01

A portable nonimaging device, the nuclear stethoscope, for measuring beat to beat ventricular time-activity curves in normal people and patients with heart disease, both at rest and during exercise, is being developed and evaluated. The latest device has several operating modes that facilitate left ventricular and background localization, measurement of transit times and automatic calculation and display of left ventricular ejection fraction. The correlation coefficient of left ventricular ejection fraction obtained with the device and with a camera-computer system was 0.92 in 35 subjects. During bicycle exercise the ejection fraction in 15 normal persons increased from 44 to 64 percent (P less than 0.001), whereas among 12 patients with heart disease it was unchanged in 5 and decreased in 7.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walpen, Thomas; Kalus, Ina; Schwaller, Juerg

Highlights: Black-Right-Pointing-Pointer Pim1{sup -/-} endothelial cell proliferation displays increased sensitivity to rapamycin. Black-Right-Pointing-Pointer mTOR inhibition by rapamycin enhances PIM1 cytosolic and nuclear protein levels. Black-Right-Pointing-Pointer Truncation of Pim1 beyond serine 276 results in nuclear localization of the kinase. Black-Right-Pointing-Pointer Nuclear PIM1 increases endothelial proliferation independent of rapamycin. -- Abstract: The PIM serine/threonine kinases and the mTOR/AKT pathway integrate growth factor signaling and promote cell proliferation and survival. They both share phosphorylation targets and have overlapping functions, which can partially substitute for each other. In cancer cells PIM kinases have been reported to produce resistance to mTOR inhibition by rapamycin. Tumormore » growth depends highly on blood vessel infiltration into the malignant tissue and therefore on endothelial cell proliferation. We therefore investigated how the PIM1 kinase modulates growth inhibitory effects of rapamycin in mouse aortic endothelial cells (MAEC). We found that proliferation of MAEC lacking Pim1 was significantly more sensitive to rapamycin inhibition, compared to wildtype cells. Inhibition of mTOR and AKT in normal MAEC resulted in significantly elevated PIM1 protein levels in the cytosol and in the nucleus. We observed that truncation of the C-terminal part of Pim1 beyond Ser 276 resulted in almost exclusive nuclear localization of the protein. Re-expression of this Pim1 deletion mutant significantly increased the proliferation of Pim1{sup -/-} cells when compared to expression of the wildtype Pim1 cDNA. Finally, overexpression of the nuclear localization mutant and the wildtype Pim1 resulted in complete resistance to growth inhibition by rapamycin. Thus, mTOR inhibition-induced nuclear accumulation of PIM1 or expression of a nuclear C-terminal PIM1 truncation mutant is sufficient to increase endothelial cell proliferation, suggesting that nuclear localization of PIM1 is important for resistance of MAEC to rapamycin-mediated inhibition of proliferation.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffith, K.D.; Read, E.J.; Carrasquillo, J.A.

Patients with metastatic melanoma undergoing therapy with cyclophosphamide (CPM), tumor-infiltrating lymphocytes (TIL), and interleukin-2 (IL-2) were studied for the ability of their 111In-labeled TIL or peripheral blood lymphocytes (PBL) to localize in sites of tumor using gamma camera imaging and biopsies. Nineteen infusions of radiolabeled TIL were given to 18 patients, while five patients received radiolabeled autologous PBL during TIL therapy. Clear tumor localization was seen on 13 of 18 nuclear scan series performed on 111In-TIL recipients, while tumor was imaged in only one of four scan sequences on patients given 111In-PBL. Nineteen paired biopsies of tumor and normal skinmore » were completed on 10 patients receiving 111In-TIL, while eight biopsies were done on three PBL patients receiving 111In-PBL. The mean percentage of total injectate activity localizing per gram of tumor tissue was 0.0049% in the TIL group and 0.0010% in the PBL group (P2 = .0004). The mean of the tumor to normal skin ratios of the 111In-TIL group was three times that for 111In-PBL (P2 = .0072). One patient was studied by nuclear scanning on three consecutive treatment courses of CPM, TIL, and IL-2. He initially demonstrated clear tumor localization by 111In-TIL at several sites, then faint localization with 111In-PBL at a single site, and subsequently positive tumor imaging on repeat 111In-TIL infusion at multiple sites. These results confirm and expand our initial data demonstrating that human TIL transferred with CPM pretreatment and followed by IL-2 preferentially localize to tumor sites and indicate that this localization is greater for TIL than PBL.« less

Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor

PubMed Central

Görner, Wolfram; Durchschlag, Erich; Wolf, Julia; Brown, Elizabeth L.; Ammerer, Gustav; Ruis, Helmut; Schüller, Christoph

2002-01-01

In yeast, environmental conditions control the transcription factor Msn2, the nuclear accumulation and function of which serve as a sensitive indicator of nutrient availablity and environmental stress load. We show here that the nuclear localization signal (NLS) of Msn2 is a direct target of cAMP-dependent protein kinase (cAPK). Genetic analysis suggests that Msn2-NLS function is inhibited by phosphorylation and activated by dephosphorylation. Msn2-NLS function is unaffected by many stress conditions that normally induce nuclear accumulation of full-length Msn2. The Msn2-NLS phosphorylation status is, however, highly sensitive to carbohydrate fluctuations during fermentative growth. Dephosphorylation occurs in >2 min after glucose withdrawal but the effect is reversed rapidly by refeeding with glucose. This response to glucose depletion is due to changes in cAPK activity rather than an increase in protein phosphatase activity. Surprisingly, the classical glucose-sensing systems are not connected to this rapid response system. Our results further imply that generic stress signals do not cause short-term depressions in cAPK activity. They operate on Msn2 by affecting an Msn5-dependent nuclear export and/or retention mechanism. PMID:11782433

The long terminal repeat-containing retrotransposon Tf1 possesses amino acids in gag that regulate nuclear localization and particle formation.

PubMed

Kim, Min-Kyung; Claiborn, Kathryn C; Levin, Henry L

2005-08-01

Tf1 is a long terminal repeat-containing retrotransposon of Schizosaccharomyces pombe that is studied to further our understanding of retrovirus propagation. One important application is to examine Tf1 as a model for how human immunodeficiency virus type 1 proteins enter the nucleus. The accumulation of Tf1 Gag in the nucleus requires an N-terminal nuclear localization signal (NLS) and the nuclear pore factor Nup124p. Here, we report that NLS activity is regulated by adjacent residues. Five mutant transposons were made, each with sequential tracts of four amino acids in Gag replaced by alanines. All five versions of Tf1 transposed with frequencies that were significantly lower than that of the wild type. Although all five made normal amounts of Gag, two of the mutations did not make cDNA, indicating that Gag contributed to reverse transcription. The localization of the Gag in the nucleus was significantly reduced by mutations A1, A2, and A3. These results identified residues in Gag that contribute to the function of the NLS. The Gags of A4 and A5 localized within the nucleus but exhibited severe defects in the formation of virus-like particles. Of particular interest was that the mutations in Gag-A4 and Gag-A5 caused their nuclear localization to become independent of Nup124p. These results suggested that Nup124p was only required for import of Tf1 Gag because of its extensive multimerization.

APE/Ref-1 is increased in nuclear fractions of human thyroid hyperfunctioning nodules.

PubMed

Russo, D; Celano, M; Bulotta, S; Bruno, R; Arturi, F; Giannasio, P; Filetti, S; Damante, G; Tell, G

2002-08-30

Apurinic/apyrimidinic endonuclease APE/Ref-1 is a multifunctional protein provided with DNA repair, transcription-factor regulation and anti-apoptotic activities. We have previously reported that, in thyroid cells, TSH regulates both the synthesis and nuclear translocation of APE/Ref-1. We have also shown that nuclear levels of this protein are reduced both in thyroid carcinoma tissues and cell lines. In the present study, APE/Ref-1 expression and cellular localization were analysed by Western blot in hyperfunctioning thyroid nodules from patients with toxic adenoma and/or toxic multinodular goiter. The total content of APE/Ref-1 protein was increased in the majority of the hyperfunctioning tissues with respect to normal adjacent tissue. There was also an increase in the nuclear levels of APE/Ref-1, suggesting enhanced cytoplasm-to-nucleus translocation of the protein in addition to its increased rate of synthesis. These results demonstrate that the phenomenon of nuclear translocation of APE/Ref-1 hypothesized on the basis of cell culture experiments does actually occur in vivo. Together with previous observations in thyroid carcinomas and tumoral cell lines, our findings suggest a two-stage model of APE/Ref-1 behaviour during malignant thyrocyte transformation: an early stage characterized by simple hyperplasia and upregulation of APE/Ref-1 in the nuclear compartment of the cell and a later stage in which nuclear levels of the protein drop to below-normal levels as the cell becomes progressively undifferentiated.

Localization of phosphorylated forms of Bcl-2 in mitosis: co-localization with Ki-67 and nucleolin in nuclear structures and on mitotic chromosomes.

PubMed

Barboule, Nadia; Truchet, Isabelle; Valette, Annie

2005-04-01

Bcl-2 phosphorylation is a normal physiological process occurring at mitosis or during mitotic arrest induced by microtubule damaging agents. The consequences of Bcl-2 phosphorylation on its function are still controversial. To better understand the role of Bcl-2 phosphorylation in mitosis, we studied the subcellular localization of phosphorylated forms of Bcl-2. Immunofluorescence experiments performed in synchronized HeLa cells indicate for the first time that mitotic phosphorylated forms of Bcl-2 can be detected in nuclear structures in prophase cells together with nucleolin and Ki-67. In later mitotic stages, as previously described, phosphorylated forms of Bcl-2 are localized on mitotic chromosomes. In addition, we demonstrate that Bcl-2 in these structures is at least in part phosphorylated on the T56 residue. Then, coimmunoprecipitation experiments reveal that, in cells synchronized at the onset of mitosis, Bcl-2 is present in a complex with nucleolin, cdc2 kinase and PP1 phosphatase. Taken together, these data further support the idea that Bcl-2 could have a new function at mitosis.

The mouse mismatch repair protein, MSH3, is a nucleoplasmic protein that aggregates into denser nuclear bodies under conditions of stress.

PubMed

Holt, Ian; Thanh Lam, Le; Tomé, Stéphanie; Wansink, Derick G; Te Riele, Hein; Gourdon, Geneviève; Morris, Glenn E

2011-06-01

The mismatch repair protein, MSH3, together with MSH2, forms the MutSβ heterodimer which recognizes and repairs base pair mismatches and larger insertion/deletion loops in DNA. Lack of specific antibodies against mouse MSH3 has hampered studies of its expression and localization. Mouse MSH3 is not immunogenic in normal mice. This problem was overcome by immunizing msh3-knockout mice and generating a panel of ten monoclonal antibodies, two of which localize MSH3 specifically in cultured mouse cells and bind to an epitope containing amino-acids 33-37. The panel also includes two antibodies that recognise both mouse and human MSH3 and bind to a conserved epitope containing amino-acids 187-194. The mouse MSH3-specific antibodies show that MSH3 is a nuclear protein with a finely-granular nucleoplasmic distribution, largely absent from areas of condensed heterochromatin. Specificity of the localization was demonstrated by absence of immunostaining in a cell line from the msh3-knockout mouse. Furthermore, we show for the first time that stress treatment of mouse cells with ethanol or hydrogen peroxide caused the re-distribution of MSH3 into nuclear bodies containing the proliferating cell nuclear antigen (PCNA), a known binding partner of MutSβ. Copyright © 2011 Wiley-Liss, Inc.

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

Expression and subcellular localization of a novel nuclear acetylcholinesterase protein.

PubMed

Santos, Susana Constantino Rosa; Vala, Inês; Miguel, Cláudia; Barata, João T; Garção, Pedro; Agostinho, Paula; Mendes, Marta; Coelho, Ana V; Calado, Angelo; Oliveira, Catarina R; e Silva, João Martins; Saldanha, Carlota

2007-08-31

Acetylcholine is found in the nervous system and also in other cell types (endothelium, lymphocytes, and epithelial and blood cells), which are globally termed the non-neuronal cholinergic system. In this study we investigated the expression and subcellular localization of acetylcholinesterase (AChE) in endothelial cells. Our results show the expression of the 70-kDa AChE in both cytoplasmic and nuclear compartments. We also describe, for the first time, a nuclear and cytoskeleton-bound AChE isoform with approximately 55 kDa detected in endothelial cells. This novel isoform is decreased in response to vascular endothelial growth factor via the proteosomes pathway, and it is down-regulated in human leukemic T-cells as compared with normal T-cells, suggesting that the decreased expression of the 55-kDa AChE protein may contribute to an angiogenic response and associate with tumorigenesis. Importantly, we show that its nuclear expression is not endothelial cell-specific but also evidenced in non-neuronal and neuronal cells. Concerning neuronal cells, we can distinguish an exclusively nuclear expression in postnatal neurons in contrast to a cytoplasmic and nuclear expression in embryonic neurons, suggesting that the cell compartmentalization of this new AChE isoform is changed during the development of nervous system. Overall, our studies suggest that the 55-kDa AChE may be involved in different biological processes such as neural development, tumor progression, and angiogenesis.

Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells.

PubMed

Shimura, Tsutomu; Hamada, Nobuyuki; Sasatani, Megumi; Kamiya, Kenji; Kunugita, Naoki

2014-01-01

Cyclin D1 is a mitogenic sensor that responds to growth signals from the extracellular environment and regulates the G 1-to-S cell cycle transition. When cells are acutely irradiated with a single dose of 10 Gy, cyclin D1 is degraded, causing cell cycle arrest at the G 1/S checkpoint. In contrast, cyclin D1 accumulates in human tumor cells that are exposed to long-term fractionated radiation (0.5 Gy/fraction of X-rays). In this study we investigated the effect of fractionated low-dose radiation exposure on cyclin D1 localization in 3 strains of normal human fibroblasts. To specifically examine the nuclear accumulation of cyclin D1, cells were treated with a hypotonic buffer containing detergent to remove cytoplasmic cyclin D1. Proliferating cell nuclear antigen (PCNA) immunofluorescence was used to identify cells in S phase. With this approach, we observed S-phase nuclear retention of cyclin D1 following low-dose fractionated exposures, and found that cyclin D1 nuclear retention increased with exposure time. Cells that retained nuclear cyclin D1 were more likely to have micronuclei than non-retaining cells, indicating that the accumulation of nuclear cyclin D1 was associated with genomic instability. Moreover, inhibition of the v-akt murine thymoma viral oncogene homolog (AKT) pathway facilitated cyclin D1 degradation and eliminated cyclin D1 nuclear retention in cells exposed to fractionated radiation. Thus, cyclin D1 may represent a useful marker for monitoring long-term effects associated with exposure to low levels of radiation.

Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells

PubMed Central

Shimura, Tsutomu; Hamada, Nobuyuki; Sasatani, Megumi; Kamiya, Kenji; Kunugita, Naoki

2014-01-01

Cyclin D1 is a mitogenic sensor that responds to growth signals from the extracellular environment and regulates the G1-to-S cell cycle transition. When cells are acutely irradiated with a single dose of 10 Gy, cyclin D1 is degraded, causing cell cycle arrest at the G1/S checkpoint. In contrast, cyclin D1 accumulates in human tumor cells that are exposed to long-term fractionated radiation (0.5 Gy/fraction of X-rays). In this study we investigated the effect of fractionated low-dose radiation exposure on cyclin D1 localization in 3 strains of normal human fibroblasts. To specifically examine the nuclear accumulation of cyclin D1, cells were treated with a hypotonic buffer containing detergent to remove cytoplasmic cyclin D1. Proliferating cell nuclear antigen (PCNA) immunofluorescence was used to identify cells in S phase. With this approach, we observed S-phase nuclear retention of cyclin D1 following low-dose fractionated exposures, and found that cyclin D1 nuclear retention increased with exposure time. Cells that retained nuclear cyclin D1 were more likely to have micronuclei than non-retaining cells, indicating that the accumulation of nuclear cyclin D1 was associated with genomic instability. Moreover, inhibition of the v-akt murine thymoma viral oncogene homolog (AKT) pathway facilitated cyclin D1 degradation and eliminated cyclin D1 nuclear retention in cells exposed to fractionated radiation. Thus, cyclin D1 may represent a useful marker for monitoring long-term effects associated with exposure to low levels of radiation. PMID:24583467

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaehler, Christian; Guenther, Anika; Uhlich, Anja

Arginine methylation is a posttranslational modification that is of importance in diverse cellular processes. Recent proteomic mass spectrometry studies reported arginine methylation of ataxin-2-like (ATXN2L), the paralog of ataxin-2, a protein that is implicated in the neurodegenerative disorder spinocerebellar ataxia type 2. Here, we investigated the methylation state of ATXN2L and its significance for ATXN2L localization. We first confirmed that ATXN2L is asymmetrically dimethylated in vivo, and observed that the nuclear localization of ATXN2L is altered under methylation inhibition. We further discovered that ATXN2L associates with the protein arginine-N-methyltransferase 1 (PRMT1). Finally, we showed that neither mutation of the arginine–glycine-richmore » motifs of ATXN2L nor methylation inhibition alters ATXN2L localization to stress granules, suggesting that methylation of ATXN2L is probably not mandatory. - Highlights: • ATXN2L is asymmetrically dimethylated in vivo. • ATXN2L interacts with PRMT1 under normal and stress conditions. • PRMT1-mediated dimethylation of ATXN2L controls its nuclear localization. • ATXN2L localization to stress granules appears independent of its methylation state.« less

In silico prediction of escherichia coli proteins targeting the host cell nucleus, with special reference to their role in colon cancer etiology.

PubMed

Khan, Abdul Arif

2014-06-01

The potential role of Escherichia coli in the development of colorectal carcinoma (CRC) has been investigated in many studies. Although the exact mechanism is not clear, chronic inflammation caused by E. coli and other related events are suggested as possible causes behind E. coli-induced colon cancer. It has been found that CRC cells, but not normal cells, are colonized by an intracellular form of E. coli. We predicted nuclear targeting of bacterial proteins in the host cell through computational tools nuclear localization signal (NLS) mapper and balanced subcellular localization predictor (BaCeILo). During intracellular E. coli residence, such targeting is highly likely and may have a possible role in colon cancer etiology. We observed that several gene expression-associated proteins of E. coli can migrate to the host nucleus during intracellular infections. This situation provides an opportunity for competitive interaction of host and pathogen proteins with similar cellular substrates, thereby increasing the chances of development of colon cancer. Moreover, the results indicated that proteins localized in the membrane of E. coli mostly act as secretary proteins in host cells. No exact correlation was observed between NLS prediction and nuclear localization prediction by BaCeILo. This is partly because of a number of reasons, including that only 30% of nuclear proteins carry NLS and that proteins <40 kDa molecular weight can passively target the host nucleus. This study concludes that detection of gene expression-specific E. coli proteins and their targeting of the nucleus may have a profound impact on CRC etiology.

mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein

PubMed Central

Kaltimbacher, Valérie; Bonnet, Crystel; Lecoeuvre, Gaëlle; Forster, Valérie; Sahel, José-Alain; Corral-Debrinski, Marisol

2006-01-01

As previously established in yeast, two sequences within mRNAs are responsible for their specific localization to the mitochondrial surface—the region coding for the mitochondrial targeting sequence and the 3′UTR. This phenomenon is conserved in human cells. Therefore, we decided to use mRNA localization as a tool to address to mitochondria, a protein that is not normally imported. For this purpose, we associated a nuclear recoded ATP6 gene with the mitochondrial targeting sequence and the 3′UTR of the nuclear SOD2 gene, which mRNA exclusively localizes to the mitochondrial surface in HeLa cells. The ATP6 gene is naturally located into the organelle and encodes a highly hydrophobic protein of the respiratory chain complex V. In this study, we demonstrated that hybrid ATP6 mRNAs, as the endogenous SOD2 mRNA, localize to the mitochondrial surface in human cells. Remarkably, fusion proteins localize to mitochondria in vivo. Indeed, ATP6 precursors synthesized in the cytoplasm were imported into mitochondria in a highly efficient way, especially when both the MTS and the 3′UTR of the SOD2 gene were associated with the re-engineered ATP6 gene. Hence, these data indicate that mRNA targeting to the mitochondrial surface represents an attractive strategy for allowing the mitochondrial import of proteins originally encoded by the mitochondrial genome without any amino acid change in the protein that could interfere with its biologic activity. PMID:16751614

Cytoplasmic Location of α1A Voltage-Gated Calcium Channel C-Terminal Fragment (Cav2.1-CTF) Aggregate Is Sufficient to Cause Cell Death

PubMed Central

Takahashi, Makoto; Obayashi, Masato; Ishiguro, Taro; Sato, Nozomu; Niimi, Yusuke; Ozaki, Kokoro; Mogushi, Kaoru; Mahmut, Yasen; Tanaka, Hiroshi; Tsuruta, Fuminori; Dolmetsch, Ricardo; Yamada, Mitsunori; Takahashi, Hitoshi; Kato, Takeo; Mori, Osamu; Eishi, Yoshinobu; Mizusawa, Hidehiro; Ishikawa, Kinya

2013-01-01

The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei. PMID:23505410

Stabilization and localization of Xist RNA are controlled by separate mechanisms and are not sufficient for X inactivation.

PubMed

Clemson, C M; Chow, J C; Brown, C J; Lawrence, J B

1998-07-13

These studies address whether XIST RNA is properly localized to the X chromosome in somatic cells where human XIST expression is reactivated, but fails to result in X inactivation (Tinker, A.V., and C.J. Brown. 1998. Nucl. Acids Res. 26:2935-2940). Despite a nuclear RNA accumulation of normal abundance and stability, XIST RNA does not localize in reactivants or in naturally inactive human X chromosomes in mouse/ human hybrid cells. The XIST transcripts are fully stabilized despite their inability to localize, and hence XIST RNA localization can be uncoupled from stabilization, indicating that these are separate steps controlled by distinct mechanisms. Mouse Xist RNA tightly localized to an active X chromosome, demonstrating for the first time that the active X chromosome in somatic cells is competent to associate with Xist RNA. These results imply that species-specific factors, present even in mature, somatic cells that do not normally express Xist, are necessary for localization. When Xist RNA is properly localized to an active mouse X chromosome, X inactivation does not result. Therefore, there is not a strict correlation between Xist localization and chromatin inactivation. Moreover, expression, stabilization, and localization of Xist RNA are not sufficient for X inactivation. We hypothesize that chromosomal association of XIST RNA may initiate subsequent developmental events required to enact transcriptional silencing.

Micropapillary Structures in Colorectal Cancer: An Anoikis-resistant Subpopulation.

PubMed

Patankar, Madhura; Väyrynen, Sara; Tuomisto, Anne; Mäkinen, Markus; Eskelinen, Sinikka; Karttunen, Tuomo J

2018-05-01

Micropapillary structures (MIPs) are focal piles of columnar cells without extracellular matrix contact, and common in serrated colorectal carcinoma (CRC). In order to characterize biology of MIPs in colorectal cancer (CRC), the proliferation and apoptosis rates, and survivin expression were compared between MIPs and other cancer epithelial cells of CRC (non-MIPs). We assessed 46 samples of normal colorectal mucosa, 62 carcinomas and 54 polyps for proliferation (Ki67), apoptosis (M30), and survivin expression by immunohistochemistry. MIPs in carcinoma showed lower rates of proliferation and apoptosis than non-MIPs. A low rate of apotosis in MIPs was associated with poor prognosis in local carcinoma. In normal crypts, nuclear-to-cytoplasmic transition of survivin indicated epithelial cell maturation. Cancer cases showed increased cytoplasmic expression of survivin than normal mucosa and polyps. However, MIPs showed lower nuclear and cytoplasmic survivin expression than non-MIPs. Our findings suggest that MIPs represent a biologically distinct subpopulation of carcinoma cells with features of anoikis resistance and possibly quiescence. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Baculovirus infection induces disruption of the nuclear lamina.

PubMed

Zhang, Xiaomei; Xu, Kaiyan; Wei, Denghui; Wu, Wenbi; Yang, Kai; Yuan, Meijin

2017-08-10

Baculovirus nucleocapsids egress from the nucleus primarily via budding at the nuclear membrane. The nuclear lamina underlying the nuclear membrane represents a substantial barrier to nuclear egress. Whether the nuclear lamina undergoes disruption during baculovirus infection remains unknown. In this report, we generated a clonal cell line, Sf9-L, that stably expresses GFP-tagged Drosophila lamin B. GFP autofluorescence colocalized with immunofluorescent anti-lamin B at the nuclear rim of Sf9-L cells, indicating GFP-lamin B was incorporated into the nuclear lamina. Meanwhile, virus was able to replicate normally in Sf9-L cells. Next, we investigated alterations to the nuclear lamina during baculovirus infection in Sf9-L cells. A portion of GFP-lamin B localized diffusely at the nuclear rim, and some GFP-lamin B was redistributed within the nucleus during the late phase of infection, suggesting the nuclear lamina was partially disrupted. Immunoelectron microscopy revealed associations between GFP-lamin B and the edges of the electron-dense stromal mattes of the virogenic stroma, intranuclear microvesicles, and ODV envelopes and nucleocapsids within the nucleus, indicating the release of some GFP-lamin B from the nuclear lamina. Additionally, GFP-lamin B phosphorylation increased upon infection. Based on these data, baculovirus infection induced lamin B phosphorylation and disruption of the nuclear lamina.

Structural hierarchy controlling dimerization and target DNA recognition in the AHR transcriptional complex.

PubMed

Seok, Seung-Hyeon; Lee, Woojong; Jiang, Li; Molugu, Kaivalya; Zheng, Aiping; Li, Yitong; Park, Sanghyun; Bradfield, Christopher A; Xing, Yongna

2017-05-23

The aryl hydrocarbon receptor (AHR) belongs to the PAS (PER-ARNT-SIM) family transcription factors and mediates broad responses to numerous environmental pollutants and cellular metabolites, modulating diverse biological processes from adaptive metabolism, acute toxicity, to normal physiology of vascular and immune systems. The AHR forms a transcriptionally active heterodimer with ARNT (AHR nuclear translocator), which recognizes the dioxin response element (DRE) in the promoter of downstream genes. We determined the crystal structure of the mammalian AHR-ARNT heterodimer in complex with the DRE, in which ARNT curls around AHR into a highly intertwined asymmetric architecture, with extensive heterodimerization interfaces and AHR interdomain interactions. Specific recognition of the DRE is determined locally by the DNA-binding residues, which discriminates it from the closely related hypoxia response element (HRE), and is globally affected by the dimerization interfaces and interdomain interactions. Changes at the interdomain interactions caused either AHR constitutive nuclear localization or failure to translocate to nucleus, underlying an allosteric structural pathway for mediating ligand-induced exposure of nuclear localization signal. These observations, together with the global higher flexibility of the AHR PAS-A and its loosely packed structural elements, suggest a dynamic structural hierarchy for complex scenarios of AHR activation induced by its diverse ligands.

Structural hierarchy controlling dimerization and target DNA recognition in the AHR transcriptional complex

PubMed Central

Lee, Woojong; Jiang, Li; Molugu, Kaivalya; Zheng, Aiping; Li, Yitong; Park, Sanghyun; Bradfield, Christopher A.; Xing, Yongna

2017-01-01

The aryl hydrocarbon receptor (AHR) belongs to the PAS (PER-ARNT-SIM) family transcription factors and mediates broad responses to numerous environmental pollutants and cellular metabolites, modulating diverse biological processes from adaptive metabolism, acute toxicity, to normal physiology of vascular and immune systems. The AHR forms a transcriptionally active heterodimer with ARNT (AHR nuclear translocator), which recognizes the dioxin response element (DRE) in the promoter of downstream genes. We determined the crystal structure of the mammalian AHR–ARNT heterodimer in complex with the DRE, in which ARNT curls around AHR into a highly intertwined asymmetric architecture, with extensive heterodimerization interfaces and AHR interdomain interactions. Specific recognition of the DRE is determined locally by the DNA-binding residues, which discriminates it from the closely related hypoxia response element (HRE), and is globally affected by the dimerization interfaces and interdomain interactions. Changes at the interdomain interactions caused either AHR constitutive nuclear localization or failure to translocate to nucleus, underlying an allosteric structural pathway for mediating ligand-induced exposure of nuclear localization signal. These observations, together with the global higher flexibility of the AHR PAS-A and its loosely packed structural elements, suggest a dynamic structural hierarchy for complex scenarios of AHR activation induced by its diverse ligands. PMID:28396409

Structural hierarchy controlling dimerization and target DNA recognition in the AHR transcriptional complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seok, Seung-Hyeon; Lee, Woojong; Jiang, Li

he aryl hydrocarbon receptor (AHR) belongs to the PAS (PER-ARNT-SIM) family transcription factors and mediates broad responses to numerous environmental pollutants and cellular metabolites, modulating diverse biological processes from adaptive metabolism, acute toxicity, to normal physiology of vascular and immune systems. The AHR forms a transcriptionally active heterodimer with ARNT (AHR nuclear translocator), which recognizes the dioxin response element (DRE) in the promoter of downstream genes. We determined the crystal structure of the mammalian AHR–ARNT heterodimer in complex with the DRE, in which ARNT curls around AHR into a highly intertwined asymmetric architecture, with extensive heterodimerization interfaces and AHR interdomainmore » interactions. Specific recognition of the DRE is determined locally by the DNA-binding residues, which discriminates it from the closely related hypoxia response element (HRE), and is globally affected by the dimerization interfaces and interdomain interactions. Changes at the interdomain interactions caused either AHR constitutive nuclear localization or failure to translocate to nucleus, underlying an allosteric structural pathway for mediating ligand-induced exposure of nuclear localization signal. These observations, together with the global higher flexibility of the AHR PAS-A and its loosely packed structural elements, suggest a dynamic structural hierarchy for complex scenarios of AHR activation induced by its diverse ligands.« less

Histomorphometric analysis of nuclear and cellular volumetric alterations in oral lichen planus, lichenoid lesions and normal oral mucosa using image analysis software.

PubMed

Venkatesiah, Sowmya S; Kale, Alka D; Hallikeremath, Seema R; Kotrashetti, Vijayalakshmi S

2013-01-01

Lichen planus is a chronic inflammatory mucocutaneous disease that clinically and histologically resembles lichenoid lesions, although the latter has a different etiology. Though criteria have been suggested for differentiating oral lichen planus from lichenoid lesions, confusion still prevails. To study the cellular and nuclear volumetric features in the epithelium of normal mucosa, lichen planus, and lichenoid lesions to determine variations if any. A retrospective study was done on 25 histologically diagnosed cases each of oral lichen planus, oral lichenoid lesions, and normal oral mucosa. Cellular and nuclear morphometric measurements were assessed on hematoxylin and eosin sections using image analysis software. Analysis of variance test (ANOVA) and Tukey's post-hoc test. The basal cells of oral lichen planus showed a significant increase in the mean nuclear and cellular areas, and in nuclear volume; there was a significant decrease in the nuclear-cytoplasmic ratio as compared to normal mucosa. The suprabasal cells showed a significant increase in nuclear and cellular areas, nuclear diameter, and nuclear and cellular volumes as compared to normal mucosa. The basal cells of oral lichenoid lesions showed significant difference in the mean cellular area and the mean nuclear-cytoplasmic ratio as compared to normal mucosa, whereas the suprabasal cells differed significantly from normal mucosa in the mean nuclear area and the nuclear and cellular volumes. Morphometry can differentiate lesions of oral lichen planus and oral lichenoid lesions from normal oral mucosa. Thus, morphometry may serve to discriminate between normal and premalignant lichen planus and lichenoid lesions. These lesions might have a high risk for malignant transformation and may behave in a similar manner with respect to malignant transformation.

Expression, fermentation and purification of a predicted intrinsically disordered region of the transcription factor, NFAT5.

PubMed

DuMond, Jenna F; He, Yi; Burg, Maurice B; Ferraris, Joan D

2015-11-01

Hypertonicity stimulates Nuclear Factor of Activated T-cells 5 (NFAT5) nuclear localization and transactivating activity. Many transcription factors are known to contain intrinsically disordered regions (IDRs) which become more structured with local environmental changes such as osmolality, temperature and tonicity. The transactivating domain of NFAT5 is predicted to be intrinsically disordered under normal tonicity, and under high NaCl, the activity of this domain is increased. To study the binding of co-regulatory proteins at IDRs a cDNA construct expressing the NFAT5 TAD was created and transformed into Escherichia coli cells. Transformed E. coli cells were mass produced by fermentation and extracted by cell lysis to release the NFAT5 TAD. The NFAT5 TAD was subsequently purified using a His-tag column, cation exchange chromatography as well as hydrophobic interaction chromatography and then characterized by mass spectrometry (MS). Published by Elsevier Inc.

Drosophila Importin-α2 Is Involved in Synapse, Axon and Muscle Development

PubMed Central

Mosca, Timothy J.; Schwarz, Thomas L.

2010-01-01

Nuclear import is required for communication between the cytoplasm and the nucleus and to enact lasting changes in gene transcription following stimuli. Binding to an Importin-α molecule in the cytoplasm is often required to mediate nuclear entry of a signaling protein. As multiple isoforms of Importin-α exist, some may be responsible for the entry of distinct cargoes rather than general nuclear import. Indeed, in neuronal systems, Importin-α isoforms can mediate very specific processes such as axonal tiling and communication of an injury signal. To study nuclear import during development, we examined the expression and function of Importin-α2 in Drosophila melanogaster. We found that Importin-α2 was expressed in the nervous system where it was required for normal active zone density at the NMJ and axonal commissure formation in the central nervous system. Other aspects of synaptic morphology at the NMJ and the localization of other synaptic markers appeared normal in importin-α2 mutants. Importin-α2 also functioned in development of the body wall musculature. Mutants in importin-α2 exhibited errors in muscle patterning and organization that could be alleviated by restoring muscle expression of Importin-α2. Thus, Importin-α2 is needed for some processes in the development of both the nervous system and the larval musculature. PMID:21151903

Requirement of Hsp105 in CoCl{sub 2}-induced HIF-1α accumulation and transcriptional activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mikami, Hiroki; Saito, Youhei, E-mail: ysaito@mb.kyoto-phu.ac.jp; Okamoto, Namiko

The mammalian stress protein Hsp105α protects cells from stress conditions. Several studies have indicated that Hsp105α is overexpressed in many types of solid tumors, which contain hypoxic microenvironments. However, the role of Hsp105α in hypoxic tumors remains largely unknown. We herein demonstrated the involvement of Hsp105α in HIF-1 functions induced by the hypoxia-mimetic agent CoCl{sub 2}. While Hsp105α is mainly localized in the cytoplasm under normal conditions, a treatment with CoCl{sub 2} induces the nuclear localization of Hsp105α, which correlated with HIF-1α expression levels. The overexpression of degradation-resistant HIF-1α enhances the nuclear localization of Hsp105α without the CoCl{sub 2} treatment.more » The CoCl{sub 2}-dependent transcriptional activation of HIF-1, which is measured using a reporter gene containing a HIF-responsive element, is reduced by the knockdown of Hsp105α. Furthermore, the CoCl{sub 2}-induced accumulation of HIF-1α is enhanced by heat shock, which results in the nuclear localization of Hsp105, and is suppressed by the knockdown of Hsp105. Hsp105 associates with HIF-1α in CoCl{sub 2}-treated cells. These results suggest that Hsp105α plays an important role in the functions of HIF-1 under hypoxic conditions, in which Hsp105α enhances the accumulation and transcriptional activity of HIF-1 through the HIF-1α-mediated nuclear localization of Hsp105α. - Highlights: • Hsp105α is required for the CoCl{sub 2}-induced transcriptional activation and accumulation of HIF-1. • Hsp105α localizes to the nucleus and interacts with HIF-1α in CoCl{sub 2}-treated cells. • Hsp105 enhances the CoCl{sub 2}-induced accumulation of HIF-1α under heat shock conditions.« less

Plasmodium berghei MAPK1 Displays Differential and Dynamic Subcellular Localizations during Liver Stage Development

PubMed Central

Wierk, Jannika Katharina; Langbehn, Annette; Kamper, Maria; Richter, Stefanie; Burda, Paul-Christian; Heussler, Volker Theo; Deschermeier, Christina

2013-01-01

Mitogen-activated protein kinases (MAPKs) regulate key signaling events in eukaryotic cells. In the genomes of protozoan Plasmodium parasites, the causative agents of malaria, two genes encoding kinases with significant homology to other eukaryotic MAPKs have been identified (mapk1, mapk2). In this work, we show that both genes are transcribed during Plasmodium berghei liver stage development, and analyze expression and subcellular localization of the PbMAPK1 protein in liver stage parasites. Live cell imaging of transgenic parasites expressing GFP-tagged PbMAPK1 revealed a nuclear localization of PbMAPK1 in the early schizont stage mediated by nuclear localization signals in the C-terminal domain. In contrast, a distinct localization of PbMAPK1 in comma/ring-shaped structures in proximity to the parasite’s nuclei and the invaginating parasite membrane was observed during the cytomere stage of parasite development as well as in immature blood stage schizonts. The PbMAPK1 localization was found to be independent of integrity of a motif putatively involved in ATP binding, integrity of the putative activation motif and the presence of a predicted coiled-coil domain in the C-terminal domain. Although PbMAPK1 knock out parasites showed normal liver stage development, the kinase may still fulfill a dual function in both schizogony and merogony of liver stage parasites regulated by its dynamic and stage-dependent subcellular localization. PMID:23544094

The tumor suppressor SHIP1 colocalizes in nucleolar cavities with p53 and components of PML nuclear bodies.

PubMed

Ehm, Patrick; Nalaskowski, Marcus M; Wundenberg, Torsten; Jücker, Manfred

2015-01-01

The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in haematopoietic cells. By converting PI(3,4,5)P3 to PtdIns(3,4)P2 at the plasma membrane, SHIP1 modifies PI3-kinase mediated signaling. We have recently demonstrated that SHIP1 is a nucleo-cytoplasmic shuttling protein and SHIP1 nuclear puncta partially colocalize with FLASH, a component of nuclear bodies. In this study, we demonstrate that endogenous SHIP1 localizes to intranucleolar regions of both normal and leukemic haematopoietic cells. In addition, we report that ectopically expressed SHIP1 accumulates in nucleolar cavities and colocalizes with the tumor suppressor protein p53 and components of PML nuclear bodies (e.g. SP100, SUMO-1 and CK2). Moreover, SHIP1 also colocalizes in nucleolar cavities with components of the ubiquitin-proteasome pathway. By using confocal microscopy data, we generated 3D-models revealing the enormous extent of the SHIP1 aggresomes in the nucleolus. Furthermore, treatment of cells with the proteasome inhibitor MG132 causes an enlargement of nucleolar SHIP1 containing structures. Unexpectedly, this accumulation can be partially prevented by treatment with the inhibitor of nuclear protein export Leptomycin B. In recent years, several proteins aggregating in nucleolar cavities were shown to be key factors of neurodegenerative diseases and cancerogenesis. Our findings support current relevance of nuclear localized SHIP1.

The tumor suppressor SHIP1 colocalizes in nucleolar cavities with p53 and components of PML nuclear bodies

PubMed Central

Ehm, Patrick; Nalaskowski, Marcus M; Wundenberg, Torsten; Jücker, Manfred

2015-01-01

The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in haematopoietic cells. By converting PI(3,4,5)P3 to PtdIns(3,4)P2 at the plasma membrane, SHIP1 modifies PI3-kinase mediated signaling. We have recently demonstrated that SHIP1 is a nucleo-cytoplasmic shuttling protein and SHIP1 nuclear puncta partially colocalize with FLASH, a component of nuclear bodies. In this study, we demonstrate that endogenous SHIP1 localizes to intranucleolar regions of both normal and leukemic haematopoietic cells. In addition, we report that ectopically expressed SHIP1 accumulates in nucleolar cavities and colocalizes with the tumor suppressor protein p53 and components of PML nuclear bodies (e.g. SP100, SUMO-1 and CK2). Moreover, SHIP1 also colocalizes in nucleolar cavities with components of the ubiquitin-proteasome pathway. By using confocal microscopy data, we generated 3D-models revealing the enormous extent of the SHIP1 aggresomes in the nucleolus. Furthermore, treatment of cells with the proteasome inhibitor MG132 causes an enlargement of nucleolar SHIP1 containing structures. Unexpectedly, this accumulation can be partially prevented by treatment with the inhibitor of nuclear protein export Leptomycin B. In recent years, several proteins aggregating in nucleolar cavities were shown to be key factors of neurodegenerative diseases and cancerogenesis. Our findings support current relevance of nuclear localized SHIP1. PMID:25723258

Structural Basis for Activation of Fatty Acid-binding Protein 4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gillilan,R.; Ayers, S.; Noy, N.

2007-01-01

Fatty acid-binding protein 4 (FABP4) delivers ligands from the cytosol to the nuclear receptor PPAR{gamma} in the nucleus, thereby enhancing the transcriptional activity of the receptor. Notably, FABP4 binds multiple ligands with a similar affinity but its nuclear translocation is activated only by specific compounds. To gain insight into the structural features that underlie the ligand-specificity in activation of the nuclear import of FABP4, we solved the crystal structures of the protein complexed with two compounds that induce its nuclear translocation, and compared these to the apo-protein and to FABP4 structures bound to non-activating ligands. Examination of these structures indicatesmore » that activation coincides with closure of a portal loop phenylalanine side-chain, contraction of the binding pocket, a subtle shift in a helical domain containing the nuclear localization signal of the protein, and a resultant change in oligomeric state that exposes the nuclear localization signal to the solution. Comparisons of backbone displacements induced by activating ligands with a measure of mobility derived from translation, libration, screw (TLS) refinement, and with a composite of slowest normal modes of the apo state suggest that the helical motion associated with the activation of the protein is part of the repertoire of the equilibrium motions of the apo-protein, i.e. that ligand binding does not induce the activated configuration but serves to stabilize it. Nuclear import of FABP4 can thus be understood in terms of the pre-existing equilibrium hypothesis of ligand binding.« less

The localization of nuclear exporters of the importin-beta family is regulated by Snf1 kinase, nutrient supply and stress.

PubMed

Quan, XinXin; Yu, Jennifer; Bussey, Howard; Stochaj, Ursula

2007-07-01

In the budding yeast Saccharomyces cerevisiae, four members of the importin-beta family of nuclear carriers, Xpo1p/Crm1p, Cse1p, Msn5p and Los1p, function as exporters of protein and tRNA. Under normal growth conditions GFP-tagged exporters are predominantly associated with nuclei. The presence of Snf1 kinase, a key regulator of cell growth and a metabolic sensor, controls the localization of GFP-exporters. Additional glucose-dependent, but Snf1-independent, mechanisms regulate carrier distribution and a switch from fermentable to non-fermentable carbon sources relocates all of the carriers, suggesting a link to the nutritional status of the cell. Moreover, stress controls the proper localization of GFP-exporters, which mislocalize upon exposure to heat, ethanol and starvation. Stress may activate the MAPK cell integrity cascade, and we tested the role of this pathway in exporter localization. Under non-stress conditions, the proper distribution of GFP-Cse1p and Xpo1p/Crm1p-GFP requires kinases of the cell integrity cascade. By contrast, Msn5p-GFP and Los1p-GFP rely on the MAPK module to relocate to the cytoplasm when cells are stressed with ethanol. Our results indicate that the association of nuclear exporters with nuclei is controlled by multiple mechanisms that are organized in a hierarchical fashion and linked to the physiological state of the cell.

Akt/p27kip1 Pathway Is Not Involved in Human Insulinoma Tumorigenesis

PubMed Central

de Lima, Andrea Paes; Garibaldi, Paula; Rubio, Maria de los Milagros; García, Florencia; Kral, Marta; Bruno, Oscar D.

2018-01-01

Insulinomas are pancreatic neuroendocrine tumors (pNET), usually benign. Akt/p27kip1 is an intracellular pathway overexpressed in many pNET. There are no data regarding its expression in human insulinomas. We aimed to investigate the expression of Akt and p27kip1 in 24 human insulinomas and to compare them to their expression in normal surrounding islets. Staining was performed on embedded paraffin tissue using polyclonal antibodies against total Akt, p-Akt, p27kip1, and pp27kip1. p-Akt was the predominant form in insulinomas; they presented lower Akt and p-Akt expression than normal islets in 83.3% and 87.5% of tumors, respectively. p27kip1 and pp27kip1 were mainly cytoplasmic in both insulinomas and normal tissue. Cytoplasmic pp27kip1 staining was higher in insulinomas and surprisingly nearly half of the insulinomas also presented nuclear p27kip1 (p = 0.029). No differences were observed in the subcellular localization of p27kip1 and activation of Akt between benign and malignant insulinomas. The low expression of Akt seen in insulinomas might explain the usual benign behavior of this type of pNET. Cytoplasmic p27kip1 in both insulinomas and normal islet cells could reflect the low rate of replication of beta cells, while nuclear p27kip1 would seem to indicate stabilization and nuclear anchoring of the cyclin D-Cdk4 complex. Our data seem to suggest that the Akt pathway is not involved in human insulinoma tumorigenesis. PMID:29853883

Developmental Regulation of p66Shc Is Altered by Bronchopulmonary Dysplasia in Baboons and Humans

PubMed Central

Lee, Matt K.; Pryhuber, Gloria S.; Schwarz, Margaret A.; Smith, Susan M.; Pavlova, Zdena; Sunday, Mary E.

2005-01-01

Rationale: The p66Shc adapter protein antagonizes mitogen-activated protein, or MAP, kinase, mediates oxidative stress, and is developmentally regulated in fetal mouse lungs. Objectives: To determine if p66Shc is similarly regulated in primates and in bronchopulmonary dysplasia (BPD), which results from oxidative injury to immature lungs. Methods: Normal and injured lungs from humans and baboons were evaluated by Western analysis and immunohistochemistry. Measurements and Main Results: In baboons, p66Shc decreased 80% between 125 and 175 days' gestation (p = 0.025), then doubled after term delivery at 185 days (p = 0.0013). In the hyperoxic 140-day fetal baboon BPD model, p66Shc expression persisted, and its localization shifted from the epithelium of gestational controls to the mesenchyme of diseased lungs, coincident with expression of proliferating cell nuclear antigen and cleaved poly(adenyl ribose) polymerase, a marker of apoptosis. Treatment with the antibombesin antibody 2A11 attenuated BPD, reduced cell proliferation, increased p66Shc expression 10.5-fold, and preserved epithelial p66Shc localization. p66Shc also decreased during normal human lung development, falling 87% between 18 and 24 weeks' gestation (p = 0.02). p66Shc was expressed throughout 18-week human lungs, became restricted to scattered epithelial cells by 24 weeks, and localized to isolated mesenchymal cells after term delivery. In contrast, p66Shc remained prominent in the epithelium of lungs with acute injury or mild BPD, and in the mesenchyme of lungs with severe disease. p66Shc localized to tissues expressing proliferating cell nuclear antigen and cleaved poly(adenyl ribose) polymerase. Conclusions: p66Shc expression, cell proliferation, and apoptosis are concomitantly altered during lung development and in BPD. PMID:15778491

Denoising and segmentation of retinal layers in optical coherence tomography images

NASA Astrophysics Data System (ADS)

Dash, Puspita; Sigappi, A. N.

2018-04-01

Optical Coherence Tomography (OCT) is an imaging technique used to localize the intra-retinal boundaries for the diagnostics of macular diseases. Due to speckle noise, low image contrast and accurate segmentation of individual retinal layers is difficult. Due to this, a method for retinal layer segmentation from OCT images is presented. This paper proposes a pre-processing filtering approach for denoising and segmentation methods for segmenting retinal layers OCT images using graph based segmentation technique. These techniques are used for segmentation of retinal layers for normal as well as patients with Diabetic Macular Edema. The algorithm based on gradient information and shortest path search is applied to optimize the edge selection. In this paper the four main layers of the retina are segmented namely Internal limiting membrane (ILM), Retinal pigment epithelium (RPE), Inner nuclear layer (INL) and Outer nuclear layer (ONL). The proposed method is applied on a database of OCT images of both ten normal and twenty DME affected patients and the results are found to be promising.

Met Nuclear Localization and Signaling in Breast Cancer

DTIC Science & Technology

2006-05-01

and in germinal regions of many tissues using 4 unique antibodies . Cell fractionation reveals a 60kDa band recognized by C-terminal Met antibodies ...cascades such as Gab1 , Grb2 and PI3K, leading to proliferation, scattering, increased motility, invasion and branching morphogenesis (reviewed in (2...Identification of Met antibodies for use on tissue microarray of normal and cancerous cells, Months 12-24 Task 2. Definition of the domain

Protein Kinase D1 attenuates tumorigenesis in colon cancer by modulating β-catenin/T cell factor activity

PubMed Central

Sundram, Vasudha; Ganju, Aditya; Hughes, Joshua E.; Khan, Sheema; Chauhan, Subhash C.; Jaggi, Meena

2014-01-01

Over 80% of colon cancer development and progression is a result of the dysregulation of β-catenin signaling pathway. Herein, for the first time, we demonstrate that a serine-threonine kinase, Protein Kinase D1 (PKD1), modulates the functions of β-catenin to suppress colon cancer growth. Analysis of normal and colon cancer tissues reveals downregulation of PKD1 expression in advanced stages of colon cancer and its co-localization with β-catenin in the colon crypts. This PKD1 downregulation corresponds with the aberrant expression and nuclear localization of β-catenin. In-vitro investigation of the PKD1-β-catenin interaction in colon cancer cells reveal that PKD1 overexpression suppresses cell proliferation and clonogenic potential and enhances cell-cell aggregation. We demonstrate that PKD1 directly interacts with β-catenin and attenuates β-catenin transcriptional activity by decreasing nuclear β-catenin levels. Additionally, we show that inhibition of nuclear β-catenin transcriptional activity is predominantly influenced by nucleus targeted PKD1. This subcellular modulation of β-catenin results in enhanced membrane localization of β-catenin and thereby increases cell-cell adhesion. Studies in a xenograft mouse model indicate that PKD1 overexpression delayed tumor appearance, enhanced necrosis and lowered tumor hypoxia. Overall, our results demonstrate a putative tumor-suppressor function of PKD1 in colon tumorigenesis via modulation of β-catenin functions in cells. PMID:25149539

The C3H-type zinc finger protein GDS1/C3H42 is a nuclear-speckle-localized protein that is essential for normal growth and development in Arabidopsis.

PubMed

Kim, Dae Won; Jeon, Su Jeong; Hwang, Sung Min; Hong, Jong Chan; Bahk, Jeong Dong

2016-09-01

Eukaryotic C3H-type zinc finger proteins (Znfs) comprise a large family of regulatory proteins involved in many aspects of plant stress response, growth and development. However, compared to mammalian, only a few plant Znfs have been functionally characterized. Here, T-DNA inserted gds1 (growth, development and splicing 1) mutant, displayed abnormal growth throughout the lifecycle owing to the reduction of cell size and number. Inverse PCR analysis revealed that the abnormal growth was caused by the disruption of At3g47120, which encodes a C3H42 protein belonging to the C-X7-C-X5-C-X3-H class of the Znf family. GDS1 was ubiquitously transcribed, but shows high levels of expression in young seedling and unexpanded new leaves. In gds1, the transcripts of many growth- and development-related genes were down-regulated, and the auxin response was dramatically reduced. A fluorescence-based assay revealed that the GDS1 protein was localized to the nucleus, prominently in the speckle compartments. Its arginine/serine dipeptide-rich-like (RS-like) domain was essential for nuclear localization. In addition, the SR1, SRm102 and U1-70K components of the U1 spliceosome interacted with GDS1 in the nuclear speckle compartments. Taken together, these suggest that GDS1, a nuclear-speckle-associated Znf, might play a significant role in splicing during plant growth and development. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP) but not PPAR-interacting protein (PRIP) is required for nuclear translocation of constitutive androstane receptor in mouse liver.

PubMed

Guo, Dongsheng; Sarkar, Joy; Ahmed, Mohamed R; Viswakarma, Navin; Jia, Yuzhi; Yu, Songtao; Sambasiva Rao, M; Reddy, Janardan K

2006-08-25

The constitutive androstane receptor (CAR) regulates transcription of phenobarbital-inducible genes that encode xenobiotic-metabolizing enzymes in liver. CAR is localized to the hepatocyte cytoplasm but to be functional, it translocates into the nucleus in the presence of phenobarbital-like CAR ligands. We now demonstrate that adenovirally driven EGFP-CAR, as expected, translocates into the nucleus of normal wild-type hepatocytes following phenobarbital treatment under both in vivo and in vitro conditions. Using this approach we investigated the role of transcription coactivators PBP and PRIP in the translocation of EGFP-CAR into the nucleus of PBP and PRIP liver conditional null mouse hepatocytes. We show that coactivator PBP is essential for nuclear translocation of CAR but not PRIP. Adenoviral expression of both PBP and EGFP-CAR restored phenobarbital-mediated nuclear translocation of exogenously expressed CAR in PBP null livers in vivo and in PBP null primary hepatocytes in vitro. CAR translocation into the nucleus of PRIP null livers resulted in the induction of CAR target genes such as CYP2B10, necessary for the conversion of acetaminophen to its hepatotoxic intermediate metabolite, N-acetyl-p-benzoquinone imine. As a consequence, PRIP-deficiency in liver did not protect from acetaminophen-induced hepatic necrosis, unlike that exerted by PBP deficiency. These results establish that transcription coactivator PBP plays a pivotal role in nuclear localization of CAR, that it is likely that PBP either enhances nuclear import or nuclear retention of CAR in hepatocytes, and that PRIP is redundant for CAR function.

Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

PubMed Central

Chen, Yi; Pirisi, Lucia; Creek, Kim E.

2013-01-01

We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. PMID:23809940

Selective Entrapment of Extrachromosomally Amplified DNA by Nuclear Budding and Micronucleation during S Phase

PubMed Central

Shimizu, Noriaki; Itoh, Nobuo; Utiyama, Hiroyasu; Wahl, Geoffrey M.

1998-01-01

Acentric, autonomously replicating extrachromosomal structures called double-minute chromosomes (DMs) frequently mediate oncogene amplification in human tumors. We show that DMs can be removed from the nucleus by a novel micronucleation mechanism that is initiated by budding of the nuclear membrane during S phase. DMs containing c-myc oncogenes in a colon cancer cell line localized to and replicated at the nuclear periphery. Replication inhibitors increased micronucleation; cell synchronization and bromodeoxyuridine–pulse labeling demonstrated de novo formation of buds and micronuclei during S phase. The frequencies of S-phase nuclear budding and micronucleation were increased dramatically in normal human cells by inactivating p53, suggesting that an S-phase function of p53 minimizes the probability of producing the broken chromosome fragments that induce budding and micronucleation. These data have implications for understanding the behavior of acentric DNA in interphase nuclei and for developing chemotherapeutic strategies based on this new mechanism for DM elimination. PMID:9508765

Expression of human telomerase reverse transcriptase protein in oral epithelial dysplasia and oral squamous cell carcinoma: An immunohistochemical study

PubMed Central

Raghunandan, Bangalore Nagarajachar; Sanjai, Karpagaselvi; Kumaraswamy, Jayalakshmi; Papaiah, Lokesh; Pandey, Bhavna; Jyothi, Bellur MadhavaRao

2016-01-01

Background: Telomerase is an RNA-dependent DNA polymerase that synthesizes TTAGGG telomeric DNA sequences and almost universally provides the molecular basis for unlimited proliferative potential. The telomeres become shorter with each cycle of replication and reach a critical limit; most cells die or enter stage of replicative senescence. Telomere length maintenance by telomerase is required for all the cells that exhibit limitless replicative potential. It has been postulated that reactivation of telomerase expression is necessary for the continuous proliferation of neoplastic cells to attain immortality. Use of immunohistochemistry (IHC) is a useful, reliable method of localizing the human telomerase reverse transcriptase (hTERT) protein in tissue sections which permits cellular localization. Although there exists a lot of information on telomerase in oral cancer, little is known about their expression in oral epithelial dysplasia and their progression to oral squamous cell carcinoma (OSCC) compared to normal oral mucosa. This study addresses this lacuna. Aims: To compare the expression of hTERT protein in oral epithelial dysplasia and OSCC with normal oral mucosa by Immunohistochemical method. Subjects and Methods: In this preliminary study, IHC was used to detect the expression of hTERT protein in OSCC (n = 20), oral epithelial dysplasia (n = 21) and normal oral mucosa (n = 10). The tissue localization of immunostain, cellular localization of immunostain, nature of stain, intensity of stain, percentage of cells stained with hTERT protein were studied. A total number of 100 cells were counted in each slide. Statistical Analysis: All the data were analyzed using SPSS software version 16.0. The tissue localization, cellular localization of cytoplasmic/nuclear/both of hTERT stain, staining intensity was compared across the groups using Pearson's Chi-square test. The mean percentage of cells stained for oral epithelial dysplasia, OSCC and normal oral mucosa were compared using analysis of variance (ANOVA). A P < 0.05 was considered to be statistically significant. Results: The mean hTERT positive cells in the study groups were as follows, 62.91% in normal oral mucosa samples, 77.06% in oral epithelial dysplasia cases, and 81.48% in OSCC. In 61.9% of oral epithelial dysplasia and 65% of OSCC in our study, staining was visualized within the nucleus predominantly in the dot like pattern. There was a statistically significant difference in the nature of nuclear stain between oral epithelial dysplasia and OSCC (P = 0.023). Conclusions: Our results suggests that the mean percentage of cells showing hTERT expression steadily increased from normal oral mucosa to oral epithelial dysplasia to OSCC. The steady trend of increase in the percentage of cells was evident in different grades of oral epithelial dysplasia group and OSCC. The nature of hTERT staining did show variations among the three groups and promise to be a potential surrogate marker for malignant transformation. Further studies using IHC on larger sample size and clinical follow-up of these patients will be ascertaining the full potential of hTERT as a surrogate marker of epithelial transformation. PMID:27194869

Intracellular distribution of Photofrin in malignant and normal endothelial cell lines.

PubMed

Saczko, J; Mazurkiewicz, M; Chwiłkowska, A; Kulbacka, J; Kramer, G; Ługowski, M; Snietura, M; Banaś, T

2007-01-01

Compared to current treatments including surgery, radiation therapy, and chemotherapy, PDT offers the advantage of an effective and selective method of destroying diseased tissues without damaging surrounding healthy tissues. One of the aspects of antitumour effectiveness of PDT is related to the distribution of photosensitizing drugs. The localization of photosensitizers in cytoplasmic organelles during PDT plays a major role in the cell destruction; therefore, intracellular localization of Ph in malignant and normal cells was investigated. The cell lines used throughout the study were: human malignant A549, MCF-7, Me45 and normal endothelial cell line HUV-EC-C. After incubation with Ph cells were examined using fluorescence and confocal microscopy to visualize the photosensitizer accumulation. For cytoplasm and mitochondria identification, cells were stained with CellTracker Green and MitoTracker Green, respectively. Distribution of Ph was different in malignant and normal cells and dependent on the incubation time. The maximal concentration of Ph in two malignant cell lines (A549 and MCF-7) was observed after 4 hours of incubation, and the most intensive signal was observed around the nuclear envelope. Intracellular distribution of Ph in the Me45 cell line showed that the fluorescence emitted by Ph overlaid that from MitoTracker. This indicates preferential accumulation of the sensitizer in mitochondria. Our results based on the mitochondrial localization support the idea that PDT can contribute to elimination of malignant cells by inducing apoptosis, which is of physiological significance.

Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bryantsev, A.L.; Chechenova, M.B.; Shelden, E.A.

During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absencemore » of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.« less

Free Radicals Generated by Ionizing Radiation Signal Nuclear Translocation of p53

NASA Technical Reports Server (NTRS)

Martinez, J. D.; Pennington, M. E.; Craven, M. T.; Warters, R. L.

1997-01-01

The p53 tumor suppressor is a transcription factor that regulates several pathways, which function collectively to maintain the integrity of the genome. Nuclear localization is critical for wild-type function. However, the signals that regulate subcellular localization of p53 have not been identified. Here, we examine the effect of ionizing radiation on the subcellular localization of p53 in two cell lines in which p63 is normally sequestered in the cytoplasm and found that ionizing radiation caused a biphasic translocation response. p53 entered the nucleus 1-2 hours postirradiation (early response), subsequently emerged from the nucleus, and then again entered the nucleus 12-24 hours after the cells had been irradiated (delayed response). These changes in subcellular localization could be completely blocked by the free radical scavenger, WR1065. By comparison, two DNA-damaging agents that do not generate free radicals, mitomycin C and doxorubicin, caused translocation only after 12-24 h of exposure to the drugs, and this effect could not be inhibited by WR1065. Hence, although all three DNA-damaging agents induced relocalization of p53 to the nucleus, only the translocation caused by radiation was sensitive to free radical scavenging. We suggest that the free radicals generated by ionizing radiation can signal p53 translocation to the nucleus.

Condensins exert force on chromatin-nuclear envelope tethers to mediate nucleoplasmic reticulum formation in Drosophila melanogaster.

PubMed

Bozler, Julianna; Nguyen, Huy Q; Rogers, Gregory C; Bosco, Giovanni

2014-12-30

Although the nuclear envelope is known primarily for its role as a boundary between the nucleus and cytoplasm in eukaryotes, it plays a vital and dynamic role in many cellular processes. Studies of nuclear structure have revealed tissue-specific changes in nuclear envelope architecture, suggesting that its three-dimensional structure contributes to its functionality. Despite the importance of the nuclear envelope, the factors that regulate and maintain nuclear envelope shape remain largely unexplored. The nuclear envelope makes extensive and dynamic interactions with the underlying chromatin. Given this inexorable link between chromatin and the nuclear envelope, it is possible that local and global chromatin organization reciprocally impact nuclear envelope form and function. In this study, we use Drosophila salivary glands to show that the three-dimensional structure of the nuclear envelope can be altered with condensin II-mediated chromatin condensation. Both naturally occurring and engineered chromatin-envelope interactions are sufficient to allow chromatin compaction forces to drive distortions of the nuclear envelope. Weakening of the nuclear lamina further enhanced envelope remodeling, suggesting that envelope structure is capable of counterbalancing chromatin compaction forces. Our experiments reveal that the nucleoplasmic reticulum is born of the nuclear envelope and remains dynamic in that they can be reabsorbed into the nuclear envelope. We propose a model where inner nuclear envelope-chromatin tethers allow interphase chromosome movements to change nuclear envelope morphology. Therefore, interphase chromatin compaction may be a normal mechanism that reorganizes nuclear architecture, while under pathological conditions, such as laminopathies, compaction forces may contribute to defects in nuclear morphology. Copyright © 2015 Bozler et al.

Condensins Exert Force on Chromatin-Nuclear Envelope Tethers to Mediate Nucleoplasmic Reticulum Formation in Drosophila melanogaster

PubMed Central

Bozler, Julianna; Nguyen, Huy Q.; Rogers, Gregory C.; Bosco, Giovanni

2014-01-01

Although the nuclear envelope is known primarily for its role as a boundary between the nucleus and cytoplasm in eukaryotes, it plays a vital and dynamic role in many cellular processes. Studies of nuclear structure have revealed tissue-specific changes in nuclear envelope architecture, suggesting that its three-dimensional structure contributes to its functionality. Despite the importance of the nuclear envelope, the factors that regulate and maintain nuclear envelope shape remain largely unexplored. The nuclear envelope makes extensive and dynamic interactions with the underlying chromatin. Given this inexorable link between chromatin and the nuclear envelope, it is possible that local and global chromatin organization reciprocally impact nuclear envelope form and function. In this study, we use Drosophila salivary glands to show that the three-dimensional structure of the nuclear envelope can be altered with condensin II-mediated chromatin condensation. Both naturally occurring and engineered chromatin-envelope interactions are sufficient to allow chromatin compaction forces to drive distortions of the nuclear envelope. Weakening of the nuclear lamina further enhanced envelope remodeling, suggesting that envelope structure is capable of counterbalancing chromatin compaction forces. Our experiments reveal that the nucleoplasmic reticulum is born of the nuclear envelope and remains dynamic in that they can be reabsorbed into the nuclear envelope. We propose a model where inner nuclear envelope-chromatin tethers allow interphase chromosome movements to change nuclear envelope morphology. Therefore, interphase chromatin compaction may be a normal mechanism that reorganizes nuclear architecture, while under pathological conditions, such as laminopathies, compaction forces may contribute to defects in nuclear morphology. PMID:25552604

Pressure dependence of coherence-incoherence crossover behavior in KFe2As2 observed by resistivity and 75As-NMR/NQR

NASA Astrophysics Data System (ADS)

Wiecki, P.; Taufour, V.; Chung, D. Y.; Kanatzidis, M. G.; Bud'ko, S. L.; Canfield, P. C.; Furukawa, Y.

2018-02-01

We present the results of 75As nuclear magnetic resonance (NMR), nuclear quadrupole resonance (NQR), and resistivity measurements in KFe2As2 under pressure (p ). The temperature dependence of the NMR shift, nuclear spin-lattice relaxation time (T1), and resistivity show a crossover between a high-temperature incoherent, local-moment behavior and a low-temperature coherent behavior at a crossover temperature (T*). T* is found to increase monotonically with pressure, consistent with increasing hybridization between localized 3 d orbital-derived bands with the itinerant electron bands. No anomaly in T* is seen at the critical pressure pc=1.8 GPa where a change of slope of the superconducting (SC) transition temperature Tc(p ) has been observed. In contrast, Tc(p ) seems to correlate with antiferromagnetic spin fluctuations in the normal state as measured by the NQR 1 /T1 data, although such a correlation cannot be seen in the replacement effects of A in the A Fe2As2 (A =K , Rb, Cs) family. In the superconducting state, two T1 components are observed at low temperatures, suggesting the existence of two distinct local electronic environments. The temperature dependence of the short T1 s indicates a nearly gapless state below Tc. On the other hand, the temperature dependence of the long component 1 /T1 L implies a large reduction in the density of states at the Fermi level due to the SC gap formation. These results suggest a real-space modulation of the local SC gap structure in KFe2As2 under pressure.

10 CFR 50.60 - Acceptance criteria for fracture prevention measures for lightwater nuclear power reactors for...

Code of Federal Regulations, 2010 CFR

2010-01-01

... lightwater nuclear power reactors for normal operation. 50.60 Section 50.60 Energy NUCLEAR REGULATORY... lightwater nuclear power reactors for normal operation. (a) Except as provided in paragraph (b) of this section, all light-water nuclear power reactors, other than reactor facilities for which the...

10 CFR 50.60 - Acceptance criteria for fracture prevention measures for lightwater nuclear power reactors for...

Code of Federal Regulations, 2011 CFR

2011-01-01

... lightwater nuclear power reactors for normal operation. 50.60 Section 50.60 Energy NUCLEAR REGULATORY... lightwater nuclear power reactors for normal operation. (a) Except as provided in paragraph (b) of this section, all light-water nuclear power reactors, other than reactor facilities for which the...

Different patterns of nuclear and mitochondrial penetration by the G3 PAMAM dendrimer and its biotin–pyridoxal bioconjugate BC-PAMAM in normal and cancer cells in vitro

PubMed Central

Uram, Łukasz; Szuster, Magdalena; Filipowicz, Aleksandra; Gargasz, Krzysztof; Wołowiec, Stanisław; Wałajtys-Rode, Elżbieta

2015-01-01

The intracellular localization and colocalization of a fluorescently labeled G3 amine-terminated cationic polyamidoamine (PAMAM) dendrimer and its biotin–pyridoxal (BC-PAMAM) bioconjugate were investigated in a concentration-dependent manner in normal human fibroblast (BJ) and squamous epithelial carcinoma (SCC-15) cell lines. After 24 hours treatment, both cell lines revealed different patterns of intracellular dendrimer accumulation depending on their cytotoxic effects. Cancer cells exhibited much higher (20-fold) tolerance for native PAMAM treatment than fibroblasts, whereas BC-PAMAM was significantly toxic only for fibroblasts at 50 µM concentration. Fibroblasts accumulated the native and bioconjugated dendrimers in a concentration-dependent manner at nontoxic range of concentration, with significantly lower bioconjugate loading. After reaching the cytotoxicity level, fluorescein isothiocyanate-PAMAM accumulation remains at high, comparable level. In cancer cells, native PAMAM loading at higher, but not cytotoxic concentrations, was kept at constant level with a sharp increase at toxic concentration. Mander’s coefficient calculated for fibroblasts and cancer cells confirmed more efficient native PAMAM penetration as compared to BC-PAMAM. Significant differences in nuclear dendrimer penetration were observed for both cell lines. In cancer cells, PAMAM signals amounted to ~25%–35% of the total nuclei area at all investigated concentrations, with lower level (15%–25%) observed for BC-PAMAM. In fibroblasts, the dendrimer nuclear signal amounted to 15% at nontoxic and up to 70% at toxic concentrations, whereas BC-PAMAM remained at a lower concentration-dependent level (0.3%–20%). Mitochondrial localization of PAMAM and BC-PAMAM revealed similar patterns in both cell lines, depending on the extracellular dendrimer concentration, and presented significantly lower signals from BC-PAMAM, which correlated well with the cytotoxicity. PMID:26379435

Target of Rapamycin Regulates Development and Ribosomal RNA Expression through Kinase Domain in Arabidopsis1[W][OA

PubMed Central

Ren, Maozhi; Qiu, Shuqing; Venglat, Prakash; Xiang, Daoquan; Feng, Li; Selvaraj, Gopalan; Datla, Raju

2011-01-01

Target of rapamycin (TOR) is a central regulator of cell growth, cell death, nutrition, starvation, hormone, and stress responses in diverse eukaryotes. However, very little is known about TOR signaling and the associated functional domains in plants. We have taken a genetic approach to dissect TOR functions in Arabidopsis (Arabidopsis thaliana) and report here that the kinase domain is essential for the role of TOR in embryogenesis and 45S rRNA expression. Twelve new T-DNA insertion mutants, spanning 14.2 kb of TOR-encoding genomic region, have been characterized. Nine of these share expression of defective kinase domain and embryo arrest at 16 to 32 cell stage. However, three T-DNA insertion lines affecting FATC domain displayed normal embryo development, indicating that FATC domain was dispensable in Arabidopsis. Genetic complementation showed that the TOR kinase domain alone in tor-10/tor-10 mutant background can rescue early embryo lethality and restore normal development. Overexpression of full-length TOR or kinase domain in Arabidopsis displayed developmental abnormalities in meristem, leaf, root, stem, flowering time, and senescence. We further show that TOR, especially the kinase domain, plays a role in ribosome biogenesis by activating 45S rRNA production. Of the six putative nuclear localization sequences in the kinase domain, nuclear localization sequence 6 was identified to confer TOR nuclear targeting in transient expression assays. Chromatin immunoprecipitation studies revealed that the HEAT repeat domain binds to 45S rRNA promoter and the 5′ external transcribed spacer elements motif. Together, these results show that TOR controls the embryogenesis, postembryonic development, and 45S rRNA production through its kinase domain in Arabidopsis. PMID:21266656

Differential prooxidative effects of the green tea polyphenol, (-)-epigallocatechin-3-gallate, in normal and oral cancer cells are related to differences in sirtuin 3 signaling.

PubMed

Tao, Ling; Park, Jong-Yung; Lambert, Joshua D

2015-02-01

We have previously reported that the green tea catechin, (-)-epigallocatechin-3-gallate (EGCG), can induce oxidative stress in oral cancer cells but exerts antioxidant effects in normal cells. Here, we report that these differential prooxidative effects are associated with sirtuin 3 (SIRT3), an important mitochondrial redox modulator. EGCG rapidly induced mitochondria-localized reactive oxygen species in human oral squamous carcinoma cells (SCC-25, SCC-9) and premalignant leukoplakia cells (MSK-Leuk1), but not in normal human gingival fibroblast cells (HGF-1). EGCG suppressed SIRT3 mRNA and protein expression, as well as, SIRT3 activity in SCC-25 cells, whereas it increased SIRT3 activity in HGF-1 cells. EGCG selectively decreased the nuclear localization of the estrogen-related receptor α (ERRα), the transcription factor regulating SIRT3 expression, in SCC-25 cells. This indicates that EGCG may regulate SIRT3 transcription in oral cancer cells via ERRα. EGCG also differentially modulated the mRNA expressions of SIRT3-associated downstream targets including glutathione peroxidase 1 and superoxide dismutase 2 in normal and oral cancer cells. SIRT3 represents a novel potential target through which EGCG exerts differential prooxidant effects in cancer and normal cells. Our results provide new biomarkers to be further explored in animal studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The compensatory G88R change is essential in restoring the normal functions of influenza A/WSN/33 virus matrix protein 1 with a disrupted nuclear localization signal.

PubMed

Xie, Hang; Lin, Zhengshi; Mosier, Philip D; Desai, Umesh R; Gao, Yamei

2013-01-01

G88R emerged as a compensatory mutation in matrix protein 1 (M1) of influenza virus A/WSN/33 when its nuclear localization signal (NLS) was disrupted by R101S and R105S substitutions. The resultant M1 triple mutant M(NLS-88R) regained replication efficiency in vitro while remaining attenuated in vivo with the potential of being a live vaccine candidate. To understand why G88R was favored by the virus as a compensatory change for the NLS loss and resultant replication deficiency, three more M1 triple mutants with an alternative G88K, G88V, or G88E change in addition to R101S and R105S substitutions in the NLS were generated. Unlike the other M1 triple mutants, M(NLS-88R) replicated more efficiently in vitro and in vivo. The G88R compensatory mutation not only restored normal functions of M1 in the presence of a disrupted NLS but also resulted in a strong association of M1 with viral ribonucleoprotein. Under a transmission electron microscope, only the M1 layer of the M(NLS-88R) virion exhibited discontinuous fingerprint-like patterns with average thicknesses close to that of wild-type A/WSN/33. Computational modeling suggested that the compensatory G88R change could reestablish the integrity of the M1 layer through new salt bridges between adjacent M1 subunits when the original interactions were interrupted by simultaneous R101S and R105S replacements in the NLS. Our results suggested that restoring the normal functions of M1 was crucial for efficient virus replication.

Metabolic Engineering of the Regulators in Nitrogen Catabolite Repression To Reduce the Production of Ethyl Carbamate in a Model Rice Wine System

PubMed Central

Zhao, Xinrui; Zou, Huijun; Fu, Jianwei; Chen, Jian

2014-01-01

Rice wine has been one of the most popular traditional alcoholic drinks in China. However, the presence of potentially carcinogenic ethyl carbamate (EC) in rice wine has raised a series of food safety issues. During rice wine production, the key reason for EC formation is urea accumulation, which occurs because of nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. NCR represses urea utilization by retaining Gln3p in the cytoplasm when preferred nitrogen sources are present. In order to increase the nuclear localization of Gln3p, some possible phosphorylation sites on the nuclear localization signal were mutated and the nuclear localization regulation signal was truncated, and the disruption of URE2 provided an additional method of reducing urea accumulation. By combining these strategies, the genes involved in urea utilization (DUR1,2 and DUR3) could be significantly activated in the presence of glutamine. During shake flask fermentations of the genetically modified strains, very little urea accumulated in the medium. Furthermore, the concentrations of urea and EC were reduced by 63% and 72%, respectively, in a model rice wine system. Examination of the normal nutrients in rice wine indicated that there were few differences in fermentation characteristics between the wild-type strain and the genetically modified strain. These results show that metabolic engineering of the NCR regulators has great potential as a method for eliminating EC during rice wine production. PMID:24185848

Metabolic engineering of the regulators in nitrogen catabolite repression to reduce the production of ethyl carbamate in a model rice wine system.

PubMed

Zhao, Xinrui; Zou, Huijun; Fu, Jianwei; Zhou, Jingwen; Du, Guocheng; Chen, Jian

2014-01-01

Rice wine has been one of the most popular traditional alcoholic drinks in China. However, the presence of potentially carcinogenic ethyl carbamate (EC) in rice wine has raised a series of food safety issues. During rice wine production, the key reason for EC formation is urea accumulation, which occurs because of nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. NCR represses urea utilization by retaining Gln3p in the cytoplasm when preferred nitrogen sources are present. In order to increase the nuclear localization of Gln3p, some possible phosphorylation sites on the nuclear localization signal were mutated and the nuclear localization regulation signal was truncated, and the disruption of URE2 provided an additional method of reducing urea accumulation. By combining these strategies, the genes involved in urea utilization (DUR1,2 and DUR3) could be significantly activated in the presence of glutamine. During shake flask fermentations of the genetically modified strains, very little urea accumulated in the medium. Furthermore, the concentrations of urea and EC were reduced by 63% and 72%, respectively, in a model rice wine system. Examination of the normal nutrients in rice wine indicated that there were few differences in fermentation characteristics between the wild-type strain and the genetically modified strain. These results show that metabolic engineering of the NCR regulators has great potential as a method for eliminating EC during rice wine production.

JOZSO, a computer code for calculating broad neutron resonances in phenomenological nuclear potentials

NASA Astrophysics Data System (ADS)

Baran, Á.; Noszály, Cs.; Vertse, T.

2018-07-01

A renewed version of the computer code GAMOW (Vertse et al., 1982) is given in which the difficulties in calculating broad neutron resonances are amended. New types of phenomenological neutron potentials with strict finite range are built in. Landscape of the S-matrix can be generated on a given domain of the complex wave number plane and S-matrix poles in the domain are localized. Normalized Gamow wave functions and trajectories of given poles can be calculated optionally.

A COMPARATIVE ANALYSIS BETWEEN FRANCE AND JAPAN ON LOCAL GOVERNMENTS' INVOLVEMENT IN NUCLEAR SAFETY GOVERNANCE

NASA Astrophysics Data System (ADS)

Sugawara, Shin-Etsu; Shiroyama, Hideaki

This paper shows a comparative analysis between France and Japan on the way of the local governments' involvement in nuclear safety governance through some interviews. In France, a law came into force that requires related local governments to establish "Commision Locale d'Information" (CLI), which means the local governments officially involve in nuclear regulatory activity. Meanwhile, in Japan, related local governments substantially involve in the operation of nuclear facilities through the "safety agreements" in spite of the lack of legal authority. As a result of comparative analysis, we can point out some institutional input from French cases as follows: to clarify the local governments' roles in the nuclear regulation system, to establish the official channels of communication among nuclear utilities, national regulatory authorities and local governments, and to stipulate explicitly the transparency as a purpose of safety regulation.

Cytoplasmic YY1 Is Associated with Increased Smooth Muscle-Specific Gene Expression

PubMed Central

Favot, Laure; Hall, Susan M.; Haworth, Sheila G.; Kemp, Paul R.

2005-01-01

Immediately after birth the adluminal vascular SMCs of the pulmonary elastic arteries undergo transient actin cytoskeletal remodeling as well as cellular de-differentiation and proliferation. Vascular smooth muscle phenotype is regulated by serum response factor, which is itself regulated in part by the negative regulator YY1. We therefore studied the subcellular localization of YY1 in arteries of normal newborn piglets and piglets affected by neonatal pulmonary hypertension. We found that YY1 localization changed during development and that expression of γ-smooth muscle actin correlated with expression of cytoplasmic rather than nuclear YY1. Analysis of the regulation of YY1 localization in vitro demonstrated that polymerized γ-actin sequestered EGFP-YY1 in the cytoplasm and that YY1 activation of c-myc promoter activity was inhibited by LIM kinase, which increases actin polymerization. Consistent with these data siRNA-mediated down-regulation of YY1 in C2C12 cells increased SM22-α expression and inhibited cell proliferation. Thus, actin polymerization controls subcellular YY1 localization, which contributes to vascular SMC proliferation and differentiation in normal pulmonary artery development. In the absence of actin depolymerization, YY1 does not relocate to the nucleus, and this lack of relocation may contribute to the pathobiology of pulmonary hypertension. PMID:16314465

Regulation of IL-10 expression by upstream stimulating factor (USF-1) in glioma-associated microglia.

PubMed

Zhang, Leying; Handel, Michelle Van; Schartner, Jill M; Hagar, Aaron; Allen, Grant; Curet, Marjorie; Badie, Behnam

2007-03-01

Understanding the local CNS immune response to neoplasms is essential in the development of immune-based treatments for malignant brain tumors. Using rodent glioma models, we have recently found tumor-associated microglia/macrophages (MG/MP) to be less responsive to known MG/MP activators such as CpG, LPS and IFN-gamma. To understand the mechanism of MG/MP suppression, nuclear extracts from rodent intracranial C6 gliomas, C6 glioma-associated MG/MP, normal brain, and normal MG/MP were obtained and studied using Electrophoretic Mobility Shift Assay (EMSA). Among the nuclear factors studied (AP-1, IRF, USF-1 and Stat-1) only USF-1, which is constitutively expressed in most cells, was down-regulated in tumor-associated MG/MP, but not normal MG/MP. Because tumor-associated MG/MP had higher expression of IL-10 (but not TNF-alpha or TGF-beta), we evaluated the role of USF-1 on IL-10 expression. siRNA mediated inhibition of USF-1 expression in primary MG/MP cultures resulted in up-regulation of IL-10 mRNA but not TNF-alpha or TGF-beta. These findings suggest that USF-1 may play a role in IL-10 regulation in MG/MP in brain tumors.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka, E-mail: kinjo@sci.hokudai.ac.jp

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 biogenesismore » 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.« less

Identification of novel nuclear localization signals of Drosophila myeloid leukemia factor.

PubMed

Sugano, Wakana; Yamaguchi, Masamitsu

2007-01-01

Myeloid leukemia factor 1 (MLF1) was first identified as part of a leukemic fusion protein produced by a chromosomal translocation, and MLF family proteins are present in many animals. In mammalian cells, MLF1 has been described as mainly cytoplasmic, but in Drosophila, one of the dMLF isoforms (dMLFA) localized mainly in the nucleus while the other isoform (dMLFB), that appears to be produced by the alternative splicing, displays both nuclear and cytoplasmic localization. To investigate the difference in subcellular localization between MLF family members, we examined the subcellular localization of deletion mutants of dMLFA isoform. The analyses showed that the C-terminal 40 amino acid region of dMLFA is necessary and sufficient for nuclear localization. Based on amino acid sequences, we hypothesized that two nuclear localization signals (NLSs) are present within the region. Site-directed mutagenesis of critical residues within the two putative NLSs leads to loss of nuclear localization, suggesting that both NLS motifs are necessary for nuclear localization.

Novel optical-based methods and analyses for elucidating cellular mechanics and dynamics

NASA Astrophysics Data System (ADS)

Koo, Peter K.

Resolving distinct biochemical interaction states by analyzing the diffusive behaviors of individual protein trajectories is challenging due to the limited statistics provided by short trajectories and experimental noise sources, which are intimately coupled into each proteins localization. In the first part of this thesis, we introduce a novel, a machine-learning based classification methodology, called perturbation expectation-maximization (pEM), which simultaneously analyzes a population of protein trajectories to uncover the system of short-time diffusive behaviors which collectively result from distinct biochemical interactions. We then discuss an experimental application of pEM to Rho GTPase, an integral regulator of cytoskeletal dynamics and cellular homeostasis, inside live cells. We also derive the maximum likelihood estimator (MLE) for driven diffusion, confined diffusion, and fractional Brownian motion. We demonstrate that MLE yields improved estimates in comparison with traditional diffusion analysis, namely mean squared displacement analysis. In addition, we also introduce mleBayes, which is an empirical Bayesian model selection scheme to classify an individual protein trajectory to a given diffusion mode. By employing mleBayes on simulated data, we demonstrate that accurate determination of the underlying diffusive properties, beyond normal diffusion, remains challenging when analyzing particle trajectories on an individual basis. To improve upon the statistical limitations of classification from analyzing trajectories on an individual basis, we extend pEM with a new version (pEMv2) to simultaneously analyzing a collection of particle trajectories to uncover the system of interactions which give rise to unique normal or non-normal diffusive states. We test the performance of pEMv2 on various sets of simulated particle trajectories which transition between various modes of normal and non-normal diffusive states to highlight considerations when employing pEMv2 analysis. We envision the presented methodologies will be applicable to a wide range of single protein tracking data where different interactions result in distinct diffusive behaviors. More generally, this study brings us an important step closer to the possibility of monitoring the endogenous biochemistry of diffusing proteins within live cells with single molecule resolution. In the second part of this thesis, the role of chromatin association to the nuclear envelope in nuclear mechanics is explored. Changes in the mechanical properties of the nucleus are increasingly found to be critical for development and disease. However, relatively little is known about the variables that cells modulate to define nuclear mechanics. The best understood player is lamin A, a protein linked to a diverse set of genetic diseases termed laminopathies. The properties of lamin A that are compromised in these diseases (and therefore underlie their pathology) remains poorly understood. One model focuses on a mechanical role for a polymeric network of lamins associated with the nuclear envelope (NE), which supports nuclear integrity. However, because heterochromatin is strongly associated with lamina, it remains unclear whether it is the lamin polymer, the associated chromatin, or both that allow the lamina to mechanically stabilize nuclei. Decoupling the impact of the lamin polymer itself from that of the associated chromatin has proven very challenging. Here, we take advantage of the model organism, S pombe, which does not express lamies, as an experimental framework in which to address the impact of chromatin and its association with the nuclear periphery on nuclear mechanics. Using a combination of new image analysis tools for in vivo imaging of nuclear dynamics and a novel optical tweezers assay capable of directly probing nuclear mechanics, we find that the association of chromatin with the NE through integral membrane proteins plays a critical role in supporting nuclear integrity. When chromatin is decoupled from the NE, nuclei are softer, undergo much larger nuclear fluctuations in vivo in response to microtubule forces, and are defective at resolving nuclear deformations. Our data further suggest that association of chromatin with the NE attenuates the flow of chromatin into nuclear fluctuations thereby preventing permanent changes in nuclear shape.

Pressure Dependence of Coherence-Incoherence Crossover Behavior in KFe 2As 2 Observed by Resistivity and 75As-NMR/NQR.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiecki, P.; Taufour, V.; Chung, D. Y.

We present the results of 75As nuclear magnetic resonance (NMR), nuclear quadrupole resonance (NQR), and resistivity measurements in KFe 2As 2 under pressure (p). The temperature dependence of the NMR shift, nuclear spin-lattice relaxation time (T1), and resistivity show a crossover between a high-temperature incoherent, local-moment behavior and a low-temperature coherent behavior at a crossover temperature (T *). T * is found to increase monotonically with pressure, consistent with increasing hybridization between localized 3d orbitalderived bands with the itinerant electron bands. No anomaly in T * is seen at the critical pressure pc = 1.8 GPa where a change ofmore » slope of the superconducting (SC) transition temperature Tc(p) has been observed. In contrast, Tc(p) seems to correlate with antiferromagnetic spin fluctuations in the normal state as measured by the NQR 1/T1 data, although such a correlation cannot be seen in the replacement effects of A in the KFe 2As 2 (A = K, Rb, Cs) family. In the superconducting state, two T1 components are observed at low temperatures, suggesting the existence of two distinct local electronic environments. The temperature dependence of the short T1s indicates a nearly gapless state below Tc. On the other hand, the temperature dependence of the long component 1/T1L implies a large reduction in the density of states at the Fermi level due to the SC gap formation. These results suggest a real-space modulation of the local SC gap structure in KFe 2As 2 under pressure.« less

Pressure dependence of coherence-incoherence crossover behavior in KFe 2 As 2 observed by resistivity and As 75 -NMR/NQR

DOE PAGES

Wiecki, P.; Taufour, V.; Chung, D. Y.; ...

2018-02-13

We present the results of 75As nuclear magnetic resonance (NMR), nuclear quadrupole resonance (NQR), and resistivity measurements in KF e2 As 2 under pressure (p). The temperature dependence of the NMR shift, nuclear spin-lattice relaxation time (T 1), and resistivity show a crossover between a high-temperature incoherent, local-moment behavior and a low-temperature coherent behavior at a crossover temperature (T*). T* is found to increase monotonically with pressure, consistent with increasing hybridization between localized 3d orbital-derived bands with the itinerant electron bands. No anomaly in T* is seen at the critical pressure p c= 1.8 GPa where a change of slopemore » of the superconducting (SC) transition temperature T c( p ) has been observed. In contrast, T c( p ) seems to correlate with antiferromagnetic spin fluctuations in the normal state as measured by the NQR 1/T 1 data, although such a correlation cannot be seen in the replacement effects of A in the AFe 2As 2 (A=K,Rb,Cs) family. In the superconducting state, two T 1 components are observed at low temperatures, suggesting the existence of two distinct local electronic environments. The temperature dependence of the short T 1s indicates a nearly gapless state below T c. On the other hand, the temperature dependence of the long component 1/T 1Limplies a large reduction in the density of states at the Fermi level due to the SC gap formation. These results suggest a real-space modulation of the local SC gap structure in KFe 2As 2 under pressure.« less

Pressure dependence of coherence-incoherence crossover behavior in KFe 2 As 2 observed by resistivity and As 75 -NMR/NQR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiecki, P.; Taufour, V.; Chung, D. Y.

We present the results of 75As nuclear magnetic resonance (NMR), nuclear quadrupole resonance (NQR), and resistivity measurements in KF e2 As 2 under pressure (p). The temperature dependence of the NMR shift, nuclear spin-lattice relaxation time (T 1), and resistivity show a crossover between a high-temperature incoherent, local-moment behavior and a low-temperature coherent behavior at a crossover temperature (T*). T* is found to increase monotonically with pressure, consistent with increasing hybridization between localized 3d orbital-derived bands with the itinerant electron bands. No anomaly in T* is seen at the critical pressure p c= 1.8 GPa where a change of slopemore » of the superconducting (SC) transition temperature T c( p ) has been observed. In contrast, T c( p ) seems to correlate with antiferromagnetic spin fluctuations in the normal state as measured by the NQR 1/T 1 data, although such a correlation cannot be seen in the replacement effects of A in the AFe 2As 2 (A=K,Rb,Cs) family. In the superconducting state, two T 1 components are observed at low temperatures, suggesting the existence of two distinct local electronic environments. The temperature dependence of the short T 1s indicates a nearly gapless state below T c. On the other hand, the temperature dependence of the long component 1/T 1Limplies a large reduction in the density of states at the Fermi level due to the SC gap formation. These results suggest a real-space modulation of the local SC gap structure in KFe 2As 2 under pressure.« less

Locus-specific gene repositioning in prostate cancer

PubMed Central

Leshner, Marc; Devine, Michelle; Roloff, Gregory W.; True, Lawrence D.; Misteli, Tom; Meaburn, Karen J.

2016-01-01

Genes occupy preferred spatial positions within interphase cell nuclei. However, positioning patterns are not an innate feature of a locus, and genes can alter their localization in response to physiological and pathological changes. Here we screen the radial positioning patterns of 40 genes in normal, hyperplasic, and malignant human prostate tissues. We find that the overall spatial organization of the genome in prostate tissue is largely conserved among individuals. We identify three genes whose nuclear positions are robustly altered in neoplastic prostate tissues. FLI1 and MMP9 position differently in prostate cancer than in normal tissue and prostate hyperplasia, whereas MMP2 is repositioned in both prostate cancer and hyperplasia. Our data point to locus-specific reorganization of the genome during prostate disease. PMID:26564800

Myosin-1C uses a novel phosphoinositide-dependent pathway for nuclear localization.

PubMed

Nevzorov, Ilja; Sidorenko, Ekaterina; Wang, Weihuan; Zhao, Hongxia; Vartiainen, Maria K

2018-02-01

Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin-dependent motor protein Myosin-1C (Myo1C) resembles the diffusion-retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

TorsinA dysfunction causes persistent neuronal nuclear pore defects.

PubMed

Pappas, Samuel S; Liang, Chun-Chi; Kim, Sumin; Rivera, CheyAnne O; Dauer, William T

2018-02-01

A critical challenge to deciphering the pathophysiology of neurodevelopmental disease is identifying which of the myriad abnormalities that emerge during CNS maturation persist to contribute to long-term brain dysfunction. Childhood-onset dystonia caused by a loss-of-function mutation in the AAA+ protein torsinA exemplifies this challenge. Neurons lacking torsinA develop transient nuclear envelope (NE) malformations during CNS maturation, but no NE defects are described in mature torsinA null neurons. We find that during postnatal CNS maturation torsinA null neurons develop mislocalized and dysfunctional nuclear pore complexes (NPC) that lack NUP358, normally added late in NPC biogenesis. SUN1, a torsinA-related molecule implicated in interphase NPC biogenesis, also exhibits localization abnormalities. Whereas SUN1 and associated nuclear membrane abnormalities resolve in juvenile mice, NPC defects persist into adulthood. These findings support a role for torsinA function in NPC biogenesis during neuronal maturation and implicate altered NPC function in dystonia pathophysiology. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Subcellular localization and cytoplasmic complex status of endogenous Keap1.

PubMed

Watai, Yoriko; Kobayashi, Akira; Nagase, Hiroko; Mizukami, Mio; McEvoy, Justina; Singer, Jeffrey D; Itoh, Ken; Yamamoto, Masayuki

2007-10-01

Keap1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin-3-based ubiquitin ligase, and a regulator of Nrf2 activity through the interaction with Nrf2 Neh2 domain. However, the mechanism(s) of Nrf2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous Keap1 molecule. Here, we report the generation of a new monoclonal antibody for the detection of endogenous Keap1 molecules. Immunocytochemical analysis of mouse embryonic fibroblasts with the antibody revealed that under normal, unstressed condition, Keap1 is localized primarily in the cytoplasm with minimal amount in the nucleus and endoplasmic reticulum. This subcellular localization profile of Keap1 appears unchanged after treatment of cells with diethyl maleate, an electrophile, and/or Leptomycin B, a nuclear export inhibitor. Subcellular fractionation analysis of mouse liver cells showed similar results. No substantial change in the subcellular distribution profile could be observed in cells isolated from butylated hydroxyanisole-treated mice. Analyses of sucrose density gradient centrifugation of mouse liver cells indicated that Keap1 appears to form multiprotein complexes in the cytoplasm. These results demonstrate that endogenous Keap1 remains mostly in the cytoplasm, and electrophiles promote nuclear accumulation of Nrf2 without altering the subcellular localization of Keap1.

Cytoplasmic Drosha Is Aberrant in Precancerous Lesions of Gastric Carcinoma and Its Loss Predicts Worse Outcome for Gastric Cancer Patients.

PubMed

Zhang, Hailong; Hou, Yixuan; Xu, Liyun; Zeng, Zongyue; Wen, Siyang; Du, Yan-E; Sun, Kexin; Yin, Jiali; Lang, Lei; Tang, Xiaoli; Liu, Manran

2016-04-01

The nuclear localization of Drosha is critical for its function as a microRNA maturation regulator. Dephosphorylation of Drosha at serine 300 and serine 302 disrupts its nuclear localization, and aberrant distribution of Drosha has been detected in some tumors. The purpose of the present study was to assess cytoplasmic/nuclear Drosha expression in gastric cancer carcinogenesis and progression. Drosha expression and its subcellular location was investigated by immunohistochemical staining of a set of tissue microarrays composed of normal adjacent tissues (374), chronic gastritis (137), precancerous lesions (94), and gastric adenocarcinoma (829) samples, and in gastric cancer cell lines with varying differentiation by immunofluorescence and western blot assay. Gradual loss of cytoplasmic Drosha was accompanied by tumor progression in both gastric cancer tissues and cell lines, and was inversely associated with tumor volume (P = 0.002), tumor grade (P < 0.001), tumor stage (P = 0.018), and distant metastasis (P = 0.026). Aberrant high levels of cytoplasmic Drosha were apparent in intestinal metaplasia and dysplasia tissues. The levels of nuclear Drosha were sharply decreased in chronic gastritis and maintained through precancerous lesions to gastric cancer. High levels of cytoplasmic Drosha predicted longer survival (LR = 7.088, P = 0.008) in gastric cancer patients. Our data provide novel insights into gastric cancer that cytoplasmic Drosha potentially plays a role in preventing carcinogenesis and tumor progression, and may be an independent predictor of patient outcome.

Serotype-specific differences in dengue virus non-structural protein 5 nuclear localization.

PubMed

Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D

2013-08-02

The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes.

Serotype-specific Differences in Dengue Virus Non-structural Protein 5 Nuclear Localization*

PubMed Central

Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D.

2013-01-01

The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes. PMID:23770669

Nuclear localization of Schizosaccharomyces pombe Mcm2/Cdc19p requires MCM complex assembly.

PubMed

Pasion, S G; Forsburg, S L

1999-12-01

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.

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

Differentiation-induced Colocalization of the KH-type Splicing Regulatory Protein with Polypyrimidine Tract Binding Protein and the c-src Pre-mRNA

PubMed Central

Hall, Megan P.; Huang, Sui; Black, Douglas L.

2004-01-01

We have examined the subcellular localization of the KH-type splicing regulatory protein (KSRP). KSRP is a multidomain RNA-binding protein implicated in a variety of cellular processes, including splicing in the nucleus and mRNA localization in the cytoplasm. We find that KSRP is primarily nuclear with a localization pattern that most closely resembles that of polypyrimidine tract binding protein (PTB). Colocalization experiments of KSRP with PTB in a mouse neuroblastoma cell line determined that both proteins are present in the perinucleolar compartment (PNC), as well as in other nuclear enrichments. In contrast, HeLa cells do not show prominent KSRP staining in the PNC, even though PTB labeling identified the PNC in these cells. Because both PTB and KSRP interact with the c-src transcript to affect N1 exon splicing, we examined the localization of the c-src pre-mRNA by fluorescence in situ hybridization. The src transcript is present in specific foci within the nucleus that are presumably sites of src transcription but are not generally perinucleolar. In normally cultured neuroblastoma cells, these src RNA foci contain PTB, but little KSRP. However, upon induced neuronal differentiation of these cells, KSRP occurs in the same foci with src RNA. PTB localization remains unaffected. This differentiation-induced localization of KSRP with src RNA correlates with an increase in src exon N1 inclusion. These results indicate that PTB and KSRP do indeed interact with the c-src transcript in vivo, and that these associations change with the differentiated state of the cell. PMID:14657238

Nuclear import of glucokinase in pancreatic beta-cells is mediated by a nuclear localization signal and modulated by SUMOylation.

PubMed

Johansson, Bente Berg; Fjeld, Karianne; Solheim, Marie Holm; Shirakawa, Jun; Zhang, Enming; Keindl, Magdalena; Hu, Jiang; Lindqvist, Andreas; Døskeland, Anne; Mellgren, Gunnar; Flatmark, Torgeir; Njølstad, Pål Rasmus; Kulkarni, Rohit N; Wierup, Nils; Aukrust, Ingvild; Bjørkhaug, Lise

2017-10-15

The localization of glucokinase in pancreatic beta-cell nuclei is a controversial issue. Although previous reports suggest such a localization, the mechanism for its import has so far not been identified. Using immunofluorescence, subcellular fractionation and mass spectrometry, we present evidence in support of glucokinase localization in beta-cell nuclei of human and mouse pancreatic sections, as well as in human and mouse isolated islets, and murine MIN6 cells. We have identified a conserved, seven-residue nuclear localization signal ( 30 LKKVMRR 36 ) in the human enzyme. Substituting the residues KK 31,32 and RR 35,36 with AA led to a loss of its nuclear localization in transfected cells. Furthermore, our data indicates that SUMOylation of glucokinase modulates its nuclear import, while high glucose concentrations do not significantly alter the enzyme nuclear/cytosolic ratio. Thus, for the first time, we provide data in support of a nuclear import of glucokinase mediated by a redundant mechanism, involving a nuclear localization signal, and which is modulated by its SUMOylation. These findings add new knowledge to the functional role of glucokinase in the pancreatic beta-cell. Copyright © 2017 Elsevier B.V. All rights reserved.

The Hsp90 Inhibitor, 17-AAG, Prevents the Ligand-Independent Nuclear Localization of Androgen Receptor in Refractory Prostate Cancer Cells

PubMed Central

Saporita, Anthony J.; Ai, Junkui; Wang, Zhou

2010-01-01

BACKGROUND Androgen receptor (AR) is the key molecule in androgen-refractory prostate cancer. Despite androgen ablative conditions, AR remains active and is necessary for the growth of androgen-refractory prostate cancer cells. Nuclear localization of AR is a prerequisite for its transcriptional activation. We examined AR localization in androgen-dependent and androgen-refractory prostate cancer cells. METHODS AND RESULTS We demonstrate increased nuclear localization of a GFP-tagged AR in the absence of hormone in androgen-refractory C4-2 cells compared to parental androgen-sensitive human prostate cancer LNCaP cells. Analysis of AR mutants impaired in ligand-binding indicates that the nuclear localization of AR in C4-2 cells is truly androgen-independent. The hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), inhibits basal PSA expression and disrupts the ligand-independent nuclear localization of AR at doses much lower than required to inhibit androgen-induced nuclear import. CONCLUSIONS Hsp90 is a key regulator of ligand-independent nuclear localization and activation of AR in androgen-refractory prostate cancer cells. PMID:17221841

Kinesin-related KIP3 of Saccharomyces cerevisiae Is Required for a Distinct Step in Nuclear Migration

PubMed Central

DeZwaan, Todd M.; Ellingson, Eric; Pellman, David; Roof, David M.

1997-01-01

Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus. PMID:9281581

Adipogenesis stimulates the nuclear localization of EWS with an increase in its O-GlcNAc glycosylation in 3T3-L1 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Qiang; Kamemura, Kazuo, E-mail: k_kamemura@nagahama-i-bio.ac.jp

2014-07-18

Highlights: • The majority of EWS localizes stably in the cytosol in 3T3-L1 preadipocytes. • Adipogenic stimuli induce the nuclear localization of EWS. • Adipogenesis promotes O-GlcNAcylation of EWS. • O-GlcNAcylation stimulates the recruitment of EWS to the nuclear periphery. - Abstract: Although the Ewing sarcoma (EWS) proto-oncoprotein is found in the nucleus and cytosol and is associated with the cell membrane, the regulatory mechanisms of its subcellular localization are still unclear. Here we found that adipogenic stimuli induce the nuclear localization of EWS in 3T3-L1 cells. Tyrosine phosphorylation in the C-terminal PY-nuclear localization signal of EWS was negative throughoutmore » adipogenesis. Instead, an adipogenesis-dependent increase in O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation of EWS was observed. Pharmacological inactivation of O-GlcNAcase in preadipocytes promoted perinuclear localization of EWS. Our findings suggest that the nuclear localization of EWS is partly regulated by the glycosylation.« less

Local dynamic nuclear polarization using quantum point contacts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wald, K.R.; Kouwenhoven, L.P.; McEuen, P.L.

1994-08-15

We have used quantum point contacts (QPCs) to locally create and probe dynamic nuclear polarization (DNP) in GaAs heterostructures in the quantum Hall regime. DNP is created via scattering between spin-polarized Landau level electrons and the Ga and As nuclear spins, and it leads to hysteresis in the dc transport characteristics. The nuclear origin of this hysteresis is demonstrated by nuclear magnetic resonance (NMR). Our results show that QPCs can be used to create and probe local nuclear spin populations, opening up new possibilities for mesoscopic NMR experiments.

Growth hormone-specific induction of the nuclear localization of porcine growth hormone receptor in porcine hepatocytes.

PubMed

Lan, H N; Hong, P; Li, R N; Shan, A S; Zheng, X

2017-10-01

The phenomenon of nuclear translocation of growth hormone receptor (GHR) in human, rat, and fish has been reported. To date, this phenomenon has not been described in a domestic animal (such as pig). In addition, the molecular mechanisms of GHR nuclear translocation have not been thoroughly elucidated. To this end, porcine hepatocytes were isolated and used as a cell model. We observed that porcine growth hormone (pGH) can induce porcine GHR's nuclear localization in porcine hepatocytes. Subsequently, the dynamics of pGH-induced pGHR's nuclear localization were analyzed and demonstrated that pGHR's nuclear localization occurs in a time-dependent manner. Next, we explored the mechanism of pGHR nuclear localization using different pGHR ligands, and we demonstrated that pGHR's nuclear translocation is GH(s)-dependent. We also observed that pGHR translocates into cell nuclei in a pGH dimerization-dependent fashion, whereas further experiments indicated that IMPα/β is involved in the nuclear translocation of the pGH-pGHR dimer. The pGH-pGHR dimer may form a pGH-GHR-JAK2 multiple complex in cell nuclei, which would suggest that similar to its function in the cell membrane, the nuclear-localized pGH-pGHR dimer might still have the ability to signal. Copyright © 2017 Elsevier Inc. All rights reserved.

Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

PubMed

Shen, Shu; Tobery, Cynthia E; Rose, Mark D

2009-05-01

Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bateman, Nicholas W.; The John P. Murtha Cancer Center, Walter Reed National Military Medical Center, 8901 Wisconsin Avenue, Bethesda 20889, MD; Shoji, Yutaka

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. - Highlights: • We have identified a bipartitemore » nuclear localization sequence (NLS) in ARID1A. • Confirmation of the NLS was performed using GFP constructs. • NLS mutant ARID1A exhibits greater stability than wild-type ARID1A.« less

Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor

PubMed Central

Savory, Joanne G. A.; Hsu, Brian; Laquian, Ian R.; Giffin, Ward; Reich, Terry; Haché, Robert J. G.; Lefebvre, Yvonne A.

1999-01-01

Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t1/2] of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin α. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1− GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export. PMID:9891038

Proximal tibial stress fracture associated with mild osteoarthritis of the knee: case report.

PubMed

Curković, Marko; Kovac, Kristina; Curković, Bozidar; Babić-Naglić, Durda; Potocki, Kristina

2011-03-01

Stress fractures are considered as multifactorial overuse injuries occurring in 0.3%-0.8% of patients suffering from rheumatic diseases, with rheumatoid arthritis being the most common underlying condition. Stress fractures can be classified according to the condition of the bone affected as: 1) fatigue stress fractures occurring when normal bone is exposed to repeated abnormal stresses; and 2) insufficiency stress fractures that occur when normal stress is applied to bone weakened by an underlying condition. Stress fractures are rarely associated with severe forms of knee osteoarthritis, accompanied with malalignment and obesity. We present a patient with a proximal tibial stress fracture associated with mild knee osteoarthritis without associated malalignment or obesity. Stress fracture should be considered when a patient with osteoarthritis presents with sudden deterioration, severe localized tenderness to palpation and localized swelling or periosteal thickening at the pain site and elevated local temperature. The diagnosis of stress fractures in patients with rheumatic diseases may often be delayed because plain film radiographs may not reveal a stress fracture soon after the symptom onset; moreover, evidence of a fracture may never appear on plain radiographs. Triple phase nuclear bone scans and magnetic resonance imaging are more sensitive in the early clinical course than plain films for initial diagnosis.

External radiation in Dolon village due to local fallout from the first USSR atomic bomb test in 1949.

PubMed

Imanaka, Tetsuji; Fukutani, Satoshi; Yamamoto, Masayoshi; Sakaguchi, Aya; Hoshi, Masaharu

2006-02-01

Dolon village, located about 60 km from the border of the Semipalatinsk Nuclear Test Site, is known to be heavily contaminated by local fallout from the first USSR atomic bomb test in 1949. External radiation in Dolon was evaluated based on recent 137Cs data in soil and calculation of temporal change in the fission product composition. After fitting a log-normal distribution to the soil data, a 137Cs deposition of 32 kBq m-2, which corresponds to the 90th-percentile of the distribution, was tentatively chosen as a value to evaluate the radiation situation in 1949. Our calculation indicated that more than 95% of the cumulative dose for 50 y had been delivered within 1 y after the deposition. The resulting cumulative dose for 1 y after the deposition, normalized to the initial contamination containing 1 kBq m-2 of 137Cs, was 15.6 mGy, assuming a fallout arrival time of 3 h and a medium level of fractionation. Finally, 0.50 Gy of absorbed dose in air was derived as our tentative estimate for 1-year cumulative external dose in Dolon due to local fallout from the first USSR test in 1949.

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

PubMed

Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

1999-03-05

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

Nuclear Transport and Accumulation of Smad Proteins Studied by Single-Molecule Microscopy.

PubMed

Li, Yichen; Luo, Wangxi; Yang, Weidong

2018-05-08

Nuclear translocation of stimulated Smad heterocomplexes is a critical step in the signal transduction of transforming growth factor β (TGF-β) from transmembrane receptors into the nucleus. Specifically, normal nuclear accumulation of Smad2/Smad4 heterocomplexes induced by TGF-β1 is involved in carcinogenesis. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-β1 remains obscure. By combining a high-speed single-molecule tracking microscopy and Förster resonance energy transfer technique, we tracked the entire TGF-β1-induced process of Smad2/Smad4 heterocomplex formation, as well as their transport through nuclear pore complexes in live cells, with a high single-molecule localization precision of 2 ms and <20 nm. Our single-molecule Förster resonance energy transfer data have revealed that in TGF-β1-treated cells, Smad2/Smad4 heterocomplexes formed in the cytoplasm, imported through the nuclear pore complexes as entireties, and finally dissociated in the nucleus. Moreover, we found that basal-state Smad2 or Smad4 cannot accumulate in the nucleus without the presence of TGF-β1, mainly because both of them have an approximately twofold higher nuclear export efficiency compared to their nuclear import. Remarkably and reversely, heterocomplexes of Smad2/Smad4 induced by TGF-β1 can rapidly concentrate in the nucleus because of their almost fourfold higher nuclear import rate in comparison with their nuclear export rate. Thus, we believe that the determined TGF-β1-dependent transport configurations and efficiencies for the basal-state Smad or stimulated Smad heterocomplexes elucidate the basic molecular mechanism to understand their nuclear transport and accumulation. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Characterization of karyopherins in androgen receptor intracellular trafficking in the yeast model

PubMed Central

Nguyen, Minh M; Harmon, Robert M; Wang, Zhou

2014-01-01

Background: Mechanisms regulating androgen receptor (AR) subcellular localization represent an essential component of AR signaling. Karyopherins are a family of nucleocytoplasmic trafficking factors. In this paper, we used the yeast model to study the effects of karyopherins on the subcellular localization of the AR. Methods: Yeast mutants deficient in different nuclear transport factors were transformed with various AR based, GFP tagged constructs and their localization was monitored using microscopy. Results: We showed that yeast can mediate androgen-induced AR nuclear localization and that in addition to the import factor, Importinα/β, this process required the import karyopherin Sxm1. We also showed that a previously identified nuclear export sequence (NESAR) in the ligand binding domain of AR does not appear to rely on karyopherins for cytoplasmic localization. Conclusions: These results suggest that while AR nuclear import relies on karyopherin activity, AR nuclear export and/or cytoplasmic localization may require other undefined mechanisms. PMID:25031696

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. Copyright © 2015 Rogers and Rose.

Kar5p Is Required for Multiple Functions in Both Inner and Outer Nuclear Envelope Fusion in Saccharomyces cerevisiae

PubMed Central

Rogers, Jason V.; Rose, Mark D.

2014-01-01

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. PMID:25467943

Type 1 IGF Receptor Localization in Paediatric Gliomas: Significant Association with WHO Grading and Clinical Outcome.

PubMed

Clément, Florencia; Martin, Ayelen; Venara, Marcela; de Luján Calcagno, Maria; Mathó, Cecilia; Maglio, Silvana; Lombardi, Mercedes García; Bergadá, Ignacio; Pennisi, Patricia A

2018-06-01

Nuclear localization of insulin-like growth factor receptor type 1 (IGF-1R) has been described as adverse prognostic factor in some cancers. We studied the expression and localization of IGF-1R in paediatric patients with gliomas, as well as its association with World Health Organization (WHO) grading and survival. We conducted a single cohort, prospective study of paediatric patients with gliomas. Samples were taken at the time of the initial surgery; IGF-1R expression and localization were characterized by immunohistochemistry (IHC), subcellular fractionation and western blotting. Tumours (47/53) showed positive staining for IGF-1R by IHC. IGF-1R nuclear labelling was observed in 10/47 cases. IGF-1R staining was mostly non-nuclear in low-grade tumours, while IGF-1R nuclear labelling was predominant in high-grade gliomas (p = 0.0001). Survival was significantly longer in patients with gliomas having non-nuclear IGF-1R localization than in patients with nuclear IGF-1R tumours (p = 0.016). In gliomas, IGF-1R nuclear localization was significantly associated with both high-grade tumours and increased risk of death. Based on a prospective design, we provide evidence of a potential usefulness of intracellular localization of IGF-1R as prognostic factor in paediatric patients with gliomas.

[Myonuclear domain and microtubule proteome during skeletal muscle maturation].

PubMed

Couturier, Nathalie; Gache, Vincent

2017-11-01

In the normal course of muscle fiber development, myonuclei actively position and adapt a precise localization in mature fibers, shaping MyoNuclear Domains (MNDs). Myonuclei positioning in fibers appears to be essential for muscle function as defects in MNDs settings are always associated with dysfunction (i.e., centronuclear myopathy, sarcopenia). Previous studies have shown that myonuclei positioning in fibers is reversible, suggesting that in pathologies presenting MNDs impairment, myonuclei could be re-addressed to the "correct" position in fibers and this could benefit to muscle function. Cytoskeleton networks, and particularly microtubules, have been implicated in early nuclei localization in myotubes. As the microtubule network is completely redesigned during muscle maturation, we hypothesized that "microtubules associated proteomes" would change between immature and mature fibers and contribute to a microtubule-dependent process resulting in MNDs setting and maintenance in mature fibers. We performed an in vitro biochemical approach to isolate microtubules partners in immature (myotubes) and mature myofibers. Using mass-spectrometry identification, we selected 244 candidates, differentially associated/expressed with microtubules during myofiber maturation and potentially controlling MNDs settings. We are currently conducting a siRNA screen approach on these candidates to decipher their respective implication in early and late phases of MNDs establishment, using an unbiased assay developed by our team allowing statistical analysis of MNDs regarding myonuclei content. This approach will lead to the identification of new pathways related to nuclear positioning and MNDs setting in normal condition and in myopathies associated to MNDs impairment such as CNMs. © 2017 médecine/sciences – Inserm.

Measurement of polarization-transfer to bound protons in carbon and its virtuality dependence

NASA Astrophysics Data System (ADS)

Izraeli, D.; Brecelj, T.; Achenbach, P.; Ashkenazi, A.; Böhm, R.; Cohen, E. O.; Distler, M. O.; Esser, A.; Gilman, R.; Kolar, T.; Korover, I.; Lichtenstadt, J.; Mardor, I.; Merkel, H.; Mihovilovič, M.; Müller, U.; Olivenboim, M.; Piasetzky, E.; Ron, G.; Schlimme, B. S.; Schoth, M.; Sfienti, C.; Širca, S.; Štajner, S.; Strauch, S.; Thiel, M.; Weber, A.; Yaron, I.; A1 Collaboration

2018-06-01

We measured the ratio Px /Pz of the transverse to longitudinal components of polarization transferred from electrons to bound protons in 12C by the 12C (e → ,e‧ p →) process at the Mainz Microtron (MAMI). We observed consistent deviations from unity of this ratio normalized to the free-proton ratio, (Px /Pz) 12C /(Px /Pz) 1H, for both s- and p-shell knocked out protons, even though they are embedded in averaged local densities that differ by about a factor of two. The dependence of the double ratio on proton virtuality is similar to the one for knocked out protons from 2H and 4He, suggesting a universal behavior. It further implies no dependence on average local nuclear density.

Increased Regional Epicardial Fat Volume Associated with Reversible Myocardial Ischemia in Patients with Suspected Coronary Artery Disease

PubMed Central

Khawaja, Tuba; Greer, Christine; Thadani, Samir R.; Kato, Tomoko S.; Bhatia, Ketan; Shimbo, Daichi; Konkak, Andrew; Bokhari, Sabahat; Einstein, Andrew J.; Schulze, P. Christian

2015-01-01

Epicardial adipose tissue is a source of pro-inflammatory cytokines and has been linked to the development of coronary artery disease. No study has systematically assessed the relationship between local epicardial fat volume (EFV) and myocardial perfusion defects. We analyzed EFV in patients undergoing SPECT myocardial perfusion imaging combined with computed tomography (CT) for attenuation correction. Low-dose CT without contrast was performed in 396 consecutive patients undergoing SPECT imaging for evaluation of coronary artery disease. Regional thickness, cross-sectional areas, and total EFV were assessed. 295 patients had normal myocardial perfusion scans and 101 had abnormal perfusion scans. Mean EFVs in normal, ischemic, and infarcted hearts were 99.8 ± 82.3 cm3, 156.4 ± 121.9 cm3, and 96.3 ± 102.1 cm3, respectively (P < 0.001). Reversible perfusion defects were associated with increased local EFV compared to normal perfusion in the distribution of the right (69.2 ± 51.5 vs 46.6 ± 32.0 cm3; P = 0.03) and left anterior descending coronary artery (87.1 ± 76.4 vs 46.7 ± 40.6 cm3; P = 0.005). Our results demonstrate increased regional epicardial fat in patients with active myocardial ischemia compared to patients with myocardial scar or normal perfusion on nuclear perfusion scans. Our results suggest a potential role for cardiac CT to improve risk stratification in patients with suspected coronary artery disease. PMID:25339129

Angiotensin II Receptor Antagonism Reduces Transforming Growth Factor Beta and Smad Signaling in Thoracic Aortic Aneurysm

PubMed Central

Nataatmadja, Maria; West, Jennifer; Prabowo, Sulistiana; West, Malcolm

2013-01-01

ABSTRACT Background The expression of transforming growth factor beta (TGF-β) and Smad3 regulates extracellular matrix homeostasis and inflammation in aortic aneurysms. The expression of Smad3 depends on signaling by angiotensin II (AngII) receptor pathways through TGF-β receptor–dependent and –independent pathways. Methods To determine the expression of AngII type 1 (AT1R) and type 2 receptors (AT2R), TGF-β, and Smad3 in thoracic aortic aneurysms, we performed immunohistochemistry testing on tissue and cultured cells derived from subjects with Marfan syndrome (MFS) and bicuspid aortic valve (BAV) malformation and from normal aortas of subjects who were organ donors. Results MFS and BAV aneurysm tissue showed enhanced accumulation of TGF-β and Smad3 in vascular smooth muscle cells (VSMCs) and in inflammatory cells in the subintimal layer and tunica media. The normal aortic wall exhibited minimal TGF-β and Smad3 staining. Cultured VSMCs from MFS and BAV samples showed nuclear Smad3 and strong cytoplasmic TGF-β expression in the cytoplasmic vesicles. In control cells, Smad3 was located mainly in the cytoplasm, and weak cytoplasmic TGF-β was distributed with a pattern similar to that of the aneurysm-derived cells. Compared to normal aorta cells, AT1R and AT2R expression was increased in both aneurysm types. Treatment of cultured VSMCs with the AT1R antagonist losartan caused both reduced TGF-β vesicle localization and nuclear expression of Smad3. Conclusions Increased TGF-β and Smad3 expression in aneurysm tissue and cultured VSMCs is consistent with aberrant TGF-β expression and the activation of Smad3 signaling. Losartan-mediated reduction in TGF-β expression and the cytoplasmic localization of Smad3 support a role for AT1R antagonism in the inhibition of aneurysm progression. PMID:23532685

The antidepressant fluoxetine normalizes the nuclear glucocorticoid receptor evoked by psychosocial stress

NASA Astrophysics Data System (ADS)

Mitić, M.; Simić, I.; Djordjević, J.; Radojčić, M. B.; Adžić, M.

2011-12-01

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the pathophysiology of depression and stress disorders. Glucocorticoids, key regulators of the stress response, exert diverse effects on cellular processes in the hippocampus. Beside non-genomic pathways, glucocorticoid effects are mediated through activation of the glucocorticoid receptor (GR), a ligand activated transcriptional factor that belongs to the nuclear hormone receptor superfamily. We analysed the GR protein levels both in the cytoplasmic and nuclear compartments of the hippocampus of Wistar rats exposed to chronic psychosocial isolation stress upon chronic fluoxetine (FLU) treatment. Under chronic stress, corticosterone levels (CORT) were decreased compared to the control, and treatment with FLU did not change its level in the stressed rats. At the molecular level, FLU normalized the level of nuclear GR protein in the hippocampus of the stressed rats. Discrepancy between normalization of nuclear GR in the hippocampus and lack of normalization of HPA axis activity judged by CORT, suggests that other brain structures such as the amygdale and prefrontal cortex that also regulate HPA axis activity, seem not to be normalized by the FLU treatment used in our study.

Semiquantitative immunohistochemical marker staining and localization in canine thyroid carcinoma and normal thyroid gland.

PubMed

Pessina, P; Castillo, V; Sartore, I; Borrego, J; Meikle, A

2016-09-01

Immunoreactive proteins in follicular cells, fibroblasts and endothelial cells were assessed in canine thyroid carcinomas and healthy thyroid glands. No differences were detected in thyrotropin receptor and thyroglobulin staining between cancer and normal tissues, but expression was higher in follicular cells than in fibroblasts. Fibroblast growth factor-2 staining was more intense in healthy follicular cells than in those of carcinomas. Follicular cells in carcinomas presented two- to three-fold greater staining intensity of thyroid transcription factor-1 and proliferating cell nuclear antigen, respectively, than healthy cells, and a similar trend was found for the latter antigen in fibroblasts. Vascular endothelial growth factor staining was more intense in the endothelial cells of tumours than in those of normal tissues. In conclusion, greater expression of factors related to proliferation and angiogenesis was demonstrated in several cell types within thyroid carcinomas compared to healthy tissues, which may represent mechanisms of tumour progression in this disease. © 2014 John Wiley & Sons Ltd.

CLOCK phosphorylation by AKT regulates its nuclear accumulation and circadian gene expression in peripheral tissues.

PubMed

Luciano, Amelia K; Zhou, Wenping; Santana, Jeans M; Kyriakides, Cleo; Velazquez, Heino; Sessa, William C

2018-06-08

C ircadian l ocomotor o utput c ycles k aput (CLOCK) is a transcription factor that activates transcription of clock-controlled genes by heterodimerizing with BMAL1 and binding to E-box elements on DNA. Although several phosphorylation sites on CLOCK have already been identified, this study characterizes a novel phosphorylation site at serine 845 (Ser-836 in humans). Here, we show that CLOCK is a novel AKT substrate in vitro and in cells, and this phosphorylation site is a negative regulator of CLOCK nuclear localization by acting as a binding site for 14-3-3 proteins. To examine the role of CLOCK phosphorylation in vivo , Clock S845A knockin mice were generated using CRISPR/Cas9 technology. Clock S845A mice are essentially normal with normal central circadian rhythms and hemodynamics. However, examination of core circadian gene expression from peripheral tissues demonstrated that Clock S845A mice have diminished expression of Per2, Reverba, Dbp, and Npas2 in skeletal muscle and Per2, Reverba, Dbp, Per1 , Rora, and Npas2 in the liver during the circadian cycle. The reduction in Dbp levels is associated with reduced H3K9ac at E-boxes where CLOCK binds despite no change in total CLOCK levels. Thus, CLOCK phosphorylation by AKT on Ser-845 regulates its nuclear translocation and the expression levels of certain core circadian genes in insulin-sensitive tissues.

The NIMA Kinase Is Required To Execute Stage-Specific Mitotic Functions after Initiation of Mitosis

PubMed Central

Govindaraghavan, Meera; Lad, Alisha A.

2014-01-01

The G2-M transition in Aspergillus nidulans requires the NIMA kinase, the founding member of the Nek kinase family. Inactivation of NIMA results in a late G2 arrest, while overexpression of NIMA is sufficient to promote mitotic events independently of cell cycle phase. Endogenously tagged NIMA-GFP has dynamic mitotic localizations appearing first at the spindle pole body and then at nuclear pore complexes before transitioning to within nuclei and the mitotic spindle and back at the spindle pole bodies at mitotic exit, suggesting that it functions sequentially at these locations. Since NIMA is indispensable for mitotic entry, it has been difficult to determine the requirement of NIMA for subaspects of mitosis. We show here that when NIMA is partially inactivated, although mitosis can be initiated, a proportion of cells fail to successfully generate two daughter nuclei. We further define the mitotic defects to show that normal NIMA function is required for the formation of a bipolar spindle, nuclear pore complex disassembly, completion of chromatin segregation, and the normal structural rearrangements of the nuclear envelope required to generate two nuclei from one. In the remaining population of cells that enter mitosis with inadequate NIMA, two daughter nuclei are generated in a manner dependent on the spindle assembly checkpoint, indicating highly penetrant defects in mitotic progression without sufficient NIMA activity. This study shows that NIMA is required not only for mitotic entry but also sequentially for successful completion of stage-specific mitotic events. PMID:24186954

Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma

PubMed Central

Marote, Georgina; Abramo, Francesca; McKay, Jenny; Thomson, Calum; Beltran, Mariana; Millar, Michael; Priestnall, Simon; Dobson, Jane; Costantino-Casas, Fernando; Petrou, Terry; McGonnell, Imelda M.; Davies, Anthony J.; Weetman, Malcolm; Garden, Oliver A.; Masters, John R.; Thrasivoulou, Christopher; Ahmed, Aamir

2016-01-01

Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease. PMID:27559731

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muha, Villo; Zagyva, Imre; Venkei, Zsolt

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 wasmore » 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.« less

Molecular cloning, expression analysis and transcript localization of testicular orphan nuclear receptor 2 in the male catfish, Clarias batrachus.

PubMed

Murugananthkumar, R; Akhila, M V; Rajakumar, A; Mamta, S K; Sudhakumari, C C; Senthilkumaran, B

2016-12-01

Testicular receptor 2 (TR2; also known as Nr2c1) is one of the first orphan nuclear receptors identified and known to regulate various physiological process with or without any ligand. In this study, we report the cloning of full length nr2c1 and its expression analysis during gonadal development, seasonal testicular cycle and after human chorionic gonadotropin (hCG) induction. In addition, in situ hybridization (ISH) was performed to localize nr2c1 transcripts in adult testis and whole catfish (1day post hatch). Tissue distribution and gonadal ontogeny studies revealed high expression of nr2c1 in developing and adult testis. Early embryonic stage-wise expression of nr2c1 seems to emphasize its importance in cellular differentiation and development. Substantial expression of nr2c1 during pre-spawning phase and localization of nr2c1 transcripts in sperm/spermatids were observed. Significant upregulation after hCG induction indicate that nr2c1 is under the regulation of gonadotropins. Whole mount ISH analysis displayed nr2c1 expression in notochord indicating its role in normal vertebrate development. Taken together, our findings suggest that nr2c1 may have a plausible role in the testicular and embryonic development of catfish. Copyright © 2015. Published by Elsevier Inc.

A gene delivery system containing nuclear localization signal: Increased nucleus import and transfection efficiency with the assistance of RanGAP1.

PubMed

Chen, Kang; Guo, Lingling; Zhang, Jiulong; Chen, Qing; Wang, Kuanglei; Li, Chenxi; Li, Weinan; Qiao, Mingxi; Zhao, Xiuli; Hu, Haiyang; Chen, Dawei

2017-01-15

In the present report, a degradable gene delivery system (PAMS/DNA/10NLS) containing nucleus location signal peptide (NLS) was prepared. The agarose gel electrophoresis, particle size and zeta potential of PAMS/DNA/10NLS were similar to those of PAMS/DNA, which proved that NLS did not affect the interaction between PAMS and DNA. PAMS/DNA/10NLS exhibited marked extracellular and intracellular degradation under acidic conditions. The degradation was believed to allow NLS to come into contact with importins easily, which was able to mediate the nucleus import. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection capability than PAMS/DNA. Moreover, the transfection of PAMS/DNA/10NLS was less dependent on the breakdown of the nucleus envelope than PAMS/DNA. Considering that GTPase-activating protein 1 (RanGAP1) was able to activate the endogenous GTPase, which was necessary for NLS-mediated nucleus import, RanGAP1 overexpressed cells (RanGAP1 cells) were produced. This result showed that RanGAP1 cells had higher GTPase activities than normal cells. Both the nucleus import and transfection efficiency of PAMS/DNA/10NLS were markedly higher in RanGAP1 cells than that in normal cells. The in vivo transfection results also showed that the transfection efficiency of PAMS/DNA/10NLS was higher in RanGAP1 pre-treated mice than that in normal mice. These findings showed that PAMS/DNA/10NLS is a promising gene delivery system with the assistance of RanGAP1. The present report describes the increased transfection efficiency of a degradable gene delivery system (PAMS/DNA/10NLS) containing nuclear location signal (NLS) with the assistance of GTPase-activating protein 1 (RanGAP1). The physicochemical properties of PAMS/DNA/10NLS were similar to those of PAMS/DNA. PAMS/DNA/10NLS exhibited great extracellular and intracellular degradations, which might allow NLS to contact with importins easily. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection capability than PAMS/DNA. The transfection of PAMS/DNA/10NLS had less dependence on the breakdown of nuclear envelope. Both the nuclear import and transfection efficiency of PAMS/DNA/10NLS were higher in RanGAP1 overexpressed cells than that in normal cells. Moreover, the transfection efficiency of PAMS/DNA/10NLS was higher in RanGAP1 pre-treated mice than that in normal mice. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Gamma thermometer based reactor core liquid level detector

DOEpatents

Burns, Thomas J.

1983-01-01

A system is provided which employs a modified gamma thermometer for determining the liquid coolant level within a nuclear reactor core. The gamma thermometer which normally is employed to monitor local core heat generation rate (reactor power), is modified by thermocouple junctions and leads to obtain an unambiguous indication of the presence or absence of coolant liquid at the gamma thermometer location. A signal processor generates a signal based on the thermometer surface heat transfer coefficient by comparing the signals from the thermocouples at the thermometer location. The generated signal is a direct indication of loss of coolant due to the change in surface heat transfer when coolant liquid drops below the thermometer location. The loss of coolant indication is independent of reactor power at the thermometer location. Further, the same thermometer may still be used for the normal power monitoring function.

Correlation of N-myc downstream-regulated gene 1 subcellular localization and lymph node metastases of colorectal neoplasms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Yan; Lv, Liyang; Du, Juan

2013-09-20

Highlights: •We clarified NDRG1 subcellular location in colorectal cancer. •We found the changes of NDRG1 distribution during colorectal cancer progression. •We clarified the correlation between NDRG1 distribution and lymph node metastasis. •It is possible that NDRG1 subcellular localization may determine its function. •Maybe NDRG1 is valuable early diagnostic markers for metastasis. -- Abstract: In colorectal neoplasms, N-myc downstream-regulated gene 1 (NDRG1) is a primarily cytoplasmic protein, but it is also expressed on the cell membrane and in the nucleus. NDRG1 is involved in various stages of tumor development in colorectal cancer, and it is possible that the different subcellular localizationsmore » may determine the function of NDRG1 protein. Here, we attempt to clarify the characteristics of NDRG1 protein subcellular localization during the progression of colorectal cancer. We examined NDRG1 expression in 49 colorectal cancer patients in cancerous, non-cancerous, and corresponding lymph node tissues. Cytoplasmic and membrane NDRG1 expression was higher in the lymph nodes with metastases than in those without metastases (P < 0.01). Nuclear NDRG1 expression in colorectal neoplasms was significantly higher than in the normal colorectal mucosa, and yet the normal colorectal mucosa showed no nuclear expression. Furthermore, our results showed higher cytoplasmic NDRG1 expression was better for differentiation, and higher membrane NDRG1 expression resulted in a greater possibility of lymph node metastasis. These data indicate that a certain relationship between the cytoplasmic and membrane expression of NDRG1 in lymph nodes exists with lymph node metastasis. NDRG1 expression may translocate from the membrane of the colorectal cancer cells to the nucleus, where it is involved in lymph node metastasis. Combination analysis of NDRG1 subcellular expression and clinical variables will help predict the incidence of lymph node metastasis.« less

Lack of anti-tumor activity with the β-catenin expression inhibitor EZN-3892 in the C57BL/6J Min/+ model of intestinal carcinogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasson, Rian M.; Briggs, Alexandra; Rizvi, Hira

2014-02-14

Highlights: • Wnt/β-catenin signaling is aberrantly activated in most colorectal cancers. • Locked nucleic acid (LNA)-based antisense is a novel tool for cancer therapy. • β-Catenin inhibition was observed in mature intestinal tissue of LNA-treated mice. • Further investigation of Wnt/β-catenin targeted therapies is warranted. - Abstract: Background: Previously, we showed that short-term inhibition of β-catenin expression and reversal of aberrant β-catenin subcellular localization by the selective COX-2 inhibitor celecoxib is associated with adenoma regression in the C57BL/6J Min/+ mouse. Conversly, long-term administration resulted in tumor resistance, leading us to investigate alternative methods for selective β-catenin chemoprevention. In this study,more » we hypothesized that disruption of β-catenin expression by EZN-3892, a selective locked nucleic acid (LNA)-based β-catenin inhibitor, would counteract the tumorigenic effect of Apc loss in Min/+ adenomas while preserving normal intestinal function. Materials and methods: C57BL/6J Apc{sup +/+} wild-type (WT) and Min/+ mice were treated with the maximum tolerated dose (MTD) of EZN-3892 (30 mg/kg). Drug effect on tumor numbers, β-catenin protein expression, and nuclear β-catenin localization were determined. Results: Although the tumor phenotype and β-catenin nuclear localization in Min/+ mice did not change following drug administration, we observed a decrease in β-catenin expression levels in the mature intestinal tissue of treated Min/+ and WT mice, providing proof of principle regarding successful delivery of the LNA-based antisense vehicle. Higher doses of EZN-3892 resulted in fatal outcomes in Min/+ mice, likely due to β-catenin ablation in the intestinal tissue and loss of function. Conclusions: Our data support the critical role of Wnt/β-catenin signaling in maintaining intestinal homeostasis and highlight the challenges of effective drug delivery to target disease without permanent toxicity to normal cellular function.« less

Maintenance of a functional higher order chromatin structure: The role of the nuclear matrix in normal and disease states

PubMed Central

Linnemann, Amelia K.; Krawetz, Stephen A.

2010-01-01

Summary The ordered packaging of DNA within the nucleus of somatic cells reflects a dynamic supportive structure that facilitates stable transcription interrupted by intermittent cycles of extreme condensation. This dynamic mode of packing and unpacking chromatin is intimately linked to the ability of the genome to specifically complex with both histones and non-histone proteins. Understanding the underlying mechanism that governs the formation of higher order chromatin structures is a key to understanding how local architecture modulates transcription. In part, the formation of these structures appears to be regulated through genomic looping that is dynamically mediated by attachment to the nuclear scaffold/matrix at S/MARs, i.e., Scaffold/Matrix Attachment Regions. Although the mechanism guiding the formation and use of these higher-ordered structures remains unknown, S/MARs continue to reveal a multitude of roles in development and the pathogenesis of disease. PMID:20948980

Maintenance of a functional higher order chromatin structure: The role of the nuclear matrix in normal and disease states.

PubMed

Linnemann, Amelia K; Krawetz, Stephen A

2009-01-01

The ordered packaging of DNA within the nucleus of somatic cells reflects a dynamic supportive structure that facilitates stable transcription interrupted by intermittent cycles of extreme condensation. This dynamic mode of packing and unpacking chromatin is intimately linked to the ability of the genome to specifically complex with both histones and non-histone proteins. Understanding the underlying mechanism that governs the formation of higher order chromatin structures is a key to understanding how local architecture modulates transcription. In part, the formation of these structures appears to be regulated through genomic looping that is dynamically mediated by attachment to the nuclear scaffold/matrix at S/MARs, i.e., Scaffold/Matrix Attachment Regions. Although the mechanism guiding the formation and use of these higher-ordered structures remains unknown, S/MARs continue to reveal a multitude of roles in development and the pathogenesis of disease.

Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

PubMed

Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina

2016-01-01

The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide.

Properties of Localized Protons in Neutron Star Matter at Finite Temperatures

NASA Astrophysics Data System (ADS)

Szmaglinski, A.; Kubis, S.; Wójcik, W.

2014-02-01

We study properties of the proton component of neutron star matter for realistic nuclear models. Vanishing of the nuclear symmetry energy implies proton-neutron separation in dense nuclear matter. Protons which form admixture tend to be localized in potential wells. Here, we extend the description of proton localization to finite temperatures. It appears that the protons are still localized at temperatures typical for hot neutron stars. That fact has important astrophysical consequences. Moreover, the temperature inclusion leads to unexpected results for the behavior of the proton localized state.

Localization of SERBP1 in stress granules and nucleoli.

PubMed

Lee, Yu-Jen; Wei, Hung-Ming; Chen, Ling-Yun; Li, Chuan

2014-01-01

SERPINE1 mRNA-binding protein 1 (SERBP1) is an arginine-methylated RNA-binding protein whose modification affects protein interaction and intracellular localization. In the present study, we show that, under normal growth conditions without stress, SERBP1 interacts with arginine-methylated and stress granule-associated proteins such as heterogeneous nuclear ribonucleoprotein A1, fragile X mental retardation protein and fragile X mental retardation syndrome-related protein 1 in an RNA-dependent manner. We also show that, after arsenite treatment, a proportion of full-length SERBP1 protein co-localizes with the typical stress granule marker T-cell intracellular antigen-1 in the cytoplasmic stress granules. Truncated SERBP1 with an N-terminal, central RG or C-terminal deletion, or single-domain segments comprising the N-terminal, central or C-terminal region, were recruited to stress granules upon arsenite treatment but with reduced efficiency. In addition, upon arsenite treatment, the localization of SERBP1 changed from a diffuse cytoplasmic localization to nuclear-dominant (concentrated in the nucleolus) A similar distribution was observed when cells were treated with the methylation inhibitor adenosine periodate, and was also detected for N- or C-terminal domain deletions and all three single-domain fragments even without stress induction. We further demonstrate that adenosine periodate treatment delays the association/dissociation of SERBP1 with stress granules. Hypomethylation retains SERBP1 in the nucleus/nucleolus regardless of arsenite treatment. Our study indicates that arginine methylation is correlated with recruitment of SERBP to stress granules and nucleoli and its retention therein. To our knowledge, this is the first report of an RNA-binding protein that is shifted simultaneously to cytoplasmic stress granules and nucleoli, two ribonucleoprotein-enriched subcellular compartments, upon stress. © 2013 FEBS.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanchez-Aparicio, Maria Teresa; Rosas, Maria Flora; Sobrino, Francisco, E-mail: fsobrino@cbm.uam.es

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 bemore » 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.« less

Cytoplasmically Retargeted HSV1-tk/GFP Reporter Gene Mutants for Optimization of Noninvasive Molecular-Genetic Imaging

PubMed Central

Ponomarev, Vladimir; Doubrovin, Michael; Serganova, Inna; Beresten, Tatiana; Vider, Jelena; Shavrin, Aleksander; Ageyeva, Ludmila; Balatoni, Julius; Blasberg, Ronald; Tjuvajev, Juri Gelovani

2003-01-01

Abstract To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSV1-tk/GFP). Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS), the addition of a nuclear export signal (NES), a combination of both mutation types, and a truncation of the first 135 bp of the native hsv1-tk coding sequence containing a “cryptic” testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improvedmetabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU). The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [131I]FIAU and a γ-camera. PMID:12869307

Dimerization of the bacterial effector protein AvrBs3 in the plant cell cytoplasm prior to nuclear import.

PubMed

Gürlebeck, Doreen; Szurek, Boris; Bonas, Ulla

2005-04-01

The effector protein AvrBs3 from the bacterial phytopathogen Xanthomonas campestris pv. vesicatoria is translocated into the plant cell where it specifically induces hypertrophy symptoms or the hypersensitive reaction. Activity of AvrBs3 depends on nuclear localization signals (NLSs) and an acidic activation domain, suggesting a role in regulation of plant transcription. Here, we show that AvrBs3 dimerizes in the plant cell prior to its nuclear import. AvrBs3 deletion derivatives were tested in the yeast two-hybrid system revealing that the repeat region, which confers specific recognition in resistant plants and is crucial for virulence function, is also essential for the self-interaction. GST pull-down assays showed that the AvrBs3-AvrBs3 interaction occurs independent of plant proteins. Coexpression of two different inactive mutant AvrBs3 derivatives in Bs3-resistant pepper plants resulted in 'trans-complementation', i.e., the induction of a hypersensitive reaction. This clearly indicates that AvrBs3-dimerization occurs in planta. Interestingly, 'trans-complementation' was not observed in susceptible plants suggesting that wild-type homodimers are needed for the AvrBs3 virulence function in plants. Furthermore, a green fluorescent protein (GFP) fusion of AvrBs3 deleted in the NLSs (AvrBs3DeltaNLS-GFP), normally localized in the cytoplasm, was imported into the nucleus upon coexpression with wild-type AvrBs3 in Nicotiana benthamiana. Thus, AvrBs3 dimerization takes place in the cytoplasm of the plant cell prior to nuclear import. Given the fact that dimerization is a common feature of transcriptional regulators, our data are consistent with the idea that AvrBs3 manipulates expression of plant genes involved in the establishment of compatible and incompatible interactions.

Nuclear localization signal regulates porcine circovirus type 2 capsid protein nuclear export through phosphorylation.

PubMed

Hou, Qiang; Hou, Shaohua; Chen, Qing; Jia, Hong; Xin, Ting; Jiang, Yitong; Guo, Xiaoyu; Zhu, Hongfei

2018-02-15

The open reading frame 2 (ORF2) of Porcine circovirus type 2 (PCV2) encodes the major Capsid (Cap) protein, which self-assembles into virus-like particle (VLP) of similar morphology to the PCV2 virion and accumulates in the nucleus through the N-terminal arginine-rich nuclear localization signal (NLS). In this study, PCV2 Cap protein and its derivates were expressed via the baculovirus expression system, and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry. Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import, and a phosphorylation site (S17) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the NLS domain to regulate Cap protein nuclear export. Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy. Moreover, the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells. Phosphorylation of NLS regulating Cap protein nuclear export provides more detailed knowledge of the PCV2 viral life cycle. Copyright © 2018 Elsevier B.V. All rights reserved.

Protein Sub-Nuclear Localization Prediction Using SVM and Pfam Domain Information

PubMed Central

Kumar, Ravindra; Jain, Sohni; Kumari, Bandana; Kumar, Manish

2014-01-01

The nucleus is the largest and the highly organized organelle of eukaryotic cells. Within nucleus exist a number of pseudo-compartments, which are not separated by any membrane, yet each of them contains only a specific set of proteins. Understanding protein sub-nuclear localization can hence be an important step towards understanding biological functions of the nucleus. Here we have described a method, SubNucPred developed by us for predicting the sub-nuclear localization of proteins. This method predicts protein localization for 10 different sub-nuclear locations sequentially by combining presence or absence of unique Pfam domain and amino acid composition based SVM model. The prediction accuracy during leave-one-out cross-validation for centromeric proteins was 85.05%, for chromosomal proteins 76.85%, for nuclear speckle proteins 81.27%, for nucleolar proteins 81.79%, for nuclear envelope proteins 79.37%, for nuclear matrix proteins 77.78%, for nucleoplasm proteins 76.98%, for nuclear pore complex proteins 88.89%, for PML body proteins 75.40% and for telomeric proteins it was 83.33%. Comparison with other reported methods showed that SubNucPred performs better than existing methods. A web-server for predicting protein sub-nuclear localization named SubNucPred has been established at http://14.139.227.92/mkumar/subnucpred/. Standalone version of SubNucPred can also be downloaded from the web-server. PMID:24897370

Decreased expression of peroxisome proliferator activated receptor gamma in cftr-/- mice.

PubMed

Ollero, Mario; Junaidi, Omer; Zaman, Munir M; Tzameli, Iphigenia; Ferrando, Adolfo A; Andersson, Charlotte; Blanco, Paola G; Bialecki, Eldad; Freedman, Steven D

2004-08-01

Some of the pathological manifestations of cystic fibrosis are in accordance with an impaired expression and/or activity of PPARgamma. We hypothesized that PPARgamma expression is altered in tissues lacking the normal cystic fibrosis transmembrane regulator protein (CFTR). PPARgamma mRNA levels were measured in colonic mucosa, ileal mucosa, adipose tissue, lung, and liver from wild-type and cftr-/- mice by quantitative RT-PCR. PPARgamma expression was decreased twofold in CFTR-regulated tissues (colon, ileum, and lung) from cftr-/- mice compared to wild-type littermates. In contrast, no differences were found in fat and liver. Immunohistochemical analysis of PPARgamma in ileum and colon revealed a predominantly nuclear localization in wild-type mucosal epithelial cells while tissues from cftr-/- mice showed a more diffuse, lower intensity labeling. A significant decrease in PPARgamma expression was confirmed in nuclear extracts of colon mucosa by Western blot analysis. In addition, binding of the PPARgamma/RXR heterodimer to an oligonucletotide containing a peroxisome proliferator responsive element (PPRE) was also decreased in colonic mucosa extracts from cftr-/- mice. Treatment of cftr-/- mice with the PPARgamma ligand rosiglitazone restored both the nuclear localization and binding to DNA, but did not increase RNA levels. We conclude that PPARgamma expression in cftr-/- mice is downregulated at the RNA and protein levels and its function diminished. These changes may be related to the loss of function of CFTR and may be relevant to the pathogenesis of metabolic abnormalities associated with cystic fibrosis in humans. Copyright 2004 Wiley-Liss, Inc.

Classic Nuclear Localization Signals and a Novel Nuclear Localization Motif Are Required for Nuclear Transport of Porcine Parvovirus Capsid Proteins

PubMed Central

Boisvert, Maude; Bouchard-Lévesque, Véronique; Fernandes, Sandra

2014-01-01

ABSTRACT Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. IMPORTANCE Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins. PMID:25078698

Heat shock modulates the subcellular localization, stability, and activity of HIPK2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Upadhyay, Mamta; Bhadauriya, Pratibha; Ganesh, Subramaniam, E-mail: sganesh@iitk.ac.in

2016-04-15

The homeodomain-interacting protein kinase-2 (HIPK2) is a highly conserved serine/threonine kinase and is involved in transcriptional regulation. HIPK2 is a highly unstable protein, and is kept at a low level under normal physiological conditions. However, exposure of cells to physiological stress – such as hypoxia, oxidative stress, or UV damage – is known to stabilize HIPK2, leading to the HIPK2-dependent activation of p53 and the cell death pathway. Therefore HIPK2 is also known as a stress kinase and as a stress-activated pro-apoptotic factor. We demonstrate here that exposure of cells to heat shock results in the stabilization of HIPK2 andmore » the stabilization is mediated via K63-linked ubiquitination. Intriguingly, a sub-lethal heat shock (42 °C, 1 h) results in the cytoplasmic localization of HIPK2, while a lethal heat shock (45 °C, 1 h) results in its nuclear localization. Cells exposed to the lethal heat shock showed significantly higher levels of the p53 activity than those exposed to the sub-lethal thermal stress, suggesting that both the level and the nuclear localization are essential for the pro-apoptotic activity of HIPK2 and that the lethal heat shock could retain the HIPK2 in the nucleus to promote the cell death. Taken together our study underscores the importance of HIPK2 in stress mediated cell death, and that the HIPK2 is a generic stress kinase that gets activated by diverse set of physiological stressors.« less

Enhanced expression of peroxisome proliferator-activated receptor gamma in epithelial ovarian carcinoma.

PubMed

Zhang, G Y; Ahmed, N; Riley, C; Oliva, K; Barker, G; Quinn, M A; Rice, G E

2005-01-17

The peroxisome proliferator-activated receptors (PPARs) belong to a subclass of nuclear hormone receptor that executes important cellular transcriptional functions. Previous studies have demonstrated the expression of PPARgamma in several tumours including colon, breast, bladder, prostate, lung and stomach. This study demonstrates the relative expression of PPARgamma in normal ovaries and different pathological grades of ovarian tumours of serous, mucinous, endometrioid, clear cell and mixed subtypes. A total of 56 ovarian specimens including 10 normal, eight benign, 10 borderline, seven grade 1, nine grade 2 and 12 grade 3 were analysed using immunohistochemistry. Immunoreactive PPARgamma was not expressed in normal ovaries. Out of eight benign and 10 borderline tumours, only one tumour in each group showed weak cytoplasmic PPARgamma expression. In contrast, 26 out of 28 carcinomas studied were positive for PPARgamma expression with staining confined to cytoplasmic and nuclear regions. An altered staining pattern of PPARgamma was observed in high-grade ovarian tumours with PPARgamma being mostly localized in the nuclei with little cytoplasmic immunoreactivity. On the other hand, predominant cytoplasmic staining was observed in lower-grade tumours. Significantly increased PPARgamma immunoreactivity was observed in malignant ovarian tumours (grade 1, 2 and 3) compared to benign and borderline tumours (chi2 = 48.80, P < 0.001). Western blot analyses showed significant elevation in the expression of immunoreactive PPARgamma in grade 3 ovarian tumours compared with that of normal ovaries and benign ovarian tumours (P < 0.01). These findings suggest an involvement of PPARgamma in the onset and development of ovarian carcinoma and provide an insight into the regulation of this molecule in the progression of the disease.

Essentials of nuclear medicine science

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hladik, W.B. III; Saha, G.B.; Study, K.T.

1987-01-01

This book contains 26 chapters. Some of the titles are: Normal Biodistribution of Diagnostic Radiopharmaceuticals; Radiopharmacokinetics in Nuclear Medicine; Nuclear Medicine Procedures for Monitoring Patient Therapy; Animal Models of Human Disease; Patient Preparation for Nuclear Medicine Studies; and Interventional Studies in Nuclear Medicine.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuan, Man I; O’Dowd, John M.; Fortunato, Elizabeth

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-introductionmore » 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. -- Highlights: •Phosphorylated nuclear lamins were inefficiently remodeled in p53KO cells. •p53KO cells expressed UL50, but it was not efficiently targeted to the nuclear rim. •UL53 was not expressed in the large majority of p53KO cells. •Cells failing to express UL53 did not localize UL50 to the nucleus. •NEC puncta/infoldings of the inner nuclear membrane were scarce in p53KO cells.« less

Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication.

PubMed

Chen, Tingting; Dong, Zhanqi; Hu, Nan; Hu, Zhigang; Dong, Feifan; Jiang, Yaming; Li, Jun; Chen, Peng; Lu, Cheng; Pan, Minhui

2017-06-15

Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences (cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a cNLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

Aberrant localization of lamin B receptor (LBR) in cellular senescence in human cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arai, Rumi; En, Atsuki; Ukekawa, Ryo

2016-05-13

5-Bromodeoxyuridine (BrdU), a thymidine analogue, induces cellular senescence in mammalian cells. BrdU induces cellular senescence probably through the regulation of chromatin because BrdU destabilizes or disrupts nucleosome positioning and decondenses heterochromatin. Since heterochromatin is tethered to the nuclear periphery through the interaction with the nuclear envelope proteins, we examined the localization of the several nuclear envelope proteins such as lamins, lamin-interacting proteins, nuclear pore complex proteins, and nuclear transport proteins in senescent cells. We have shown here that lamin B receptor (LBR) showed a change in localization in both BrdU-induced and replicative senescent cells.

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.

Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

PubMed Central

Hsu, Hong-Ming; Lee, Yu; Indra, Dharmu; Wei, Shu-Yi; Liu, Hsing-Wei; Chang, Lung-Chun; Chen, Chinpan; Ong, Shiou-Jeng

2012-01-01

In Trichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNA-binding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals. PMID:23042127

Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yi; Pirisi, Lucia; Creek, Kim E., E-mail: creekk@sccp.sc.edu

2013-09-15

We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cellmore » proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.« less

LLAMA: normal star formation efficiencies of molecular gas in the centres of luminous Seyfert galaxies

NASA Astrophysics Data System (ADS)

Rosario, D. J.; Burtscher, L.; Davies, R. I.; Koss, M.; Ricci, C.; Lutz, D.; Riffel, R.; Alexander, D. M.; Genzel, R.; Hicks, E. H.; Lin, M.-Y.; Maciejewski, W.; Müller-Sánchez, F.; Orban de Xivry, G.; Riffel, R. A.; Schartmann, M.; Schawinski, K.; Schnorr-Müller, A.; Saintonge, A.; Shimizu, T.; Sternberg, A.; Storchi-Bergmann, T.; Sturm, E.; Tacconi, L.; Treister, E.; Veilleux, S.

2018-02-01

Using new Atacama Pathfinder Experiment and James Clerk Maxwell Telescope spectroscopy of the CO 2→1 line, we undertake a controlled study of cold molecular gas in moderately luminous (Lbol = 1043-44.5 erg s-1) active galactic nuclei (AGN) and inactive galaxies from the Luminous Local AGN with Matched Analogs (LLAMA) survey. We use spatially resolved infrared photometry of the LLAMA galaxies from 2MASS, the Wide-field Infrared Survey Explorer the Infrared Astronomical Satellite and the Herschel Space Observatory (Herschel), corrected for nuclear emission using multicomponent spectral energy distribution fits, to examine the dust-reprocessed star formation rates, molecular gas fractions and star formation efficiencies (SFEs) over their central 1-3 kpc. We find that the gas fractions and central SFEs of both active and inactive galaxies are similar when controlling for host stellar mass and morphology (Hubble type). The equivalent central molecular gas depletion times are consistent with the discs of normal spiral galaxies in the local Universe. Despite energetic arguments that the AGN in LLAMA should be capable of disrupting the observable cold molecular gas in their central environments, our results indicate that nuclear radiation only couples weakly with this phase. We find a mild preference for obscured AGN to contain higher amounts of central molecular gas, which suggests connection between AGN obscuration and the gaseous environment of the nucleus. Systems with depressed SFEs are not found among the LLAMA AGN. We speculate that the processes that sustain the collapse of molecular gas into dense pre-stellar cores may also be a prerequisite for the inflow of material on to AGN accretion discs.

Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus

PubMed Central

Flather, Dylan; Semler, Bert L.

2015-01-01

The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review. PMID:26150805

Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes.

PubMed

Ward, Diane McVey; Shiflett, Shelly L; Huynh, Dinh; Vaughn, Michael B; Prestwich, Glenn; Kaplan, Jerry

2003-06-01

The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size.

Characterization of a nuclear export signal within the human T cell leukemia virus type I transactivator protein Tax.

PubMed

Alefantis, Timothy; Barmak, Kate; Harhaj, Edward W; Grant, Christian; Wigdahl, Brian

2003-06-13

Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I transactivator protein Tax plays an integral role in the etiology of adult T cell leukemia, as expression of Tax in T lymphocytes has been shown to result in immortalization. In addition, Tax is known to interface with numerous transcription factor families, including activating transcription factor/cAMP response element-binding protein and nuclear factor-kappaB, requiring Tax to localize to both the nucleus and cytoplasm. In this report, the nucleocytoplasmic localization of Tax was examined in Jurkat, HeLa, and U-87 MG cells. The results reported herein indicate that Tax contains a leucine-rich nuclear export signal (NES) that, when fused to green fluorescent protein (GFP), can direct nuclear export via the CRM-1 pathway, as determined by leptomycin B inhibition of nuclear export. However, cytoplasmic localization of full-length Tax was not altered by treatment with leptomycin B, suggesting that native Tax utilizes another nuclear export pathway. Additional support for the presence of a functional NES has also been shown because the NES mutant Tax(L200A)-GFP localized to the nuclear membrane in the majority of U-87 MG cells. Evidence has also been provided suggesting that the Tax NES likely exists as a conditionally masked signal because the truncation mutant TaxDelta214-GFP localized constitutively to the cytoplasm. These results suggest that Tax localization may be directed by specific changes in Tax conformation or by specific interactions with cellular proteins leading to changes in the availability of the Tax NES and nuclear localization signal.

Rac1 Dosage Is Crucial for Normal Endochondral Bone Growth.

PubMed

Suzuki, Dai; Bush, Jason R; Bryce, Dawn-Marie; Kamijo, Ryutaro; Beier, Frank

2017-10-01

Rac1, a member of the small Rho GTPase family, plays multiple cellular roles. Studies of mice conditionally lacking Rac1 have revealed essential roles for Rac1 in various tissues, including cartilage and limb mesenchyme, where Rac1 loss produces dwarfism and long bone shortening. To gain further insight into the role of Rac1 in skeletal development, we have used transgenic mouse lines to express a constitutively active (ca) Rac1 mutant protein in a Cre recombinase-dependent manner. Overexpression of caRac1 in limb bud mesenchyme or chondrocytes leads to reduced body weight and shorter bones compared with control mice. Histological analysis of growth plates showed that caRac1;Col2-Cre mice displayed ectopic hypertrophic chondrocytes in the proliferative zone and enlarged hypertrophic zones. These mice also displayed a reduced proportion of proliferating cell nuclear antigen-positive cells in the proliferative zone and nuclear β-catenin localization in the ectopic hypertrophic chondrocytes. Importantly, overexpression of caRac1 partially rescued the phenotypes of Rac1fl/fl;Col2-Cre and Rac1fl/fl;Prx1-Cre conditional knockout mice, including body weight, bone length, and growth plate disorganization. These results suggest that tight regulation of Rac1 activity is necessary for normal cartilage development. Copyright © 2017 Endocrine Society.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myre, Michael A.; O'Day, Danton H.

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 patchesmore » 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.« less

Used Nuclear Fuel Loading and Structural Performance Under Normal Conditions of Transport- Demonstration of Approach and Results on Used Fuel Performance Characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adkins, Harold; Geelhood, Ken; Koeppel, Brian

2013-09-30

This document addresses Oak Ridge National Laboratory milestone M2FT-13OR0822015 Demonstration of Approach and Results on Used Nuclear Fuel Performance Characterization. This report provides results of the initial demonstration of the modeling capability developed to perform preliminary deterministic evaluations of moderate-to-high burnup used nuclear fuel (UNF) mechanical performance under normal conditions of storage (NCS) and normal conditions of transport (NCT) conditions. This report also provides results from the sensitivity studies that have been performed. Finally, discussion on the long-term goals and objectives of this initiative are provided.

A Role for Caenorhabditis elegans Importin IMA-2 in Germ Line and Embryonic Mitosis

PubMed Central

Geles, Kenneth G.; Johnson, Jeffrey J.; Jong, Sena; Adam, Stephen A.

2002-01-01

The importin α family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin α proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin α proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype. ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis. PMID:12221121

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke

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 inmore » 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.« less

Biophysical assays to probe the mechanical properties of the interphase cell nucleus: substrate strain application and microneedle manipulation.

PubMed

Lombardi, Maria L; Zwerger, Monika; Lammerding, Jan

2011-09-14

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.(1) On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.(2) Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.(3) To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

A Highly Organized Structure Mediating Nuclear Localization of a Myb2 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis ▿ †

PubMed Central

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

2011-01-01

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. PMID:22021237

Nuclear import of human MLH1, PMS2, and MutLalpha: redundancy is the key.

PubMed

Leong, Vivian; Lorenowicz, Jessica; Kozij, Natalie; Guarné, Alba

2009-08-01

DNA mismatch repair maintains genomic stability by correcting errors that have escaped polymerase proofreading. Defects on mismatch repair genes lead to an increased mutation rate, microsatellite instability and predisposition to human non-polyposis colorectal cancer (HNPCC). Human MutLalpha is a heterodimer formed by the interaction of MLH1 and PMS2 that coordinates a series of key events in mismatch repair. It has been proposed that nuclear import of MutLalpha may be the first regulatory step on the activation of the mismatch repair pathway. Using confocal microscopy and mismatch repair deficient cells, we have identified the sequence determinants that drive nuclear import of human MLH1, PMS2, and MutLalpha. Transient transfection of the individual proteins reveals that MLH1 has a bipartite and PMS2 has a single monopartite nuclear localization signal. Although dimerization is not required for nuclear localization, the MutLalpha heterodimer is imported more efficiently than the MLH1 or PMS2 monomers. Interestingly, the bipartite localization signal of MLH1 can direct import of MutLalpha even when PMS2 encompasses a mutated localization signal. Hence we conclude that the presence of redundant nuclear localization signals guarantees nuclear transport of MutLalpha and, consequently, efficient mismatch repair.

Functional Reorganization of Promyelocytic Leukemia Nuclear Bodies during BK Virus Infection

PubMed Central

Jiang, Mengxi; Entezami, Pouya; Gamez, Monica; Stamminger, Thomas; Imperiale, Michael J.

2011-01-01

BK virus (BKV) is the causative agent for polyomavirus-associated nephropathy, a severe disease found in renal transplant patients due to reactivation of a persistent BKV infection. BKV replication relies on the interactions of BKV with many nuclear components, and subnuclear structures such as promyelocytic leukemia nuclear bodies (PML-NBs) are known to play regulatory roles during a number of DNA virus infections. In this study, we investigated the relationship between PML-NBs and BKV during infection of primary human renal proximal tubule epithelial (RPTE) cells. While the levels of the major PML-NB protein components remained unchanged, BKV infection of RPTE cells resulted in dramatic alterations in both the number and the size of PML-NBs. Furthermore, two normally constitutive components of PML-NBs, Sp100 and hDaxx, became dispersed from PML-NBs. To define the viral factors responsible for this reorganization, we examined the cellular localization of the BKV large tumor antigen (TAg) and viral DNA. TAg colocalized with PML-NBs during early infection, while a number of BKV chromosomes were adjacent to PML-NBs during late infection. We demonstrated that TAg alone was not sufficient to reorganize PML-NBs and that active viral DNA replication is required. Knockdown of PML protein did not dramatically affect BKV growth in culture. BKV infection, however, was able to rescue the growth of an ICP0-null herpes simplex virus 1 mutant whose growth defect was partially due to its inability to disrupt PML-NBs. We hypothesize that the antiviral functions of PML-NBs are inactivated through reorganization during normal BKV infection. PMID:21304169

Tissue distribution of aryl hydrocarbon receptor in the intestine: Implication of putative roles in tumor suppression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikuta, Togo, E-mail: togo@cancer-c.pref.saitama.jp; Kurosumi, Masafumi, E-mail: mkurosumi@cancer-c.pref.saitama.jp; Yatsuoka, Toshimasa, E-mail: yatsuoka-gi@umin.ac.jp

Intestinal homeostasis is maintained by complex interactions between intestinal microorganisms and the gut immune system. Dysregulation of gut immunity may lead to inflammatory disorders and tumorigenesis. We previously have shown the tumor suppressive effects of aryl hydrocarbon receptor (AhR) in intestinal carcinogenesis. In the present study, we investigated AhR distribution in the mouse and human intestine by histochemical analysis. In the normal intestine, AhR was mainly localized in the stroma containing immune cells in the lamina propria and lymphoid follicles. On the other hand, in the tumor tissue from human colon cancer and that developed in Apc{sup Min/+}mice, AhR expressionmore » was elevated. AhR immunostaining was found in both stromal and tumor cells. Although AhR was localized in the cytoplasm of tumor cells in most cases, nuclear AhR was also observed in some. AhR knockdown using siRNA resulted in significant promotion of cell growth in colon cancer cell lines. Furthermore, AhR activation by AhR ligands supplemented in culture medium suppressed cell growth. Our study results suggest that tumor suppressive roles of AhR are estimated in two distinct ways: in normal tissue, AhR is associated with tumor prevention by regulating gut immunity, whereas in tumor cells, it is involved in growth suppression. - Highlights: • In the normal intestine, AhR was mainly localized in stroma containing immune cells. • In the tumor tissue, AhR expression was found in both stromal and tumor cells. • AhR knockdown promoted cell growth in colon cancer cell lines.« less

Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khalil, Mohamed I., E-mail: mkhalil2@stanford.edu; Department of Molecular Biology, National Research Centre, El-Buhouth St., Cairo; Che, Xibing

VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This studymore » mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication. - Highlights: • Mutation of IE62 domain I did not affect VZV replication in melanoma cells. • IE62 domain II and III are important for VZV replication in melanoma cells. • Mutations of IE62 domain II (DBD) were lethal for virus replication. • Mutations of IE62 NLS and phosphorylation sites inhibited VZV replication. • NLS and S686A/S722A mutations altered localization of IE62 during early and late infection.« less

Inhibition of transforming growth factor-beta1-induced signaling and epithelial-to-mesenchymal transition by the Smad-binding peptide aptamer Trx-SARA.

PubMed

Zhao, Bryan M; Hoffmann, F Michael

2006-09-01

Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.

Karyopherin-mediated nuclear import of the homing endonuclease VMA1-derived endonuclease is required for self-propagation of the coding region.

PubMed

Nagai, Yuri; Nogami, Satoru; Kumagai-Sano, Fumi; Ohya, Yoshikazu

2003-03-01

VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute central regulators of cell differentiation in S. cerevisiae, and by nutrient depletion. A functional genomic approach revealed that at least two karyopherins, Srp1p and Kap142p, are required for the nuclear localization pattern. Genetic and physical interactions between Srp1p and VDE imply direct involvement of karyopherin-mediated nuclear transport in this process. Inactivation of TOR signaling or acquisition of an extra nuclear localization signal in the VDE coding region leads to artificial nuclear localization of VDE and thereby induces homing even during mitosis. These results serve as evidence that VDE utilizes the host systems of nutrient signal transduction and nucleocytoplasmic transport to ensure the propagation of its coding region.

Karyopherin-Mediated Nuclear Import of the Homing Endonuclease VMA1-Derived Endonuclease Is Required for Self-Propagation of the Coding Region

PubMed Central

Nagai, Yuri; Nogami, Satoru; Kumagai-Sano, Fumi; Ohya, Yoshikazu

2003-01-01

VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute central regulators of cell differentiation in S. cerevisiae, and by nutrient depletion. A functional genomic approach revealed that at least two karyopherins, Srp1p and Kap142p, are required for the nuclear localization pattern. Genetic and physical interactions between Srp1p and VDE imply direct involvement of karyopherin-mediated nuclear transport in this process. Inactivation of TOR signaling or acquisition of an extra nuclear localization signal in the VDE coding region leads to artificial nuclear localization of VDE and thereby induces homing even during mitosis. These results serve as evidence that VDE utilizes the host systems of nutrient signal transduction and nucleocytoplasmic transport to ensure the propagation of its coding region. PMID:12588991

Identification of multiple nuclear localization signals in murine Elf3, an ETS transcription factor.

PubMed

Do, Hyun-Jin; Song, Hyuk; Yang, Heung-Mo; Kim, Dong-Ku; Kim, Nam-Hyung; Kim, Jin-Hoi; Cha, Kwang-Yul; Chung, Hyung-Min; Kim, Jae-Hwan

2006-03-20

We investigated nuclear localization signal (NLS) determinants within the AT-hook and ETS DNA-binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium-specific ETS transcription factors. Deletion mutants containing the AT-hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT-hook domain, four basic residues (244KRKR247) were critical for strong NLS activity, and two potent bipartite NLS motifs (236-252 and 249-267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues (318KKK320) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport.

Same-Day Imaging Using Small Proteins: Clinical Experience and Translational Prospects in Oncology.

PubMed

Krasniqi, Ahmet; D'Huyvetter, Matthias; Devoogdt, Nick; Frejd, Fredrik Y; Sörensen, Jens; Orlova, Anna; Keyaerts, Marleen; Tolmachev, Vladimir

2018-06-01

Imaging of expression of therapeutic targets may enable stratification of patients for targeted treatments. The use of small radiolabeled probes based on the heavy-chain variable region of heavy-chain-only immunoglobulins or nonimmunoglobulin scaffolds permits rapid localization of radiotracers in tumors and rapid clearance from normal tissues. This makes high-contrast imaging possible on the day of injection. This mini review focuses on small proteins for radionuclide-based imaging that would allow same-day imaging, with the emphasis on clinical applications and promising preclinical developments within the field of oncology. © 2018 by the Society of Nuclear Medicine and Molecular Imaging.

Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

PubMed

Anderson, Gavin; Jang, Chanyong; Wang, Renyuan; Goodin, Michael

2018-05-01

The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

Acute Zonal Cone Photoreceptor Outer Segment Loss.

PubMed

Aleman, Tomas S; Sandhu, Harpal S; Serrano, Leona W; Traband, Anastasia; Lau, Marisa K; Adamus, Grazyna; Avery, Robert A

2017-05-01

The diagnostic path presented narrows down the cause of acute vision loss to the cone photoreceptor outer segment and will refocus the search for the cause of similar currently idiopathic conditions. To describe the structural and functional associations found in a patient with acute zonal occult photoreceptor loss. A case report of an adolescent boy with acute visual field loss despite a normal fundus examination performed at a university teaching hospital. Results of a complete ophthalmic examination, full-field flash electroretinography (ERG) and multifocal ERG, light-adapted achromatic and 2-color dark-adapted perimetry, and microperimetry. Imaging was performed with spectral-domain optical coherence tomography (SD-OCT), near-infrared (NIR) and short-wavelength (SW) fundus autofluorescence (FAF), and NIR reflectance (REF). The patient was evaluated within a week of the onset of a scotoma in the nasal field of his left eye. Visual acuity was 20/20 OU, and color vision was normal in both eyes. Results of the fundus examination and of SW-FAF and NIR-FAF imaging were normal in both eyes, whereas NIR-REF imaging showed a region of hyporeflectance temporal to the fovea that corresponded with a dense relative scotoma noted on light-adapted static perimetry in the left eye. Loss in the photoreceptor outer segment detected by SD-OCT co-localized with an area of dense cone dysfunction detected on light-adapted perimetry and multifocal ERG but with near-normal rod-mediated vision according to results of 2-color dark-adapted perimetry. Full-field flash ERG findings were normal in both eyes. The outer nuclear layer and inner retinal thicknesses were normal. Localized, isolated cone dysfunction may represent the earliest photoreceptor abnormality or a distinct entity within the acute zonal occult outer retinopathy complex. Acute zonal occult outer retinopathy should be considered in patients with acute vision loss and abnormalities on NIR-REF imaging, especially if multimodal imaging supports an intact retinal pigment epithelium and inner retina but an abnormal photoreceptor outer segment.

Transcription factor YY1 can control AID-mediated mutagenesis in mice.

PubMed

Zaprazna, Kristina; Basu, Arindam; Tom, Nikola; Jha, Vibha; Hodawadekar, Suchita; Radova, Lenka; Malcikova, Jitka; Tichy, Boris; Pospisilova, Sarka; Atchison, Michael L

2018-02-01

Activation-induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off-target non-Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock-out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID-mediated mutations. Using mice that overexpress AID (IgκAID yy1 f/f ) or that express normal AID levels (yy1 f/f ), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the Sμ switch region, and reduced AID-mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1-AID physical interaction which blocks AID ubiquitination. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Autosomal recessive mutations in nuclear transport factor KPNA7 are associated with infantile spasms and cerebellar malformation.

PubMed

Paciorkowski, Alex R; Weisenberg, Judy; Kelley, Joshua B; Spencer, Adam; Tuttle, Emily; Ghoneim, Dalia; Thio, Liu Lin; Christian, Susan L; Dobyns, William B; Paschal, Bryce M

2014-05-01

Nuclear import receptors of the KPNA family recognize the nuclear localization signal in proteins and together with importin-β mediate translocation into the nucleus. Accordingly, KPNA family members have a highly conserved architecture with domains that contact the nuclear localization signal and bind to importin-β. Here, we describe autosomal recessive mutations in KPNA7 found by whole exome sequencing in a sibling pair with severe developmental disability, infantile spasms, subsequent intractable epilepsy consistent with Lennox-Gastaut syndrome, partial agenesis of the corpus callosum, and cerebellar vermis hypoplasia. The mutations mapped to exon 7 in KPNA7 result in two amino-acid substitutions, Pro339Ala and Glu344Gln. On the basis of the crystal structure of the paralog KPNA2 bound to a bipartite nuclear localization signal from the retinoblastoma protein, the amino-acid substitutions in the affected subjects were predicted to occur within the seventh armadillo repeat that forms one of the two nuclear localization signal-binding sites in KPNA family members. Glu344 is conserved in all seven KPNA proteins, and we found that the Glu354Gln mutation in KPNA2 is sufficient to reduce binding to the retinoblastoma nuclear localization signal to approximately one-half that of wild-type protein. Our data show that compound heterozygous mutations in KPNA7 are associated with a human neurodevelopmental disease, and provide the first example of a human disease associated with mutation of a nuclear transport receptor.

Autosomal recessive mutations in nuclear transport factor KPNA7 are associated with infantile spasms and cerebellar malformation

PubMed Central

Paciorkowski, Alex R; Weisenberg, Judy; Kelley, Joshua B; Spencer, Adam; Tuttle, Emily; Ghoneim, Dalia; Thio, Liu Lin; Christian, Susan L; Dobyns, William B; Paschal, Bryce M

2014-01-01

Nuclear import receptors of the KPNA family recognize the nuclear localization signal in proteins and together with importin-β mediate translocation into the nucleus. Accordingly, KPNA family members have a highly conserved architecture with domains that contact the nuclear localization signal and bind to importin-β. Here, we describe autosomal recessive mutations in KPNA7 found by whole exome sequencing in a sibling pair with severe developmental disability, infantile spasms, subsequent intractable epilepsy consistent with Lennox–Gastaut syndrome, partial agenesis of the corpus callosum, and cerebellar vermis hypoplasia. The mutations mapped to exon 7 in KPNA7 result in two amino-acid substitutions, Pro339Ala and Glu344Gln. On the basis of the crystal structure of the paralog KPNA2 bound to a bipartite nuclear localization signal from the retinoblastoma protein, the amino-acid substitutions in the affected subjects were predicted to occur within the seventh armadillo repeat that forms one of the two nuclear localization signal-binding sites in KPNA family members. Glu344 is conserved in all seven KPNA proteins, and we found that the Glu354Gln mutation in KPNA2 is sufficient to reduce binding to the retinoblastoma nuclear localization signal to approximately one-half that of wild-type protein. Our data show that compound heterozygous mutations in KPNA7 are associated with a human neurodevelopmental disease, and provide the first example of a human disease associated with mutation of a nuclear transport receptor. PMID:24045845

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Subcellular localization of celery mannitol dehydrogenase. A cytosolic metabolic enzyme in nuclei.

PubMed Central

Yamamoto, Y T; Zamski, E; Williamson, J D; Conkling, M A; Pharr, D M

1997-01-01

Mannitol dehydrogenase (MTD) is the first enzyme in mannitol catabolism in celery (Apium graveolens L. var dulce [Mill] Pers. cv Florida 638). Mannitol is an important photoassimilate, as well as providing plants with resistance to salt and osmotic stress. Previous work has shown that expression of the celery Mtd gene is regulated by many factors, such as hexose sugars, salt and osmotic stress, and salicylic acid. Furthermore, MTD is present in cells of sink organs, phloem cells, and mannitol-grown suspension cultures. Immunogold localization and biochemical analyses presented here demonstrate that celery MTD is localized in the cytosol and nuclei. Although the cellular density of MTD varies among different cell types, densities of nuclear and cytosolic MTD in a given cell are approximately equal. Biochemical analyses of nuclear extracts from mannitol-grown cultured cells confirmed that the nuclear-localized MTD is enzymatically active. The function(s) of nuclear-localized MTD is unknown. PMID:9414553

Normal central retinal function and structure preserved in retinitis pigmentosa.

PubMed

Jacobson, Samuel G; Roman, Alejandro J; Aleman, Tomas S; Sumaroka, Alexander; Herrera, Waldo; Windsor, Elizabeth A M; Atkinson, Lori A; Schwartz, Sharon B; Steinberg, Janet D; Cideciyan, Artur V

2010-02-01

To determine whether normal function and structure, as recently found in forms of Usher syndrome, also occur in a population of patients with nonsyndromic retinitis pigmentosa (RP). Patients with simplex, multiplex, or autosomal recessive RP (n = 238; ages 9-82 years) were studied with static chromatic perimetry. A subset was evaluated with optical coherence tomography (OCT). Co-localized visual sensitivity and photoreceptor nuclear layer thickness were measured across the central retina to establish the relationship of function and structure. Comparisons were made to patients with Usher syndrome (n = 83, ages 10-69 years). Cross-sectional psychophysical data identified patients with RP who had normal rod- and cone-mediated function in the central retina. There were two other patterns with greater dysfunction, and longitudinal data confirmed that progression can occur from normal rod and cone function to cone-only central islands. The retinal extent of normal laminar architecture by OCT corresponded to the extent of normal visual function in patients with RP. Central retinal preservation of normal function and structure did not show a relationship with age or retained peripheral function. Usher syndrome results were like those in nonsyndromic RP. Regional disease variation is a well-known finding in RP. Unexpected was the observation that patients with presumed recessive RP can have regions with functionally and structurally normal retina. Such patients will require special consideration in future clinical trials of either focal or systemic treatment. Whether there is a common molecular mechanism shared by forms of RP with normal regions of retina warrants further study.

Nup124p Is a Nuclear Pore Factor of Schizosaccharomyces pombe That Is Important for Nuclear Import and Activity of Retrotransposon Tf1

PubMed Central

Balasundaram, David; Benedik, Michael J.; Morphew, Mary; Dang, Van-Dinh; Levin, Henry L.

1999-01-01

The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag. PMID:10409764

Nup124p is a nuclear pore factor of Schizosaccharomyces pombe that is important for nuclear import and activity of retrotransposon Tf1.

PubMed

Balasundaram, D; Benedik, M J; Morphew, M; Dang, V D; Levin, H L

1999-08-01

The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag.

Emerin and histone deacetylase 3 (HDAC3) cooperatively regulate expression and nuclear positions of MyoD, Myf5, and Pax7 genes during myogenesis

PubMed Central

Demmerle, Justin; Koch, Adam J.; Holaska, James M.

2016-01-01

The spatial organization of chromatin is critical in establishing cell-type dependent gene expression programs. The inner nuclear membrane protein emerin has been implicated in regulating global chromatin architecture. We show emerin associates with genomic loci of muscle differentiation promoting factors in murine myogenic progenitors, including Myf5 and MyoD. Prior to their transcriptional activation Myf5 and MyoD loci localized to the nuclear lamina in proliferating progenitors and moved to the nucleoplasm upon transcriptional activation during differentiation. The Pax7 locus, which is transcribed in proliferating progenitors, localized to the nucleoplasm and Pax7 moved to the nuclear lamina upon repression during differentiation. Localization of Myf5, MyoD, and Pax7 to the nuclear lamina and proper temporal expression of these genes required emerin and HDAC3. Interestingly, activation of HDAC3 catalytic activity rescued both Myf5 localization to the nuclear lamina and its expression. Collectively, these data support a model whereby emerin facilitates repressive chromatin formation at the nuclear lamina by activating the catalytic activity of HDAC3 to regulate the coordinated spatiotemporal expression of myogenic differentiation genes. PMID:24062260

Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization

PubMed Central

Näf, Dieter; Kupfer, Gary M.; Suliman, Ahmed; Lambert, Kathleen; D’Andrea, Alan D.

1998-01-01

Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and cellular hypersensitivity to DNA-cross-linking agents. Eight complementation groups of FA (FA-A through FA-H) have been identified. Two FA genes, corresponding to complementation groups FA-A and FA-C, have been cloned, but the functions of the encoded FAA and FAC proteins remain unknown. We have recently demonstrated that FAA and FAC interact to form a nuclear complex. In this study, we have analyzed a series of mutant forms of the FAA protein with respect to functional activity, FAC binding, and nuclear localization. Mutation or deletion of the amino-terminal nuclear localization signal (NLS) of FAA results in loss of functional activity, loss of FAC binding, and cytoplasmic retention of FAA. Replacement of the NLS sequence with a heterologous NLS sequence, derived from the simian virus 40 T antigen, results in nuclear localization but does not rescue functional activity or FAC binding. Nuclear localization of the FAA protein is therefore necessary but not sufficient for FAA function. Mutant forms of FAA which fail to bind to FAC also fail to promote the nuclear accumulation of FAC. In addition, wild-type FAC promotes the accumulation of wild-type FAA in the nucleus. Our results suggest that FAA and FAC perform a concerted function in the cell nucleus, required for the maintenance of chromosomal stability. PMID:9742112

[Analysis of nuclear localization and signal function of MITF protein predisposing to Warrdenburg syndrome].

PubMed

Zhang, Hua; Feng, Juan; Chen, Hongsheng; Li, Jiada; Luo, Hunjin; Feng, Yong

2015-12-01

To study the role of dysfunction of nuclear localization signals (NLS) of MITF protein in the pathogenesis of Waardenburg syndrome. Eukaryotic expression plasmid pCMV-MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITF△NLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase (TYR). The oligonucleotide 5'-GAACGAAGAAGAAGATTT-3' was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfected separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subcellular distribution was observed by immunoflorescence assays. Expression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITF△NLS were successfully generated. Compared with the wild-type MITF, MITF△NLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus. This study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.

Syndecan-4 Regulates Muscle Differentiation and Is Internalized from the Plasma Membrane during Myogenesis.

PubMed

Rønning, Sissel B; Carlson, Cathrine R; Stang, Espen; Kolset, Svein O; Hollung, Kristin; Pedersen, Mona E

2015-01-01

The cell surface proteoglycan syndecan-4 has been reported to be crucial for muscle differentiation, but the molecular mechanisms still remain to be fully understood. During in vitro differentiation of bovine muscle cells immunocytochemical analyses showed strong labelling of syndecan-4 intracellularly, in close proximity with Golgi structures, in membranes of intracellular vesicles and finally, in the nuclear area including the nuclear envelope. Chase experiments showed that syndecan-4 was internalized from the plasma membrane during this process. Furthermore, when syndecan-4 was knocked down by siRNA more myotubes were formed, and the expression of myogenic transcription factors, β1-integrin and actin was influenced. However, when bovine muscle cells were treated with a cell-penetrating peptide containing the cytoplasmic region of syndecan-4, myoblast fusion and thus myotube formation was blocked, both in normal cells and in syndecan-4 knock down cells. Altogether this suggests that the cytoplasmic domain of syndecan-4 is important in regulation of myogenesis. The internalization of syndecan-4 from the plasma membrane during muscle differentiation and the nuclear localization of syndecan-4 in differentiated muscle cells may be part of this regulation, and is a novel aspect of syndecan biology which merits further studies.

Cycloheximide- and puromycin-induced heat resistance: different effects on cytoplasmic and nuclear luciferases

PubMed Central

Michels, Annemieke A; Kanon, Bart; Konings, Antonius W.T; Bensaude, Olivier; Kampinga, Harm H

2000-01-01

Inhibition of translation can result in cytoprotection against heat shock. The mechanism of this protection has remained elusive so far. Here, the thermoprotective effects of the translation inhibitor cycloheximide (CHX) and puromycin were investigated, using as reporter firefly luciferase localized either in the nucleus or in the cytoplasm. A short preincubation of O23 cells with either translation inhibitor was found to attenuate the heat inactivation of a luciferase directed into the cytoplasm, whereas the heat sensitivity of a nuclear-targeted luciferase remained unaffected. After a long-term CHX pretreatment, both luciferases were more heat resistant. Both the cytoplasmic and the nuclear luciferase are protected against heat-induced inactivation in thermotolerant cells and in cells overexpressing heat shock protein (Hsp)70. CHX incubations further attenuated cytoplasmic luciferase inactivation in thermotolerant and in Hsp70 overexpressing cells, even when Hsp70-mediated protection was saturated. It is concluded that protection by translation inhibition is unlikely due to an increase in the pool of free Hsps normally engaged in translation and released from the nascent polypeptide chains on the ribosomes. Rather, a decrease in nascent chains and thermolabile polypeptides may account for the heat resistance promoted by inhibitors of translation. PMID:11005376

Expression and localization of peroxisome proliferator-activated receptors and nuclear factor kappaB in normal and lesional psoriatic skin.

PubMed

Westergaard, Majken; Henningsen, Jeanette; Johansen, Claus; Rasmussen, Sofie; Svendsen, Morten Lyhne; Jensen, Uffe Birk; Schrøder, Henrik Daa; Staels, Bart; Iversen, Lars; Bolund, Lars; Kragballe, Knud; Kristiansen, Karsten

2003-11-01

Abnormal epidermal proliferation and differentiation characterize the inflammatory skin disease psoriasis. Here we demonstrate that expression of PPARdelta mRNA and protein is markedly upregulated in psoriatic lesions and that lipoxygenase products accumulating in psoriatic lesions are potent activators of PPARdelta. The expression levels of NF-kappaB p50 and p65 were not significantly altered in lesional compared with nonlesional psoriatic skin. In the basal layer of normal epidermis both p50 and p65 were sequestered in the cytoplasm, whereas p50, but not p65, localized to nuclei in the suprabasal layers, and this distribution was maintained in lesional psoriatic skin. In normal human keratinocytes PPAR agonists neither impaired IL-1beta-induced translocation of p65 nor IL-1beta-induced NF-kappaB DNA binding. We show that PPARdelta physically interacts with the N-terminal Rel homology domain of p65. Irrespective of the presence of agonists none of the PPAR subtypes decreased p65-mediated transactivation in keratinocytes. In contrast p65, but not p50, was a potent repressor of PPAR-mediated transactivation. The p65-dependent repression of PPARdelta- but not PPARalpha- or PPARgamma-mediated transactivation was partially relieved by forced expression of the coactivators p300 or CBP. We suggest that deficient NF-kappaB activation in chronic psoriatic plaques permitting unabated PPARdelta-mediated transactivation contributes to the pathologic phenotype of psoriasis.

Phosphatidic acid interacts with a MYB transcription factor and regulates its nuclear localization and function in Arabidopsis.

PubMed

Yao, Hongyan; Wang, Geliang; Guo, Liang; Wang, Xuemin

2013-12-01

Phosphatidic acid (PA) has emerged as a class of cellular mediators involved in various cellular and physiological processes, but little is known about its mechanism of action. Here we show that PA interacts with werewolf (WER), a R2R3 MYB transcription factor involved in root hair formation. The PA-interacting region is confined to the end of the R2 subdomain. The ablation of the PA binding motif has no effect on WER binding to DNA, but abolishes its nuclear localization and its function in regulating epidermal cell fate. Inhibition of PA production by phospholipase Dζ also suppresses WER's nuclear localization, root hair formation, and elongation. These results suggest a role for PA in promoting protein nuclear localization.

Phosphatidic Acid Interacts with a MYB Transcription Factor and Regulates Its Nuclear Localization and Function in Arabidopsis[C][W

PubMed Central

Yao, Hongyan; Wang, Geliang; Guo, Liang; Wang, Xuemin

2013-01-01

Phosphatidic acid (PA) has emerged as a class of cellular mediators involved in various cellular and physiological processes, but little is known about its mechanism of action. Here we show that PA interacts with WEREWOLF (WER), a R2R3 MYB transcription factor involved in root hair formation. The PA-interacting region is confined to the end of the R2 subdomain. The ablation of the PA binding motif has no effect on WER binding to DNA, but abolishes its nuclear localization and its function in regulating epidermal cell fate. Inhibition of PA production by phospholipase Dζ also suppresses WER’s nuclear localization, root hair formation, and elongation. These results suggest a role for PA in promoting protein nuclear localization. PMID:24368785

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paterson, Carolyn P.; Ayalew, Lisanework E.; Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada

The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acidmore » 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.« less

10 CFR 50.65 - Requirements for monitoring the effectiveness of maintenance at nuclear power plants.

Code of Federal Regulations, 2012 CFR

2012-01-01

... maintenance at nuclear power plants. 50.65 Section 50.65 Energy NUCLEAR REGULATORY COMMISSION DOMESTIC... Construction Permits § 50.65 Requirements for monitoring the effectiveness of maintenance at nuclear power..., including normal shutdown operations. (a)(1) Each holder of an operating license for a nuclear power plant...

10 CFR 50.65 - Requirements for monitoring the effectiveness of maintenance at nuclear power plants.

Code of Federal Regulations, 2013 CFR

2013-01-01

... maintenance at nuclear power plants. 50.65 Section 50.65 Energy NUCLEAR REGULATORY COMMISSION DOMESTIC... Construction Permits § 50.65 Requirements for monitoring the effectiveness of maintenance at nuclear power..., including normal shutdown operations. (a)(1) Each holder of an operating license for a nuclear power plant...

10 CFR 50.65 - Requirements for monitoring the effectiveness of maintenance at nuclear power plants.

Code of Federal Regulations, 2011 CFR

2011-01-01

... maintenance at nuclear power plants. 50.65 Section 50.65 Energy NUCLEAR REGULATORY COMMISSION DOMESTIC... Construction Permits § 50.65 Requirements for monitoring the effectiveness of maintenance at nuclear power..., including normal shutdown operations. (a)(1) Each holder of an operating license for a nuclear power plant...

10 CFR 50.65 - Requirements for monitoring the effectiveness of maintenance at nuclear power plants.

Code of Federal Regulations, 2014 CFR

2014-01-01

... maintenance at nuclear power plants. 50.65 Section 50.65 Energy NUCLEAR REGULATORY COMMISSION DOMESTIC... Construction Permits § 50.65 Requirements for monitoring the effectiveness of maintenance at nuclear power..., including normal shutdown operations. (a)(1) Each holder of an operating license for a nuclear power plant...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Young, I.T.; Vanderlaan, M.; Kromhout, L.

Improved early detection of neoplasia by screening of urothelial cells requires an understanding of the features distinguishing normal and neoplastic cell populations. The authors have begun a program of study based upon a rate model system for the controlled observation of early-stage lesions produced by the carcinogen N-butyl-N-(4-hydroxybutyl)- nitrosamine. Cells dissociated directly from normal and malignant urothelium were characterized by conventional cytopathology techniques and by quantitative microscopy (for nuclear texture and nuclear and cytoplasmic size, shape, and stain content) to derive a comprehensive picture of bladder tumor development. By following the changes that occur in the dissociated urothelial cells themore » authors have found that the nuclear area, total nuclear stain, nuclear shape, and the nuclear chromatin change significantly over a 48-wk interval as the lesions progress toward malignancy. 24 references, 10 figures, 1 table.« less

Unique nuclear localization of Nile tilapia (Oreochromis niloticus) Neu4 sialidase is regulated by nuclear transport receptor importin α/β.

PubMed

Honda, Akinobu; Chigwechokha, Petros Kingstone; Kamada-Futagami, Yuko; Komatsu, Masaharu; Shiozaki, Kazuhiro

2018-06-01

Sialidase catalyzes the removal of sialic acids from glycoconjugates. Different from Neu1 and Neu3 sialidases, Neu4 enzymatic properties such as substrate specificity and subcellular localization are not well-conserved among vertebrates. In fish only zebrafish and medaka neu4 genes have been cloned and their polypeptides have been characterized so far. Thus, characterization of Neu4 from other fish species is necessary to evaluate Neu4 physiological functions. Here, Nile tilapia was chosen for the characterization of Neu4 polypeptide considering that it is one of the major cultured fish all over the world and that its genomic sequences are now available. Coding DNA sequence of tilapia Neu4 was identified as 1,497 bp and its recombinant protein showed broad substrate specificity and optimal sialidase enzyme activity pH at 4.0. Neu4 activity was sustained even in neutral and alkali pH. Interestingly, immunofluorescence analysis revealed that major subcellular localization of tilapia Neu4 was nuclear, quite distinct from zebrafish (ER) and medaka Neu4 (lysosome). Bioinformatic analysis showed the existence of putative nuclear localization signal (NLS) in tilapia Neu4. In general, it is known that importin families bind to several proteins via NLS and transfer them into nucleus. Therefore, to determine the involvement of putative NLS in Neu4 nuclear localization, Neu4 mutant deleting NLS was constructed and expressed in cultured cells. As a result, NLS deletion significantly diminished the nuclear localization. Furthermore, treatment of importazole, interrupter of binding importin β and RanGTP, significantly suppressed Neu4 nuclear localization. In summary, tilapia Neu4 is a unique sialidase localized at nucleus and its transport system into nucleus is regulated by importin. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Role of the mRNA export factor Sus1 in oxidative stress tolerance in Candida albicans.

PubMed

Xiao, Chenpeng; Yu, Qilin; Zhang, Bing; Li, Jianrong; Zhang, Dan; Li, Mingchun

2018-02-05

In eukaryotes, the nuclear export of mRNAs is essential for gene expression. However, little is known about the role of mRNA nuclear export in the important fungal pathogen, Candida albicans. In this study, we identified C. albicans Sus1, a nucleus-localized protein that is required for mRNA export. Interestingly, the sus1Δ/Δ displayed hyper-sensitivity to extracellular oxidative stress, enhanced ROS accumulation and severe oxidative stress-related cell death. More strikingly, although the mutant exhibited normal activation of the expression of oxidative stress response (OSR) genes, it had attenuated activity of ROS scavenging system, which may be attributed to the defect in OSR mRNA export in this mutant. In addition, the virulence of the sus1Δ/Δ was seriously attenuated. Taken together, our findings provide evidence that the mRNA export factor Sus1 plays an important role in oxidative stress tolerance and pathogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

Altered peroxisome-proliferator activated receptors expression in human endometrial cancer.

PubMed

Knapp, Paweł; Chabowski, Adrian; Błachnio-Zabielska, Agnieszka; Jarząbek, Katarzyna; Wołczyński, Sławomir

2012-01-01

Peroxisome proliferator-activated receptors (PPARs) belong to a family of nuclear hormone receptors acting as transcriptional factors, recently involved also in carcinogenesis. Present study was undertaken to evaluate the presence and subcellular localization of different PPAR isoforms (α, β, γ) in healthy endometrial tissue (n = 10) and endometrial carcinoma (FIGO I, endometrioides type, G1, n = 35). We sought to analyze PPARs mRNA content as well as protein immunohistochemical expression that was further quantified by Western Blot technique. For both PPARα and PPARβ, protein expression was significantly higher in endometrial cancers compared to normal endometrial mucosa. In opposite, PPARγ protein expression was lower in endometrial cancer cells. In each case, immunohistochemical reaction was confined to the perinuclear and/or nuclear region. At the transcriptional level, the content of mRNA of all PPAR subunits did not follow the protein pattern of changes. These results provide evidence for altered PPAR's protein expression and disregulation of posttranslational processes in endometrial cancers.

[Nuclear protein matrix from giant nuclei of Chironomus plumosus determinates polythene chromosome organization].

PubMed

Makarov, M S; Chentsov, Iu S

2010-01-01

Giant nuclei from salivary glands of Chironomus plumosus were treated in situ with detergent, 2 M NaCl and nucleases in order to reveal residual nuclear matrix proteins (NMP). It was shown, that preceding stabilization of non-histone proteins with 2 mM CuCl2 allowed to visualize the structure of polythene chromosomes at every stage of the extraction of histones and DNA. Stabilized NPM of polythene chromosomes maintains their morphology and banding patterns, which is observed by light and electron microscopy, whereas internal fibril net or residual nucleoli are not found. In stabilized NPM of polythene chromosomes, topoisomerase IIalpha and SMC1 retain their localization that is typical of untreated chromosomes. NPM of polythene chromosomes also includes sites of DNA replication, visualized with BrDU incubation, and some RNA-components. So, we can conclude that structure of NPM from giant nuclei is equal to NPM from normal interphase nuclei, and that morphological features of polythene chromosomes depend on the presence of NMP.

Stroma Targeting Nuclear Imaging and Radiopharmaceuticals

PubMed Central

Shetty, Dinesh; Jeong, Jae-Min; Shim, Hyunsuk

2012-01-01

Malignant transformation of tumor accompanies profound changes in the normal neighboring tissue, called tumor stroma. The tumor stroma provides an environment favoring local tumor growth, invasion, and metastatic spreading. Nuclear imaging (PET/SPECT) measures biochemical and physiologic functions in the human body. In oncology, PET/SPECT is particularly useful for differentiating tumors from postsurgical changes or radiation necrosis, distinguishing benign from malignant lesions, identifying the optimal site for biopsy, staging cancers, and monitoring the response to therapy. Indeed, PET/SPECT is a powerful, proven diagnostic imaging modality that displays information unobtainable through other anatomical imaging, such as CT or MRI. When combined with coregistered CT data, [18F]fluorodeoxyglucose ([18F]FDG)-PET is particularly useful. However, [18F]FDG is not a target-specific PET tracer. This paper will review the tumor microenvironment targeting oncologic imaging such as angiogenesis, invasion, hypoxia, growth, and homing, and also therapeutic radiopharmaceuticals to provide a roadmap for additional applications of tumor imaging and therapy. PMID:22685650

Nuclear medicine in clinical neurology: an update

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oldendorf, W.H.

1981-01-01

Isotope scanning using technetium 99m pertechnetate has fallen into disuse since the advent of x-ray computerized tomography. Regional brain blood flow studies have been pursued on a research basis. Increased regional blood flow during focal seizure activity has been demonstrated and is of use in localizing such foci. Cisternography as a predictive tool in normal pressure hydrocephalus is falling into disuse. Positron tomographic scanning is a potent research tool that can demonstrate both regional glycolysis and blood flow. Unfortunately, it is extremely expensive and complex to apply in a clinical setting. With support from the National Institutes of Health, sevenmore » extramural centers have been funded to develop positron tomographic capabilities, and they will greatly advance our knowledge of stroke pathophysiology, seizure disorders, brain tumors, and various degenerative diseases. Nuclear magnetic resonance imaging is a potentially valuable tool since it creates tomographic images representing the distribution of brain water. No tissue ionization is produced, and images comparable to second-generation computerized tomographic scans are already being produced in humans.« less

The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji

2014-01-17

Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains amore » highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription.« less

Genomic organization of the human mi-er1 gene and characterization of alternatively spliced isoforms: regulated use of a facultative intron determines subcellular localization.

PubMed

Paterno, Gary D; Ding, Zhihu; Lew, Yuan-Y; Nash, Gord W; Mercer, F Corinne; Gillespie, Laura L

2002-07-24

mi-er1 (previously called er1) is a fibroblast growth factor-inducible early response gene activated during mesoderm induction in Xenopus embryos and encoding a nuclear protein that functions as a transcriptional activator. The human orthologue of mi-er1 was shown to be upregulated in breast carcinoma cell lines and breast tumours when compared to normal breast cells. In this report, we investigate the structure of the human mi-er1 (hmi-er1) gene and characterize the alternatively spliced transcripts and protein isoforms. hmi-er1 is a single copy gene located at 1p31.2 and spanning 63 kb. It contains 17 exons and includes one skipped exon, a facultative intron and three polyadenylation signals to produce 12 transcripts encoding six distinct proteins. hmi-er1 transcripts were expressed at very low levels in most human adult tissues and the mRNA isoform pattern varied with the tissue. The 12 transcripts encode proteins containing a common internal sequence with variable N- and C-termini. Three distinct N- and two distinct C-termini were identified, giving rise to six protein isoforms. The two C-termini differ significantly in size and sequence and arise from alternate use of a facultative intron to produce hMI-ER1alpha and hMI-ER1beta. In all tissues except testis, transcripts encoding the beta isoform were predominant. hMI-ER1alpha lacks the predicted nuclear localization signal and transfection assays revealed that, unlike hMI-ER1beta, it is not a nuclear protein, but remains in the cytoplasm. Our results demonstrate that alternate use of a facultative intron regulates the subcellular localization of hMI-ER1 proteins and this may have important implications for hMI-ER1 function.

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-NLS-L-NPs). Results indicate that a higher NLS density does not result in maximum protein nuclear localization and that a universal optimal density for NPs of different sizes does not exist.

Death-domain associated protein-6 (DAXX) mediated apoptosis in hantavirus infection is counter-balanced by activation of interferon-stimulated nuclear transcription factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khaiboullina, Svetlana F., E-mail: sv.khaiboullina@gmail.com; Morzunov, Sergey P.; Boichuk, Sergei V.

2013-09-01

Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20 kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirusmore » triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.« less

Stronger activation of SREBP-1a by nucleus-localized HBx

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Qi; Qiao, Ling; Yang, Jian

2015-05-08

We previously showed that hepatitis B virus (HBV) X protein activates the sterol regulatory element-binding protein-1a (SREBP-1a). Here we examined the role of nuclear localization of HBx in this process. In comparison to the wild-type and cytoplasmic HBx, nuclear HBx had stronger effects on SREBP-1a and fatty acid synthase transcription activation, intracellular lipid accumulation and cell proliferation. Furthermore, nuclear HBx could activate HBV enhancer I/X promoter and was more effective on up-regulating HBV mRNA level in the context of HBV replication than the wild-type HBx, while the cytoplasmic HBx had no effect. Our results demonstrate the functional significance of themore » nucleus-localized HBx in regulating host lipogenic pathway and HBV replication. - Highlights: • Nuclear HBx is more effective on activating SREBP-1a and FASN transcription. • Nuclear HBx is more effective on enhancing intracellular lipid accumulation. • Nuclear HBx is more effective on enhancing cell proliferation. • Nuclear HBx up-regulates HBV enhancer I/X promoter activity. • Nuclear HBx increases HBV mRNA level in the context of HBV replication.« less

Nuclear phytochrome A signaling promotes phototropism in Arabidopsis.

PubMed

Kami, Chitose; Hersch, Micha; Trevisan, Martine; Genoud, Thierry; Hiltbrunner, Andreas; Bergmann, Sven; Fankhauser, Christian

2012-02-01

Phototropin photoreceptors (phot1 and phot2 in Arabidopsis thaliana) enable responses to directional light cues (e.g., positive phototropism in the hypocotyl). In Arabidopsis, phot1 is essential for phototropism in response to low light, a response that is also modulated by phytochrome A (phyA), representing a classical example of photoreceptor coaction. The molecular mechanisms underlying promotion of phototropism by phyA remain unclear. Most phyA responses require nuclear accumulation of the photoreceptor, but interestingly, it has been proposed that cytosolic phyA promotes phototropism. By comparing the kinetics of phototropism in seedlings with different subcellular localizations of phyA, we show that nuclear phyA accelerates the phototropic response, whereas in the fhy1 fhl mutant, in which phyA remains in the cytosol, phototropic bending is slower than in the wild type. Consistent with this data, we find that transcription factors needed for full phyA responses are needed for normal phototropism. Moreover, we show that phyA is the primary photoreceptor promoting the expression of phototropism regulators in low light (e.g., PHYTOCHROME KINASE SUBSTRATE1 [PKS1] and ROOT PHOTO TROPISM2 [RPT2]). Although phyA remains cytosolic in fhy1 fhl, induction of PKS1 and RPT2 expression still occurs in fhy1 fhl, indicating that a low level of nuclear phyA signaling is still present in fhy1 fhl.

Nuclear Phytochrome A Signaling Promotes Phototropism in Arabidopsis[W][OA

PubMed Central

Kami, Chitose; Hersch, Micha; Trevisan, Martine; Genoud, Thierry; Hiltbrunner, Andreas; Bergmann, Sven; Fankhauser, Christian

2012-01-01

Phototropin photoreceptors (phot1 and phot2 in Arabidopsis thaliana) enable responses to directional light cues (e.g., positive phototropism in the hypocotyl). In Arabidopsis, phot1 is essential for phototropism in response to low light, a response that is also modulated by phytochrome A (phyA), representing a classical example of photoreceptor coaction. The molecular mechanisms underlying promotion of phototropism by phyA remain unclear. Most phyA responses require nuclear accumulation of the photoreceptor, but interestingly, it has been proposed that cytosolic phyA promotes phototropism. By comparing the kinetics of phototropism in seedlings with different subcellular localizations of phyA, we show that nuclear phyA accelerates the phototropic response, whereas in the fhy1 fhl mutant, in which phyA remains in the cytosol, phototropic bending is slower than in the wild type. Consistent with this data, we find that transcription factors needed for full phyA responses are needed for normal phototropism. Moreover, we show that phyA is the primary photoreceptor promoting the expression of phototropism regulators in low light (e.g., PHYTOCHROME KINASE SUBSTRATE1 [PKS1] and ROOT PHOTO TROPISM2 [RPT2]). Although phyA remains cytosolic in fhy1 fhl, induction of PKS1 and RPT2 expression still occurs in fhy1 fhl, indicating that a low level of nuclear phyA signaling is still present in fhy1 fhl. PMID:22374392

NUCLEOPORINS NPP-1, NPP-3, NPP-4, NPP-11 and NPP-13 ARE REQUIRED FOR PROPER SPINDLE ORIENTATION IN C. ELEGANS

PubMed Central

Schetter, Aaron; Askjaer, Peter; Piano, Fabio; Mattaj, Iain; Kemphues, Kenneth

2006-01-01

Nucleoporins are components of the nuclear pore, which is required for nucleo-cytoplasmic transport. We report a role for a subclass of nucleoporins in orienting the mitotic spindle in C. elegans embryos. RNAi-mediated depletion of any of five putative nucleoporins npp-1, npp-3, npp-4, npp-11, and npp-13 leads to indistinguishable spindle orientation defects. Transgenic worms expressing NPP-1::GFP or NPP-11::GFP show GFP localization at the nuclear envelope, consistent with their predicted function. NPP-1 interacts with the other nucleoporins in yeast two-hybrid assays suggesting that the proteins affect spindle orientation by a common process. The failed orientation phenotype of npp-1(RNAi) is at least partially epistatic to the ectopic spindle rotation in the AB blastomere of par-3 mutant embryos. This suggests that NPP-1 contributes to the mechanics of spindle orientation. However, NPP-1 is also required for PAR-6 asymmetry at the two-cell stage, indicating that nucleoporins may be required to define cortical domains in the germ line blasotmere P1. Nuclear envelope structure is abnormal in npp-1(RNAi) embryos but the envelope maintains its integrity and most nuclear proteins we assayed accumulate normally. These findings raise the possibility that these nucleoporins may have direct roles in orienting the mitotic spindle and the maintenance of cell polarity. PMID:16325795

Nuclear export of the small ribosomal subunit requires the Ran–GTPase cycle and certain nucleoporins

PubMed Central

Moy, Terence I.; Silver, Pamela A.

1999-01-01

After their assembly in the nucleolus, ribosomal subunits are exported from the nucleus to the cytoplasm. After export, the 20S rRNA in the small ribosomal subunit is cleaved to yield 18S rRNA and the small 5′ ITS1 fragment. The 5′ ITS1 RNA is normally degraded by the cytoplasmic Xrn1 exonuclease, but in strains lacking XRN1, the 5′ ITS1 fragment accumulates in the cytoplasm. Using the cytoplasmic localization of the 5′ ITS1 fragment as an indicator for the export of the small ribosomal subunit, we have identified genes that are required for ribosome export. Ribosome export is dependent on the Ran–GTPase as mutations in Ran or its regulators caused 5′ ITS1 to accumulate in the nucleoplasm. Mutations in the genes encoding the nucleoporin Nup82 and in the NES exporter Xpo1/Crm1 also caused the nucleoplasmic accumulation of 5′ ITS1. Mutants in a subset of nucleoporins and in the nuclear transport factors Srp1, Kap95, Pse1, Cse1, and Mtr10 accumulate the 5′ ITS1 in the nucleolus and affect ribosome assembly. In contrast, we did not detect nuclear accumulation of 5′ ITS1 in 28 yeast strains that have mutations in other genes affecting nuclear trafficking. PMID:10465789

Irradiation with heavy-ion particles changes the cellular distribution of human histone acetyltransferase HAT1.

PubMed

Lebel, Emily A; Boukamp, Petra; Tafrov, Stefan T

2010-06-01

Hat1 was the first histone acetyltransferase identified; however, its biological function is still unclear. In this report, it is shown for the first time that human Hat1 has two isoforms. Isoform a has 418 amino acids (aa) and is localized exclusively in the nuclear matrix of normal human keratinocytes (NHKs). Isoform b has 334 aa and is located in the cytoplasm, the nucleoplasm, attached to the chromatin and to the nuclear matrix. Immunohistochemical analyses revealed that the bulk of Hat1 is confined to the nucleus, with much lesser amounts in the cytoplasm. Cells undergoing mitotic division have an elevated amount of Hat1 compared to those that are non-mitotic. Senescent cells, however, exhibit a higher concentration of Hat1 in the cytoplasm compare to proliferating cells and the amount of Hat1 in the nucleus decreases with the progression of senescence. NHKs exposed to hydrogen peroxide (H(2)O(2)) or to a beam of high mass and energy ion particles displayed bright nuclear staining for Hat1, a phenotype that was not observed in NHKs exposed to gamma-rays. We established that the enhanced nuclear staining for Hat1 in response to these treatments is regulated by the PI3K and the mitogen-activated protein kinase signaling pathways. Our observations clearly implicate Hat1 in the cellular response assuring the survival of the treated cells.

Cytoplasmatic and Nuclear YAP1 and pYAP1 Staining in Urothelial Bladder Cancer.

PubMed

Latz, Stefan; Umbach, Tine; Goltz, Diane; Kristiansen, Glen; Müller, Stephan C; Ellinger, Jörg

2016-01-01

Yes-associated protein 1 (YAP1), the nuclear effector of the Hippo pathway, plays an important role in many tumor entities. We evaluated staining and clinical significance of YAP1 and phosphorylated YAP1 (pYAP1) in urothelial bladder cancer (BCA). We used a tissue micorarray with samples of patients with muscle-invasive bladder cancer (MIBC, n = 192), non-muscle-invasive bladder cancer (NMIBC, n = 192) and normal urothelial bladder tissue (CTRL, n = 38) to determine the immunhistochemical staining of YAP1 and pYAP1. Cytoplasmatic and nuclear levels were evaluated. The t test was used for comparative analysis. Overall survival and progression-free survival were evaluated by Kaplan-Meier estimates and the Cox proportional hazard regression model. Nuclear YAP1 as well as cytoplasmatic pYAP1 levels were higher in CTRL than in BCA, whereby both--NMIBC and MIBC--had lower levels than CTRL. Among patients with MIBC, cytoplasmatic YAP1 and pYAP1 staining decreased with advanced stage. YAP1 and pYAP1 staining did not correlate with the recurrence rate, progression-free, cancer-specific or overall survival. Immunhistochemical staining and subcellular localization of YAP1 and pYAP1 are different for BCA, NMIBC, MIBC and CTRL, indicating that the Hippo pathway is involved in urothelial carcinogenesis. © 2015 S. Karger AG, Basel.

NUP-1 Is a Large Coiled-Coil Nucleoskeletal Protein in Trypanosomes with Lamin-Like Functions

PubMed Central

DuBois, Kelly N.; Alsford, Sam; Holden, Jennifer M.; Buisson, Johanna; Swiderski, Michal; Bart, Jean-Mathieu; Ratushny, Alexander V.; Wan, Yakun; Bastin, Philippe; Barry, J. David; Navarro, Miguel; Horn, David; Aitchison, John D.; Rout, Michael P.; Field, Mark C.

2012-01-01

A unifying feature of eukaryotic nuclear organization is genome segregation into transcriptionally active euchromatin and transcriptionally repressed heterochromatin. In metazoa, lamin proteins preserve nuclear integrity and higher order heterochromatin organization at the nuclear periphery, but no non-metazoan lamin orthologues have been identified, despite the likely presence of nucleoskeletal elements in many lineages. This suggests a metazoan-specific origin for lamins, and therefore that distinct protein elements must compose the nucleoskeleton in other lineages. The trypanosomatids are highly divergent organisms and possess well-documented but remarkably distinct mechanisms for control of gene expression, including polycistronic transcription and trans-splicing. NUP-1 is a large protein localizing to the nuclear periphery of Trypanosoma brucei and a candidate nucleoskeletal component. We sought to determine if NUP-1 mediates heterochromatin organization and gene regulation at the nuclear periphery by examining the influence of NUP-1 knockdown on morphology, chromatin positioning, and transcription. We demonstrate that NUP-1 is essential and part of a stable network at the inner face of the trypanosome nuclear envelope, since knockdown cells have abnormally shaped nuclei with compromised structural integrity. NUP-1 knockdown also disrupts organization of nuclear pore complexes and chromosomes. Most significantly, we find that NUP-1 is required to maintain the silenced state of developmentally regulated genes at the nuclear periphery; NUP-1 knockdown results in highly specific mis-regulation of telomere-proximal silenced variant surface glycoprotein (VSG) expression sites and procyclin loci, indicating a disruption to normal chromatin organization essential to life-cycle progression. Further, NUP-1 depletion leads to increased VSG switching and therefore appears to have a role in control of antigenic variation. Thus, analogous to vertebrate lamins, NUP-1 is a major component of the nucleoskeleton with key roles in organization of the nuclear periphery, heterochromatin, and epigenetic control of developmentally regulated loci. PMID:22479148

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

Relativistically corrected nuclear magnetic resonance chemical shifts calculated with the normalized elimination of the small component using an effective potential-NMR chemical shifts of molybdenum and tungsten

NASA Astrophysics Data System (ADS)

Filatov, Michael; Cremer, Dieter

2003-07-01

A new method for relativistically corrected nuclear magnetic resonance (NMR) chemical shifts is developed by combining the individual gauge for the localized orbital approach for density functional theory with the normalized elimination of a small component using an effective potential. The new method is used for the calculation of the NMR chemical shifts of 95Mo and 183W in various molybdenum and tungsten compounds. It is shown that quasirelativistic corrections lead to an average improvement of calculated NMR chemical shift values by 300 and 120 ppm in the case of 95Mo and 183W, respectively, which is mainly due to improvements in the paramagnetic contributions. The relationship between electronic structure of a molecule and the relativistic paramagnetic corrections is discussed. Relativistic effects for the diamagnetic part of the magnetic shielding caused by a relativistic contraction of the s,p orbitals in the core region concern only the shielding values, however, have little consequence for the shift values because of the large independence from electronic structure and a cancellation of these effects in the shift values. It is shown that the relativistic corrections can be improved by level shift operators and a B3LYP hybrid functional, for which Hartree-Fock exchange is reduced to 15%.

Thyroid Hormone, Cancer, and Apoptosis.

PubMed

Lin, Hung-Yun; Chin, Yu-Tan; Yang, Yu-Chen S H; Lai, Husan-Yu; Wang-Peng, Jacqueline; Liu, Leory F; Tang, Heng-Yuan; Davis, Paul J

2016-06-13

Thyroid hormones play important roles in regulating normal metabolism, development, and growth. They also stimulate cancer cell proliferation. Their metabolic and developmental effects and growth effects in normal tissues are mediated primarily by nuclear hormone receptors. A cell surface receptor for the hormone on integrin [alpha]vβ3 is the initiation site for effects on tumor cells. Clinical hypothyroidism may retard cancer growth, and hyperthyroidism was recently linked to the prevalence of certain cancers. Local levels of thyroid hormones are controlled through activation and deactivation of iodothyronine deiodinases in different organs. The relative activities of different deiodinases that exist in tissues or organs also affect the progression and development of specific types of cancers. In this review, the effects of thyroid hormone on signaling pathways in breast, brain, liver, thyroid, and colon cancers are discussed. The importance of nuclear thyroid hormone receptor isoforms and of the hormone receptor on the extracellular domain of integrin [alpha]vβ3 as potential cancer risk factors and therapeutic targets are addressed. We analyze the intracellular signaling pathways activated by thyroid hormones in cancer progression in hyperthyroidism or at physiological concentrations in the euthyroid state. Determining how to utilize the deaminated thyroid hormone analog (tetrac), and its nanoparticulate derivative to reduce risks of cancer progression, enhance therapeutic outcomes, and prevent cancer recurrence is also deliberated. © 2016 American Physiological Society. Compr Physiol 6:1221-1237, 2016. Copyright © 2016 John Wiley & Sons, Inc.

Differential Expression and Immunolocalization of Antioxidant Enzymes in Entamoeba histolytica Isolates during Metronidazole Stress

PubMed Central

Iyer, Lakshmi Rani; Singh, Nishant; Verma, Anil Kumar; Paul, Jaishree

2014-01-01

Entamoeba histolytica infections are endemic in the Indian subcontinent. Five to eight percent of urban population residing under poor sanitary conditions suffers from Entamoeba infections. Metronidazole is the most widely prescribed drug used for amoebiasis. In order to understand the impact of metronidazole stress on the parasite, we evaluated the expression of two antioxidant enzymes, peroxiredoxin and FeSOD, in Entamoeba histolytica isolates during metronidazole stress. The results reveal that, under metronidazole stress, the mRNA expression levels of these enzymes did not undergo any significant change. Interestingly, immunolocalization studies with antibodies targeting peroxiredoxin indicate differential localization of the protein in the cell during metronidazole stress. In normal conditions, all the Entamoeba isolates exhibit presence of peroxiredoxin in the nucleus as well as in the membrane; however with metronidazole stress the protein localized mostly to the membrane. The change in the localization pattern was more pronounced when the cells were subjected to short term metronidazole stress compared to cells adapted to metronidazole. The protein localization to the cell membrane could be the stress response mechanism in these isolates. Colocalization pattern of peroxiredoxin with CaBp1, a cytosolic protein, revealed that the membrane and nuclear localization was specific to peroxiredoxin during metronidazole stress. PMID:25013795

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

PubMed Central

Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

1996-01-01

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 Fig. 7 Fig. 8 PMID:8632983

Par-4 dependent modulation of cellular β-catenin by medicinal plant natural product derivative 3-azido Withaferin A.

PubMed

Amin, Hina; Nayak, Debasis; Ur Rasool, Reyaz; Chakraborty, Souneek; Kumar, Anmol; Yousuf, Khalid; Sharma, Parduman Raj; Ahmed, Zabeer; Sharma, Neelam; Magotra, Asmita; Mukherjee, Debaraj; Kumar, Lekha Dinesh; Goswami, Anindya

2016-05-01

Here, we provide evidences that natural product derivative 3-azido Withaferin A (3-AWA) abrogated EMT and invasion by modulating β-catenin localization and its transcriptional activity in the prostate as well as in breast cancer cells. This study, for the first time, reveals 3-AWA treatment consistently sequestered nuclear β-catenin and augmented its cytoplasmic pool as evidenced by reducing β-catenin transcriptional activity in these cells. Moreover, 3-AWA treatment triggered robust induction of pro-apoptotic intracellular Par-4, attenuated Akt activity and rescued Phospho-GSK3β (by Akt) to promote β-catenin destabilization. Further, our in vitro studies demonstrate that 3-AWA treatment amplified E-cadherin expression along with sharp downregulation of c-Myc and cyclin D1 proteins. Strikingly, endogenous Par-4 knock down by siRNA underscored 3-AWA mediated inhibition of nuclear β-catenin was Par-4 dependent and suppression of Par-4 activity, either by Bcl-2 or by Ras transfection, restored the nuclear β-catenin level suggesting Par-4 mediated β-catenin regulation was not promiscuous. In vivo results further demonstrated that 3-AWA was effective inhibitor of tumor growth and immunohistochemical studies indicated that increased expression of total β-catenin and decreased expression of phospho-β-catenin and Par-4 in breast cancer tissues as compared to normal breast tissue suggesting Par-4 and β-catenin proteins are mutually regulated and inversely co-related in normal as well as cancer condition. Thus, strategic regulation of intracellular Par-4 by 3-AWA in diverse cancers could be an effective tool to control cancer cell metastasis. Conclusively, this report puts forward a novel approach of controlling deregulated β-catenin signaling by 3-AWA induced Par-4 protein. © 2015 Wiley Periodicals, Inc.

Nuclear distribution of the Trypanosoma cruzi RNA Pol I subunit RPA31 during growth and metacyclogenesis, and characterization of its nuclear localization signal.

PubMed

Canela-Pérez, Israel; López-Villaseñor, Imelda; Cevallos, Ana María; Hernández, Roberto

2018-03-01

Trypanosoma cruzi is the aetiologic agent of Chagas disease. Our research group studies ribosomal RNA (rRNA) gene transcription and nucleolus dynamics in this species of trypanosomes. RPA31 is an essential subunit of RNA polymerase I (Pol I) whose presence is apparently restricted to trypanosomes. Using fluorescent-tagged versions of this protein (TcRPA31-EGFP), we describe its nuclear distribution during growth and metacyclogenesis. Our findings indicate that TcRPA31-EGFP alters its nuclear presence from concentrated nucleolar localization in exponentially growing epimastigotes to a dispersed granular distribution in the nucleoplasm of stationary epimastigotes and metacyclic trypomastigotes. These changes likely reflect a structural redistribution of the Pol I transcription machinery in quiescent cellular stages where downregulation of rRNA synthesis is known to occur. In addition, and related to the nuclear internalization of this protein, the presence of a classical bipartite-type nuclear localization signal was identified towards its C-terminal end. The functionality of this motif was demonstrated by its partial or total deletion in recombinant versions of the tagged fluorescent protein. Moreover, ivermectin inhibited the nuclear localization of the labelled chimaera, suggesting the involvement of the importin α/β transport system.

NUCLEAR REACTOR CONTROL SYSTEM

DOEpatents

Epler, E.P.; Hanauer, S.H.; Oakes, L.C.

1959-11-01

A control system is described for a nuclear reactor using enriched uranium fuel of the type of the swimming pool and other heterogeneous nuclear reactors. Circuits are included for automatically removing and inserting the control rods during the course of normal operation. Appropriate safety circuits close down the nuclear reactor in the event of emergency.

Nuclear Import of the Retrotransposon Tf1 Is Governed by a Nuclear Localization Signal That Possesses a Unique Requirement for the FXFG Nuclear Pore Factor Nup124p

PubMed Central

Dang, Van-Dinh; Levin, Henry L.

2000-01-01

Retroviruses, such as human immunodeficiency virus, that infect nondividing cells generate integration precursors that must cross the nuclear envelope to reach the host genome. As a model for retroviruses, we investigated the nuclear entry of Tf1, a long-terminal-repeat-containing retrotransposon of the fission yeast Schizosaccharomyces pombe. Because the nuclear envelope of yeasts remains intact throughout the cell cycle, components of Tf1 must be transported through the envelope before integration can occur. The nuclear localization of the Gag protein of Tf1 is different from that of other proteins tested in that it has a specific requirement for the FXFG nuclear pore factor, Nup124p. Using extensive mutagenesis, we found that Gag contained three nuclear localization signals (NLSs) which, when included individually in a heterologous protein, were sufficient to direct nuclear import. In the context of the intact transposon, mutations in the NLS that mapped to the first 10 amino acid residues of Gag significantly impaired Tf1 retrotransposition and abolished nuclear localization of Gag. Interestingly, this NLS activity in the heterologous protein was specifically dependent upon the presence of Nup124p. Deletion analysis of heterologous proteins revealed the surprising result that the residues in Gag with the NLS activity were independent from the residues that conveyed the requirement for Nup124p. In fact, a fragment of Gag that lacked NLS activity, residues 10 to 30, when fused to a heterologous protein, was sufficient to cause the classical NLS of simian virus 40 to require Nup124p for nuclear import. Within the context of the current understanding of nuclear import, these results represent the novel case of a short amino acid sequence that specifies the need for a particular nuclear pore complex protein. PMID:11003674

Nuclear import of the retrotransposon Tf1 is governed by a nuclear localization signal that possesses a unique requirement for the FXFG nuclear pore factor Nup124p.

PubMed

Dang, V D; Levin, H L

2000-10-01

Retroviruses, such as human immunodeficiency virus, that infect nondividing cells generate integration precursors that must cross the nuclear envelope to reach the host genome. As a model for retroviruses, we investigated the nuclear entry of Tf1, a long-terminal-repeat-containing retrotransposon of the fission yeast Schizosaccharomyces pombe. Because the nuclear envelope of yeasts remains intact throughout the cell cycle, components of Tf1 must be transported through the envelope before integration can occur. The nuclear localization of the Gag protein of Tf1 is different from that of other proteins tested in that it has a specific requirement for the FXFG nuclear pore factor, Nup124p. Using extensive mutagenesis, we found that Gag contained three nuclear localization signals (NLSs) which, when included individually in a heterologous protein, were sufficient to direct nuclear import. In the context of the intact transposon, mutations in the NLS that mapped to the first 10 amino acid residues of Gag significantly impaired Tf1 retrotransposition and abolished nuclear localization of Gag. Interestingly, this NLS activity in the heterologous protein was specifically dependent upon the presence of Nup124p. Deletion analysis of heterologous proteins revealed the surprising result that the residues in Gag with the NLS activity were independent from the residues that conveyed the requirement for Nup124p. In fact, a fragment of Gag that lacked NLS activity, residues 10 to 30, when fused to a heterologous protein, was sufficient to cause the classical NLS of simian virus 40 to require Nup124p for nuclear import. Within the context of the current understanding of nuclear import, these results represent the novel case of a short amino acid sequence that specifies the need for a particular nuclear pore complex protein.

The Defective Nuclear Lamina in Hutchinson-Gilford Progeria Syndrome Disrupts the Nucleocytoplasmic Ran Gradient and Inhibits Nuclear Localization of Ubc9▿

PubMed Central

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.

2011-01-01

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. PMID:21670151

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.

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. © FASEB.

RECQL4-deficient cells are hypersensitive to oxidative stress/damage: Insights for osteosarcoma prevalence and heterogeneity in Rothmund-Thomson syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werner, Sean R.; Prahalad, Agasanur K.; Yang Jieping

2006-06-23

Rothmund-Thomson syndrome (RTS) is a heterogeneous disease, associated with increased prevalence of osteosarcoma in very young patients with a mutated RECQL4 gene. In this study, we tested the ability of RECQL4 deficient fibroblasts, derived from a RTS patient to recover from hydrogen peroxide (H{sub 2}O{sub 2})-induced oxidative stress/damage. Immunoperoxidase staining for 8-oxo-deoxyguanosine (8-oxo-dG) formation in RTS and normal human fibroblasts were compared to assess DNA damage. We determined DNA synthesis, cell growth, cell cycle distribution, and viability in RTS and normal human fibroblasts before and after H{sub 2}O{sub 2} treatment. H{sub 2}O{sub 2} induces 8-oxo-dG formation in both RTS andmore » normal fibroblasts. In normal human fibroblasts, RECQL4 was predominantly localized to cytoplasm; nuclear translocation and foci formation occurred in response to oxidant stimulation. After recovery from oxidant exposure, viable RTS fibroblasts showed irreversible growth arrest compared to normal fibroblasts. DNA synthesis decreased significantly in treated RTS cells, with concomitant reduction of cells in the S-phase. These results suggest that enhanced oxidant sensitivity in RECQL4 deficient fibroblasts derived from RTS patients could be attributed to abnormal DNA metabolism and proliferation failure. The ramifications of these findings on osteosarcoma prevalence and heterogeneity in RTS are discussed.« less

A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

PubMed

Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

2015-12-02

The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV. Copyright © 2015 Elsevier B.V. All rights reserved.

Piwi Nuclear Localization and Its Regulatory Mechanism in Drosophila Ovarian Somatic Cells.

PubMed

Yashiro, Ryu; Murota, Yukiko; Nishida, Kazumichi M; Yamashiro, Haruna; Fujii, Kaede; Ogai, Asuka; Yamanaka, Soichiro; Negishi, Lumi; Siomi, Haruhiko; Siomi, Mikiko C

2018-06-19

In Drosophila ovarian somatic cells (OSCs), Piwi represses transposons transcriptionally to maintain genome integrity. Piwi nuclear localization requires the N terminus and PIWI-interacting RNA (piRNA) loading of Piwi. However, the underlying mechanism remains unknown. Here, we show that Importinα (Impα) plays a pivotal role in Piwi nuclear localization and that Piwi has a bipartite nuclear localization signal (NLS). Impα2 and Impα3 are highly expressed in OSCs, whereas Impα1 is the least expressed. Loss of Impα2 or Impα3 forces Piwi to be cytoplasmic, which is rectified by overexpression of any Impα members. Extension of Piwi-NLS with an additional Piwi-NLS leads Piwi to be imported to the nucleus in a piRNA-independent manner, whereas replacement of Piwi-NLS with SV40-NLS fails. Limited proteolysis analysis suggests that piRNA loading onto Piwi triggers conformational change, exposing the N terminus to the environment. These results suggest that Piwi autoregulates its nuclear localization by exposing the NLS to Impα upon piRNA loading. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Antibody to the rheumatoid arthritis nuclear antigen. Its relationship to in vivo Epstein-Barr virus infection.

PubMed

Catalano, M A; Carson, D A; Niederman, J C; Feorino, P; Vaughan, J H

1980-05-01

Most patients with seropositive rheumatoid arthritis, and a variable but lesser percentage of normal subjects, have precipitating antibodies to a nuclear antigen, rheumatoid arthritis nuclear antigen, present in Epstein-Barr virus-infected human B lymphoblastoid cells. We have used a sensitive indirect immunofluorescence assay for antibody to rheumatoid arthritis nuclear antigen in a study of patients with infectious mononucleosis and healthy control subjects. Of 110 sera from normal, college-age cadets, 58 were from individuals without prior Epstein-Barr virus infection, as indicated by the lack of antibody to viral capsid antigen. All of these also lacked activity to rheumatoid arthritis nuclear antigen. 52 sera were positive for antibody to viral capsid antigen, and antibody to rheumatoid arthritis nuclear antigen was present in 26 (50%) of these. In 67 sequential sera from 11 college-age students with infectious mononucleosis who became positive for antibody to rheumatoid arthritis nuclear antigen, only 2 were positive during the 1 mo. Thereafter the incidence and titers increased progressively through the 1st yr after infection. This time-course resembled that for the development of antibody to Epstein-Barr nuclear antigen, another transformation antigen in Epstein-Barr virus-infected B lymphocytes. The development of positivity for both was much later than that of antibody to the structural viral capsid antigen, which in the current study was always positive by 1 wk. Thus, antibody to rheumatoid arthritis nuclear antigen is present in a large proportion of normal individuals and can now be clearly ascribed, from both in vivo and in vitro studies, to prior infection with Epstein-Barr virus.

Large-Scale Paraphrasing for Natural Language Understanding

DTIC Science & Technology

2018-04-01

to manufacture , use, or sell any patented invention that may relate to them. This report is the result of contracted fundamental research deemed...station contaminated local fish populations Atomic power generation in Springfield polluted indigenous seafood stocks Radioactive power generation...from PPDB. Springfield’s nuclear power plant contaminated local fish populations nuclear power station nuclear plant power plant fish stocks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Piccioli, Zachary; McKee, Courtney H.; Leszczynski, Anna

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 bymore » 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.« less

Tau regulates the subcellular localization of calmodulin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barreda, Elena Gomez de; Avila, Jesus, E-mail: javila@cbm.uam.es; CIBER de Enfermedades Neurodegenerativas, 28031 Madrid

Highlights: {yields} In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in amore » change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.« less

Nuclear Lipids in the Nervous System: What they do in Health and Disease.

PubMed

Garcia-Gil, Mercedes; Albi, Elisabetta

2017-02-01

In the last 20 years it has been widely demonstrated that cell nucleus contains neutral and polar lipids localized in nuclear membranes, nucleoli, nuclear matrix and chromatin. Nuclear lipids may show specific organization forming nuclear lipid microdomains and have both structural and functional roles. Depending on their localization, nuclear lipids play different roles such as the regulation of nuclear membrane and nuclear matrix fluidity but they also can act as platforms for vitamin and hormone function, for active chromatin anchoring, and for the regulation of gene expression, DNA duplication and transcription. Crosstalk among different kinds of lipid signalling pathways influence the physiopathology of numerous cell types. In neural cells the nuclear lipids are involved in cell proliferation, differentiation, inflammation, migration and apoptosis. Abnormal metabolism of nuclear lipids might be closely associated with tumorigenesis and neurodegenerative diseases such as Alzheimer disease and Parkinson disease among others.

Roles of brca2 (fancd1) in oocyte nuclear architecture, gametogenesis, gonad tumors, and genome stability in zebrafish.

PubMed

Rodríguez-Marí, Adriana; Wilson, Catherine; Titus, Tom A; Cañestro, Cristian; BreMiller, Ruth A; Yan, Yi-Lin; Nanda, Indrajit; Johnston, Adam; Kanki, John P; Gray, Erin M; He, Xinjun; Spitsbergen, Jan; Schindler, Detlev; Postlethwait, John H

2011-03-01

Mild mutations in BRCA2 (FANCD1) cause Fanconi anemia (FA) when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd)-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53) rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture.

Roles of brca2 (fancd1) in Oocyte Nuclear Architecture, Gametogenesis, Gonad Tumors, and Genome Stability in Zebrafish

PubMed Central

Rodríguez-Marí, Adriana; Wilson, Catherine; Titus, Tom A.; Cañestro, Cristian; BreMiller, Ruth A.; Yan, Yi-Lin; Nanda, Indrajit; Johnston, Adam; Kanki, John P.; Gray, Erin M.; He, Xinjun; Spitsbergen, Jan; Schindler, Detlev; Postlethwait, John H.

2011-01-01

Mild mutations in BRCA2 (FANCD1) cause Fanconi anemia (FA) when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd)-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53) rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture. PMID:21483806

The Selenocysteine-Specific Elongation Factor Contains Unique Sequences That Are Required for Both Nuclear Export and Selenocysteine Incorporation.

PubMed

Dubey, Aditi; Copeland, Paul R

2016-01-01

Selenocysteine (Sec) is a critical residue in at least 25 human proteins that are essential for antioxidant defense and redox signaling in cells. Sec is inserted into proteins cotranslationally by the recoding of an in-frame UGA termination codon to a Sec codon. In eukaryotes, this recoding event requires several specialized factors, including a dedicated, Sec-specific elongation factor called eEFSec, which binds Sec-tRNASec with high specificity and delivers it to the ribosome for selenoprotein production. Unlike most translation factors, including the canonical elongation factor eEF1A, eEFSec readily localizes to the nucleus of mammalian cells and shuttles between the cytoplasmic and nuclear compartments. The functional significance of eEFSec's nuclear localization has remained unclear. In this study, we have examined the subcellular localization of eEFSec in the context of altered Sec incorporation to demonstrate that reduced selenoprotein production does not correlate with changes in the nuclear localization of eEFSec. In addition, we identify several novel sequences of the protein that are essential for localization as well as Sec insertion activity, and show that eEFSec utilizes CRM1-mediated nuclear export pathway. Our findings argue for two distinct pools of eEFSec in the cell, where the cytoplasmic pool participates in Sec incorporation and the nuclear pool may be involved in an as yet unknown function.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sekhri, Palak; Tao, Tao; Kaplan, Feige

As the sole E2 enzyme for SUMOylation, Ubc9 is predominantly nuclear. However, the underlying mechanisms of Ubc9 nuclear localization are still not well understood. Here we show that RNAi-depletion of Imp13, an importin known to mediate Ubc9 nuclear import, reduces both Ubc9 nuclear accumulation and global SUMOylation. Furthermore, Ubc9-R13A or Ubc9-H20D mutation previously shown to interrupt the interaction of Ubc9 with nucleus-enriched SUMOs reduces the nuclear enrichment of Ubc9, suggesting that the interaction of Ubc9 with the nuclear SUMOs may enhance Ubc9 nuclear retention. Moreover, Ubc9-R17E mutation, which is known to disrupt the interaction of Ubc9 with both SUMOs andmore » Imp13, causes a greater decrease in Ubc9 nuclear accumulation than Ubc9-R13A or Ubc9-H20D mutation. Lastly, Ubc9-K74A/S89D mutations that perturb the interaction of Ubc9 with nucleus-enriched SUMOylation-consensus motifs has no effect on Ubc9 nuclear localization. Altogether, our results have elucidated that the amino acid residues within the N-terminal region of Ubc9 play a pivotal role in regulation of Ubc9 nuclear localization. - Highlights: • Imp13-mediated nuclear import of Ubc9 is critical for global SUMOylation. • Ubc9 mutations disrupting Ubc9-SUMO interaction decrease Ubc9 nuclear accumulation. • N-terminal amino acid residues of Ubc9 are critical for Ubc9 nuclear enrichment.« less

Tale taming radioactive fears: Linking nuclear waste disposal to the "continuum of the good".

PubMed

Yli-Kauhaluoma, Sari; Hänninen, Hannu

2014-04-01

We examine how the constructor of the world's first repository for the final disposal of spent nuclear fuel in Eurajoki, Finland, aims to shape lay understanding of the facility's risks and to tame the nuclear fears of the local community by producing positive associations, imagery and tales. Our empirical material consists of the constructor's newsletters targeted mainly at the local residents. In the narrative analysis, we identified a storyline where the construction of the repository is linked into the "continuum of the good" in the municipality of the construction site and the surrounding areas. The storyline consists of five different themes all emphasizing the "continuum of the good" in the area: cultural heritage, well-being, developing expertise, natural environment, and local families. Our study contributes to the literature on pro-nuclear storytelling by showing how the inclination is towards narratives that are constructed around local symbols, cultural landmarks, and institutions.

Nuclear location of a chromatin insulator in Drosophila melanogaster.

PubMed

Xu, Qinghao; Li, Mo; Adams, Jessica; Cai, Haini N

2004-03-01

Chromatin-related functions are associated with spatial organization in the nucleus. We have investigated the relationship between the enhancer-blocking activity and subnuclear localization of the Drosophila melanogaster suHw insulator. Using fluorescent in situ hybridization, we observed that genomic loci containing the gypsy retrotransposon were distributed closer to the nuclear periphery than regions without the gypsy retrotransposon. However, transgenes containing a functional 340 bp suHw insulator did not exhibit such biased distribution towards the nuclear periphery, which suggests that the suHw insulator sequence is not responsible for the peripheral localization of the gypsy retrotransposon. Antibody stains showed that the two proteins essential for the suHw insulator activity, SUHW and MOD(MDG4), are not restricted to the nuclear periphery. The enhancer-blocking activity of suHw remained intact under the heat shock conditions, which was shown to disrupt the association of gypsy, SUHW and MOD(MDG4) with the nuclear periphery. Our results indicate that the suHw insulator can function in the nuclear interior, possibly through local interactions with chromatin components or other nuclear structures.

Nuclear phenomena in low-energy nuclear reaction research.

PubMed

Krivit, Steven B

2013-09-01

This is a comment on Storms E (2010) Status of Cold Fusion, Naturwissenschaften 97:861-881. This comment provides the following remarks to other nuclear phenomena observed in low-energy nuclear reactions aside from helium-4 make significant contributions to the overall energy balance; and normal hydrogen, not just heavy hydrogen, produces excess heat.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cassell, Geoffrey D.; Weitzman, Matthew D.

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}more » 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.« less

Nuclear localization of coactivator RAC3 is mediated by a bipartite NLS and importin {alpha}3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeung, Percy Luk; Zhang, Aihua; Chen, J. Don

2006-09-15

The nuclear receptor coactivator RAC3 (also known as SRC-3/ACTR/AIB1/p/CIP/TRAM-1) belongs to the p160 coactivator family, which are involved in several physiological processes and diseases. Here we have investigated how RAC3 is translocated into the nucleus and show that it is mediated through a bipartite NLS and importin {alpha}3. This bipartite NLS is located within the conserved bHLH domain, and its mutation abolished nuclear localization. The NLS is also sufficient to cause nuclear import of EGFP, and the activity requires basic amino acids within the NLS. RAC3 binds strongly to importin {alpha}3, which also depends on the basic amino acids. Functionally,more » RAC3 cytoplasmic mutant loses its ability to enhance transcription, suggesting that nuclear localization is essential for coactivator function. Together, these results reveal a previous unknown mechanism for nuclear translocation of p160 coactivators and a critical function of the conserved bHLH within the coactivator.« less

Unusual broad-line Mg II emitters among luminous galaxies in the baryon oscillation spectroscopic survey

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roig, Benjamin; Blanton, Michael R.; Ross, Nicholas P.

2014-02-01

Many classes of active galactic nuclei (AGNs) have been observed and recorded since the discovery of Seyfert galaxies. In this paper, we examine the sample of luminous galaxies in the Baryon Oscillation Spectroscopic Survey. We find a potentially new observational class of AGNs, one with strong and broad Mg II λ2799 line emission, but very weak emission in other normal indicators of AGN activity, such as the broad-line Hα, Hβ, and the near-ultraviolet AGN continuum, leading to an extreme ratio of broad Hα/Mg II flux relative to normal quasars. Meanwhile, these objects' narrow-line flux ratios reveal AGN narrow-line regions withmore » levels of activity consistent with the Mg II fluxes and in agreement with that of normal quasars. These AGN may represent an extreme case of the Baldwin effect, with very low continuum and high equivalent width relative to typical quasars, but their ratio of broad Mg II to broad Balmer emission remains very unusual. They may also be representative of a class of AGN where the central engine is observed indirectly with scattered light. These galaxies represent a small fraction of the total population of luminous galaxies (≅ 0.1%), but are more likely (about 3.5 times) to have AGN-like nuclear line emission properties than other luminous galaxies. Because Mg II is usually inaccessible for the population of nearby galaxies, there may exist a related population of broad-line Mg II emitters in the local universe which is currently classified as narrow-line emitters (Seyfert 2 galaxies) or low ionization nuclear emission-line regions.« less

Myocardial regeneration in adriamycin cardiomyopathy by nuclear expression of GLP1 using ultrasound targeted microbubble destruction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Shuyuan; Chen, Jiaxi; Huang, Pintong

Recently GLP-1 was found to have cardioprotective effects independent of those attributable to tight glycemic control. Methods and results: We employed ultrasound targeted microbubble destruction (UTMD) to deliver piggybac transposon plasmids encoding the GLP-1 gene with a nuclear localizing signal to rat hearts with adriamycin cardiomyopathy. After a single UTMD treatment, overexpression of transgenic GLP-1 was found in nuclei of rat heart cells with evidence that transfected cardiac cells had undergone proliferation. UTMD-GLP-1 gene therapy restored LV mass, fractional shortening index, and LV posterior wall diameter to nearly normal. Nuclear overexpression of GLP-1 by inducing phosphorylation of FoxO1-S256 and translocationmore » of FoxO1 from the nucleus to the cytoplasm significantly inactivated FoxO1 and activated the expression of cyclin D1 in nuclei of cardiac muscle cells. Reversal of adriamycin cardiomyopathy appeared to be mediated by dedifferentiation and proliferation of nuclear FoxO1-positive cardiac muscle cells with evidence of embryonic stem cell markers (OCT4, Nanog, SOX2 and c-kit), cardiac early differentiation markers (NKX2.5 and ISL-1) and cellular proliferation markers (BrdU and PHH3) after UTMD with GLP-1 gene therapy. Conclusions: Intranuclear myocardial delivery of the GLP-1gene can reverse established adriamycin cardiomyopathy by stimulating myocardial regeneration. - Highlights: • The activation of nuclear FoxO1 in cardiac muscle cells associated with adriamycin cardiomyopathy. • Myocardial nuclear GLP-1 stimulates myocardial regeneration and reverses adriamycin cardiomyopathy. • The process of myocardial regeneration associated with dedifferentiation and proliferation.« less

The role of myosin II in glioma invasion: A mathematical model

PubMed Central

Lee, Wanho; Lim, Sookkyung; Kim, Yangjin

2017-01-01

Gliomas are malignant tumors that are commonly observed in primary brain cancer. Glioma cells migrate through a dense network of normal cells in microenvironment and spread long distances within brain. In this paper we present a two-dimensional multiscale model in which a glioma cell is surrounded by normal cells and its migration is controlled by cell-mechanical components in the microenvironment via the regulation of myosin II in response to chemoattractants. Our simulation results show that the myosin II plays a key role in the deformation of the cell nucleus as the glioma cell passes through the narrow intercellular space smaller than its nuclear diameter. We also demonstrate that the coordination of biochemical and mechanical components within the cell enables a glioma cell to take the mode of amoeboid migration. This study sheds lights on the understanding of glioma infiltration through the narrow intercellular spaces and may provide a potential approach for the development of anti-invasion strategies via the injection of chemoattractants for localization. PMID:28166231

Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function

PubMed Central

Lerit, Dorothy A.; Jordan, Holly A.; Poulton, John S.; Fagerstrom, Carey J.; Galletta, Brian J.; Peifer, Mark

2015-01-01

Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle–dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability. PMID:26150390

Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function.

PubMed

Lerit, Dorothy A; Jordan, Holly A; Poulton, John S; Fagerstrom, Carey J; Galletta, Brian J; Peifer, Mark; Rusan, Nasser M

2015-07-06

Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle-dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability.

Microscopic investigation of the weakly correlated noncentrosymmetric superconductor SrAuSi3

NASA Astrophysics Data System (ADS)

Barbero, N.; Biswas, P. K.; Isobe, M.; Amato, A.; Morenzoni, E.; Hillier, A. D.; Ott, H.-R.; Mesot, J.; Shiroka, T.

2018-01-01

SrAuSi3 is a noncentrosymmetric superconductor (NCS) with Tc=1.54 K, which to date has been studied only via macroscopic techniques. By combining nuclear-magnetic-resonance and muon-spin-rotation measurements, we investigate both the normal and the superconducting phase of SrAuSi3 at a local level. In the normal phase, our data indicate a standard metallic behavior with weak electron correlations and a Korringa constant Sexp=1.31 ×10-5 sK. The latter, twice the theoretical value, can be justified by the Moriya theory of exchange enhancement. In the superconducting phase, the material exhibits conventional BCS-type superconductivity with a weak-coupling s -wave pairing, a gap value Δ (0 )=0.213 (2 ) meV, and a magnetic penetration depth λ (0 )=398 (2 ) nm. The experimental proof of weak correlations in SrAuSi3 implies that correlation effects can be decoupled from those of antisymmetric spin-orbit coupling, thus enabling accurate band-structure calculations in the weakly correlated NCSs.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 situatedmore » 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.« less

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

Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene.

PubMed

Moon, Dong Chan; Choi, Chul Hee; Lee, Su Man; Lee, Jung Hwa; Kim, Seung Il; Kim, Dong Sun; Lee, Je Chul

2012-01-01

Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), (225)RKRKRK(230). Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.

Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha

2014-07-18

Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated thatmore » TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations.« less

COP1 is required for UV-B–induced nuclear accumulation of the UVR8 photoreceptor

PubMed Central

Skvortsova, Mariya Y.; Loubéry, Sylvain

2016-01-01

The UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) promotes UV-B acclimation and tolerance in Arabidopsis thaliana. UVR8 localizes to both cytosol and nucleus, but its main activity is assumed to be nuclear. UV-B photoreception stimulates nuclear accumulation of UVR8 in a presently unknown manner. Here, we show that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is required for UV-B–induced nuclear accumulation of UVR8, but bypassing the COP1 requirement for UVR8 nuclear accumulation did not rescue the cop1 mutant UV-B phenotype. Using a glucocorticoid receptor (GR)-based fusion protein system to conditionally localize GR-UVR8 to the nucleus, we have demonstrated that both photoactivation and nuclear localization of UVR8 are required for UV-B–induced photomorphogenic responses. In contrast, there was no UV-B response when UV-B–activated UVR8 was artificially retained in the cytosol. In agreement with a predominantly nuclear activity, constitutively active UVR8W285A accumulated in the nucleus also in the absence of UV-B. Furthermore, GR-COP1 expression lines suggested that UV-B–activated UVR8 can be coimported into the nucleus by COP1. Our data strongly support localization of UVR8 signaling in the nucleus and a dual role for COP1 in the regulation of UV-B–induced UVR8 nuclear accumulation and in UVR8-mediated UV-B signaling. PMID:27407149

Local vibrational modes of the water dimer - Comparison of theory and experiment

NASA Astrophysics Data System (ADS)

Kalescky, R.; Zou, W.; Kraka, E.; Cremer, D.

2012-12-01

Local and normal vibrational modes of the water dimer are calculated at the CCSD(T)/CBS level of theory. The local H-bond stretching frequency is 528 cm-1 compared to a normal mode stretching frequency of just 143 cm-1. The adiabatic connection scheme between local and normal vibrational modes reveals that the lowering is due to mass coupling, a change in the anharmonicity, and coupling with the local HOH bending modes. The local mode stretching force constant is related to the strength of the H-bond whereas the normal mode stretching force constant and frequency lead to an erroneous underestimation of the H-bond strength.

The Human Polycomb Group Complex Associates with Pericentromeric Heterochromatin to Form a Novel Nuclear Domain

PubMed Central

Saurin, Andrew J.; Shiels, Carol; Williamson, Jill; Satijn, David P.E.; Otte, Arie P.; Sheer, Denise; Freemont, Paul S.

1998-01-01

The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila. Recently, a mammalian PcG complex has been identified with several PcG proteins implicated in the regulation of Hox gene expression. Although the mammalian PcG complex appears analogous to the complex in Drosophila, the molecular mechanisms and functions for the mammalian PcG complex remain unknown. Here we describe a detailed characterization of the human PcG complex in terms of cellular localization and chromosomal association. By using antibodies that specifically recognize three human PcG proteins— RING1, BMI1, and hPc2—we demonstrate in a number of human cell lines that the PcG complex forms a unique discrete nuclear structure that we term PcG bodies. PcG bodies are prominent novel nuclear structures with the larger PcG foci generally localized near the centromeres, as visualized with a kinetochore antibody marker. In both normal fetal and adult fibroblasts, PcG bodies are not randomly dispersed, but appear clustered into defined areas within the nucleus. We show in three different human cell lines that the PcG complex can tightly associate with large pericentromeric heterochromatin regions (1q12) on chromosome 1, and with related pericentromeric sequences on different chromosomes, providing evidence for a mammalian PcG–heterochromatin association. Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions. We discuss these results in terms of the known function of the PcG complex as a transcriptional repression complex. PMID:9722603

Accumulation of the sigma-1 receptor is common to neuronal nuclear inclusions in various neurodegenerative diseases.

PubMed

Miki, Yasuo; Mori, Fumiaki; Kon, Tomoya; Tanji, Kunikazu; Toyoshima, Yasuko; Yoshida, Mari; Sasaki, Hidenao; Kakita, Akiyoshi; Takahashi, Hitoshi; Wakabayashi, Koichi

2014-04-01

The sigma-1 receptor (SIGMAR1) is now known to be one of the endoplasmic reticulum (ER) chaperones, which participate in the degradation of misfolded proteins in cells via the ER-related degradation machinery linked to the ubiquitin-proteasome pathway. Mutations of the SIGMAR1 gene are implicated in the pathogenesis of familial frontotemporal lobar degeneration and motor neuron disease. Involvement of ER dysfunction in the formation of inclusion bodies in various neurodegenerative diseases has also become evident. We performed immunohistochemical staining to clarify the localization of SIGMAR1 in the brains of patients with neurodegenerative disorders, including trans-activation response DNA protein 43 (TDP-43) proteinopathy, tauopathy, α-synucleinopathy, polyglutamine disease and intranuclear inclusion body disease (INIBD). Double-immunocytofluorescence and Western blot analyses of cultured cells were also performed to investigate the role of SIGMAR1 using a specific exportin 1 inhibitor, leptomycin B and an ER stress inducer, thapsigargin. SIGMAR1 was consistently shown to be co-localized with neuronal nuclear inclusions in TDP-43 proteinopathy, five polyglutamine diseases and INIBD, as well as in intranuclear Marinesco bodies in aged normal controls. Cytoplasmic inclusions in neurons and glial cells were unreactive for SIGMAR1. In cultured cells, immunocytofluorescent study showed that leptomycin B and thapsigargin were shown to sequester SIGMAR1 within the nucleus, acting together with p62. This finding was also supported by immunoblot analysis. These results indicate that SIGMAR1 might shuttle between the nucleus and the cytoplasm. Neurodegenerative diseases characterized by neuronal nuclear inclusions might utilize the ER-related degradation machinery as a common pathway for the degradation of aberrant proteins. © 2013 Japanese Society of Neuropathology.

Nuclear Matrix Proteins in Disparity of Prostate Cancer

DTIC Science & Technology

2013-09-01

nuclear coactivator-3 (NCOA3). 5 Methods Patients and Prostate Cancer Specimens Fresh, flash -frozen specimens were obtained from age- (50 to...for reliable data interpretation. Gene Array Analysis Total RNA isolated from LCM-procured normal epithelium and tumor cells from flash -frozen...PCR Briefly, RNA was extracted from matched LCM procured normal epithelium and tumor cells of age-, tumor grade-matched flash -frozen sections (n=24

Nuclear Power Plant environment`s surveillance by satellite remote sensing and in-situ monitoring data

NASA Astrophysics Data System (ADS)

Zoran, Maria

The main environmental issues affecting the broad acceptability of nuclear power plant are the emission of radioactive materials, the generation of radioactive waste, and the potential for nuclear accidents. All nuclear fission reactors, regardless of design, location, operator or regulator, have the potential to undergo catastrophic accidents involving loss of control of the reactor core, failure of safety systems and subsequent widespread fallout of hazardous fission products. Risk is the mathematical product of probability and consequences, so lowprobability and high-consequence accidents, by definition, have a high risk. NPP environment surveillance is a very important task in frame of risk assessment. Satellite remote sensing data had been applied for dosimeter levels first time for Chernobyl NPP accident in 1986. Just for a normal functioning of a nuclear power plant, multitemporal and multispectral satellite data in complementarily with field data are very useful tools for NPP environment surveillance and risk assessment. Satellite remote sensing is used as an important technology to help environmental research to support research analysis of spatio-temporal dynamics of environmental features nearby nuclear facilities. Digital processing techniques applied to several LANDSAT, MODIS and QuickBird data in synergy with in-situ data are used to assess the extent and magnitude of radiation and non-radiation effects on the water, near field soil, vegetation and air. As a test case the methodology was applied for for Nuclear Power Plant (NPP) Cernavoda, Romania. Thermal discharge from nuclear reactors cooling is dissipated as waste heat in Danube-Black -Sea Canal and Danube River. Water temperatures captured in thermal IR imagery are correlated with meteorological parameters. If during the winter thermal plume is localized to an area of a few km of NPP, the temperature difference between the plume and non-plume areas being about 1.5 oC, during summer and fall , is a larger thermal plume up to 5-6 km far along Danube Black Sea Canal ,the temperature change is about 1.0 oC.

Nuclear-specific AR-V7 Protein Localization is Necessary to Guide Treatment Selection in Metastatic Castration-resistant Prostate Cancer.

PubMed

Scher, Howard I; Graf, Ryon P; Schreiber, Nicole A; McLaughlin, Brigit; Lu, David; Louw, Jessica; Danila, Daniel C; Dugan, Lyndsey; Johnson, Ann; Heller, Glenn; Fleisher, Martin; Dittamore, Ryan

2017-06-01

Circulating tumor cells (CTCs) expressing AR-V7 protein localized to the nucleus (nuclear-specific) identify metastatic castration-resistant prostate cancer (mCRPC) patients with improved overall survival (OS) on taxane therapy relative to the androgen receptor signaling inhibitors (ARSi) abiraterone acetate, enzalutamide, and apalutamide. To evaluate if expanding the positivity criteria to include both nuclear and cytoplasmic AR-V7 localization ("nuclear-agnostic") identifies more patients who would benefit from a taxane over an ARSi. The study used a cross-sectional cohort. Between December 2012 and March 2015, 193 pretherapy blood samples, 191 of which were evaluable, were collected and processed from 161 unique mCRPC patients before starting a new line of systemic therapy for disease progression at the Memorial Sloan Kettering Cancer Center. The association between two AR-V7 scoring criteria, post-therapy prostate-specific antigen (PSA) change (PTPC) and OS following ARSi or taxane treatment, was explored. One criterion required nuclear-specific AR-V7 localization, and the other required an AR-V7 signal but was agnostic to protein localization in CTCs. Correlation of AR-V7 status to PTPC and OS was investigated. Relationships with survival were analyzed using multivariable Cox regression and log-rank analyses. A total of 34 (18%) samples were AR-V7-positive using nuclear-specific criteria, and 56 (29%) were AR-V7-positive using nuclear-agnostic criteria. Following ARSi treatment, none of the 16 nuclear-specific AR-V7-positive samples and six of the 32 (19%) nuclear-agnostic AR-V7-positive samples had ≥50% PTPC at 12 weeks. The strongest baseline factor influencing OS was the interaction between the presence of nuclear-specific AR-V7-positive CTCs and treatment with a taxane (hazard ratio 0.24, 95% confidence interval 0.078-0.79; p=0.019). This interaction was not significant when nuclear-agnostic criteria were used. To reliably inform treatment selection using an AR-V7 protein biomarker in CTCs, nuclear-specific localization is required. We analyzed outcomes for patients with metastatic castration-resistant prostate cancer on androgen receptor signaling inhibitors and standard chemotherapy. Patients with circulating tumor cells that had AR-V7 protein in the cellular nuclei were very likely to survive longer on taxane-based chemotherapy, and tests unable to distinguish where the protein is located in the cell are not as predictive of benefit. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Liquid uranium alloy-helium fission reactor

DOEpatents

Minkov, Vladimir

1986-01-01

This invention teaches a nuclear fission reactor having a core vessel and at least one tandem heat exchanger vessel coupled therewith across upper and lower passages to define a closed flow loop. Nuclear fuel such as a uranium alloy in its liquid phase fills these vessels and flow passages. Solid control elements in the reactor core vessel are adapted to be adjusted relative to one another to control fission reaction of the liquid fuel therein. Moderator elements in the other vessel and flow passages preclude fission reaction therein. An inert gas such as helium is bubbled upwardly through the heat exchanger vessel operable to move the liquid fuel upwardly therein and unidirectionally around the closed loop and downwardly through the core vessel. This helium gas is further directed to heat conversion means outside of the reactor vessels to utilize the heat from the fission reaction to generate useful output. The nuclear fuel operates in the 1200.degree.-1800.degree. C. range, and even higher to 2500.degree. C., limited only by the thermal effectiveness of the structural materials, increasing the efficiency of power generation from the normal 30-35% with 300.degree.-500.degree. C. upper limit temperature to 50-65%. Irradiation of the circulating liquid fuel, as contrasted to only localized irradiation of a solid fuel, provides improved fuel utilization.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th

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 ofmore » 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.« less

The relationship of the oestrogen and progestin receptors in the abnormal uterus of the adult anovulatory rat. Effects of neonatal treatment with testosterone propionate or clomiphene citrate.

PubMed Central

White, J O; Moore, P A; Elder, M G; Lim, L

1981-01-01

The neonatal administration of testosterone propionate to Wistar rats resulted in anovulatory adults in persistent vaginal oestrus. Clomiphene citrate had a similar effect. In both groups of adults, hyperplasia of the uterine epithelium and occasional metaplasia was observed. The uterine nuclear and cytosol oestrogen and progestin receptors of these anovulatory rats were found to have affinities for their respective ligands similar to those of normal females. The nuclear oestrogen receptor comprised occupied and unoccupied components, as in normal females. The content of the nuclear oestrogen receptor was comparable with that of females in the late dioestrous or pro-oestrous phase. This content was higher in the clomiphene-treated group. Despite the relatively high nuclear oestrogen receptor content the content of progestin receptors, a putative index of the oestrogenic response, was lower in the treated rats than in normal adult females throughout the cycle. Administration of oestradiol to both treatment groups resulted in depletion of cytosol oestrogen receptor content 1 h later, which, however, was not reflected by an increase in the content of nuclear oestrogen receptors. There was no measurable increase in progesterone receptor content in treated rats after daily administration of oestrogen (5 microgram/rat) for 3 days. These changes in sex-hormone-receptor interactions involving an impairment of the normal oestrogenic response may be associated with the abnormal differentiation of the uterus in these sterile, anovulatory animals. Images Fig. 1. Fig. 2. PMID:7316994

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.

Investigating resonances above and below the threshold in nuclear reactions of astrophysical interest and beyond

DOE Office of Scientific and Technical Information (OSTI.GOV)

La Cognata, M., E-mail: lacognata@lns.infn.it; Kiss, G. G.; Mukhamedzhanov, A. M.

2015-10-15

Resonances in nuclear cross sections dramatically change their trends. Therefore, the presence of unexpected resonances might lead to unpredicted consequences on astrophysics and nuclear physics. In nuclear physics, resonances allow one to study states in the intermediate compound systems, to evaluate their cluster structure, for instance, especially in the energy regions approaching particle decay thresholds. In astrophysics, resonances might lead to changes in the nucleosynthesis flow, determining different isotopic compositions of the nuclear burning ashes. For these reasons, the Trojan Horse method has been modified to investigate resonant reactions. Thanks to this novel approach, for the first time normalization tomore » direct data might be avoided. Moreover, in the case of sub threshold resonances, the Trojan Horse method modified to investigate resonances allows one to deduce the asymptotic normalization coefficient, showing the close connection between the two indirect approaches.« less

Acute Zonal Cone Photoreceptor Outer Segment Loss

PubMed Central

Sandhu, Harpal S.; Serrano, Leona W.; Traband, Anastasia; Lau, Marisa K.; Adamus, Grazyna; Avery, Robert A.

2017-01-01

Importance The diagnostic path presented narrows down the cause of acute vision loss to the cone photoreceptor outer segment and will refocus the search for the cause of similar currently idiopathic conditions. Objective To describe the structural and functional associations found in a patient with acute zonal occult photoreceptor loss. Design, Setting, and Participants A case report of an adolescent boy with acute visual field loss despite a normal fundus examination performed at a university teaching hospital. Main Outcomes and Measures Results of a complete ophthalmic examination, full-field flash electroretinography (ERG) and multifocal ERG, light-adapted achromatic and 2-color dark-adapted perimetry, and microperimetry. Imaging was performed with spectral-domain optical coherence tomography (SD-OCT), near-infrared (NIR) and short-wavelength (SW) fundus autofluorescence (FAF), and NIR reflectance (REF). Results The patient was evaluated within a week of the onset of a scotoma in the nasal field of his left eye. Visual acuity was 20/20 OU, and color vision was normal in both eyes. Results of the fundus examination and of SW-FAF and NIR-FAF imaging were normal in both eyes, whereas NIR-REF imaging showed a region of hyporeflectance temporal to the fovea that corresponded with a dense relative scotoma noted on light-adapted static perimetry in the left eye. Loss in the photoreceptor outer segment detected by SD-OCT co-localized with an area of dense cone dysfunction detected on light-adapted perimetry and multifocal ERG but with near-normal rod-mediated vision according to results of 2-color dark-adapted perimetry. Full-field flash ERG findings were normal in both eyes. The outer nuclear layer and inner retinal thicknesses were normal. Conclusions and Relevance Localized, isolated cone dysfunction may represent the earliest photoreceptor abnormality or a distinct entity within the acute zonal occult outer retinopathy complex. Acute zonal occult outer retinopathy should be considered in patients with acute vision loss and abnormalities on NIR-REF imaging, especially if multimodal imaging supports an intact retinal pigment epithelium and inner retina but an abnormal photoreceptor outer segment. PMID:28384671

Germline disruption of Pten localization causes enhanced sex-dependent social motivation and increased glial production.

PubMed

Tilot, Amanda K; Gaugler, Mary K; Yu, Qi; Romigh, Todd; Yu, Wanfeng; Miller, Robert H; Frazier, Thomas W; Eng, Charis

2014-06-15

PTEN Hamartoma Tumor Syndrome (PHTS) is an autosomal-dominant genetic condition underlying a subset of autism spectrum disorder (ASD) with macrocephaly. Caused by germline mutations in PTEN, PHTS also causes increased risks of multiple cancers via dysregulation of the PI3K and MAPK signaling pathways. Conditional knockout models have shown that neural Pten regulates social behavior, proliferation and cell size. Although much is known about how the intracellular localization of PTEN regulates signaling in cancer cell lines, we know little of how PTEN localization influences normal brain physiology and behavior. To address this, we generated a germline knock-in mouse model of cytoplasm-predominant Pten and characterized its behavioral and cellular phenotypes. The homozygous Pten(m3m4) mice have decreased total Pten levels including a specific drop in nuclear Pten and exhibit region-specific increases in brain weight. The Pten(m3m4) model displays sex-specific increases in social motivation, poor balance and normal recognition memory-a profile reminiscent of some individuals with high functioning ASD. The cytoplasm-predominant protein caused cellular hypertrophy limited to the soma and led to increased NG2 cell proliferation and accumulation of glia. The animals also exhibit significant astrogliosis and microglial activation, indicating a neuroinflammatory phenotype. At the signaling level, Pten(m3m4) mice show brain region-specific differences in Akt activation. These results demonstrate that differing alterations to the same autism-linked gene can cause distinct behavioral profiles. The Pten(m3m4) model is the first murine model of inappropriately elevated social motivation in the context of normal cognition and may expand the range of autism-related behaviors replicated in animal models. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Phosphorylation of a conserved serine in the deoxyribonucleic acid binding domain of nuclear receptors alters intracellular localization.

PubMed

Sun, Kai; Montana, Vedrana; Chellappa, Karthikeyani; Brelivet, Yann; Moras, Dino; Maeda, Yutaka; Parpura, Vladimir; Paschal, Bryce M; Sladek, Frances M

2007-06-01

Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4alpha (HNF4alpha). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4alpha and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4alpha (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4alpha as well as decreased endogenous HNF4alpha protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor alpha, retinoid X receptor alpha, and thyroid hormone receptor beta) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor alpha and peroxisome proliferator-activated receptor alpha. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.

Boron Neutron Capture Therapy - A Literature Review

PubMed Central

Nedunchezhian, Kavitaa; Thiruppathy, Manigandan; Thirugnanamurthy, Sarumathi

2016-01-01

Boron Neutron Capture Therapy (BNCT) is a radiation science which is emerging as a hopeful tool in treating cancer, by selectively concentrating boron compounds in tumour cells and then subjecting the tumour cells to epithermal neutron beam radiation. BNCT bestows upon the nuclear reaction that occurs when Boron-10, a stable isotope, is irradiated with low-energy thermal neutrons to yield α particles (Helium-4) and recoiling lithium-7 nuclei. A large number of 10 Boron (10B) atoms have to be localized on or within neoplastic cells for BNCT to be effective, and an adequate number of thermal neutrons have to be absorbed by the 10B atoms to maintain a lethal 10B (n, α) lithium-7 reaction. The most exclusive property of BNCT is that it can deposit an immense dose gradient between the tumour cells and normal cells. BNCT integrates the fundamental focusing perception of chemotherapy and the gross anatomical localization proposition of traditional radiotherapy. PMID:28209015

Analysis of Mancos shale failure in light of localization theory for transversely isotropic materials.

NASA Astrophysics Data System (ADS)

Ingraham, M. D.; Dewers, T. A.; Heath, J. E.

2016-12-01

Utilizing the localization conditions laid out in Rudnicki 2002, the failure of a series of tests performed on Mancos shale has been analyzed. Shale specimens were tested under constant mean stress conditions in an axisymmetric stress state, with specimens cored both parallel and perpendicular to bedding. Failure data indicates that for the range of pressures tested the failure surface is well represented by a Mohr- Coulomb failure surface with a friction angle of 34.4 for specimens cored parallel to bedding, and 26.5 for specimens cored perpendicular to bedding. There is no evidence of a yield cap up to 200 MPa mean stress. Comparison with the theory shows that the best agreement in terms of band angles comes from assuming normality of the plastic strain increment. 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.

Normal values and standardization of parameters in nuclear cardiology: Japanese Society of Nuclear Medicine working group database.

PubMed

Nakajima, Kenichi; Matsumoto, Naoya; Kasai, Tokuo; Matsuo, Shinro; Kiso, Keisuke; Okuda, Koichi

2016-04-01

As a 2-year project of the Japanese Society of Nuclear Medicine working group activity, normal myocardial imaging databases were accumulated and summarized. Stress-rest with gated and non-gated image sets were accumulated for myocardial perfusion imaging and could be used for perfusion defect scoring and normal left ventricular (LV) function analysis. For single-photon emission computed tomography (SPECT) with multi-focal collimator design, databases of supine and prone positions and computed tomography (CT)-based attenuation correction were created. The CT-based correction provided similar perfusion patterns between genders. In phase analysis of gated myocardial perfusion SPECT, a new approach for analyzing dyssynchrony, normal ranges of parameters for phase bandwidth, standard deviation and entropy were determined in four software programs. Although the results were not interchangeable, dependency on gender, ejection fraction and volumes were common characteristics of these parameters. Standardization of (123)I-MIBG sympathetic imaging was performed regarding heart-to-mediastinum ratio (HMR) using a calibration phantom method. The HMRs from any collimator types could be converted to the value with medium-energy comparable collimators. Appropriate quantification based on common normal databases and standard technology could play a pivotal role for clinical practice and researches.

Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants

PubMed Central

Martel, Catherine; Macchi, Paolo; Furic, Luc; Kiebler, Michael A.; Desgroseillers, Luc

2005-01-01

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport. PMID:16162096

RASCAL is a new human cytomegalovirus-encoded protein that localizes to the nuclear lamina and in cytoplasmic vesicles at late times postinfection.

PubMed

Miller, Matthew S; Furlong, Wendy E; Pennell, Leesa; Geadah, Marc; Hertel, Laura

2010-07-01

The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear rim-associated cytomegaloviral protein, or RASCAL. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.

ORF73 LANA homologs of RRV and MneRV2 contain an extended RGG/RG-rich nuclear and nucleolar localization signal that interacts directly with importin β1 for non-classical nuclear import.

PubMed

Howard, Kellie; Cherezova, Lidia; DeMaster, Laura K; Rose, Timothy M

2017-11-01

The latency-associated nuclear antigens (LANA) of KSHV and macaque RFHVMn, members of the RV1 rhadinovirus lineage, are closely related with conservation of complex nuclear localization signals (NLS) containing bipartite KR-rich motifs and RG-rich domains, which interact distinctly with importins α and ß1 for nuclear import via classical and non-classical pathways, respectively. RV1 LANAs are expressed in the nucleus of latently-infected cells where they inhibit replication and establish a dominant RV1 latency. Here we show that LANA homologs of macaque RRV and MneRV2 from the more distantly-related RV2 lineage, lack the KR-rich NLS, and instead have a large RG-rich NLS with multiple RG dipeptides and a conserved RGG motif. The RG-NLS interacts uniquely with importin β1, which mediates nuclear import and accumulation of RV2 LANA in the nucleolus. The alternative nuclear import and localization of RV2 LANA homologs may contribute to the dominant RV2 lytic replication phenotype. Copyright © 2017. Published by Elsevier Inc.

Scaling the Drosophila Wing: TOR-Dependent Target Gene Access by the Hippo Pathway Transducer Yorkie

PubMed Central

Parker, Joseph; Struhl, Gary

2015-01-01

Organ growth is controlled by patterning signals that operate locally (e.g., Wingless/Ints [Wnts], Bone Morphogenetic Proteins [BMPs], and Hedgehogs [Hhs]) and scaled by nutrient-dependent signals that act systemically (e.g., Insulin-like peptides [ILPs] transduced by the Target of Rapamycin [TOR] pathway). How cells integrate these distinct inputs to generate organs of the appropriate size and shape is largely unknown. The transcriptional coactivator Yorkie (Yki, a YES-Associated Protein, or YAP) acts downstream of patterning morphogens and other tissue-intrinsic signals to promote organ growth. Yki activity is regulated primarily by the Warts/Hippo (Wts/Hpo) tumour suppressor pathway, which impedes nuclear access of Yki by a cytoplasmic tethering mechanism. Here, we show that the TOR pathway regulates Yki by a separate and novel mechanism in the Drosophila wing. Instead of controlling Yki nuclear access, TOR signaling governs Yki action after it reaches the nucleus by allowing it to gain access to its target genes. When TOR activity is inhibited, Yki accumulates in the nucleus but is sequestered from its normal growth-promoting target genes—a phenomenon we term “nuclear seclusion.” Hence, we posit that in addition to its well-known role in stimulating cellular metabolism in response to nutrients, TOR also promotes wing growth by liberating Yki from nuclear seclusion, a parallel pathway that we propose contributes to the scaling of wing size with nutrient availability. PMID:26474042

UL31 and UL34 Proteins of Herpes Simplex Virus Type 1 Form a Complex That Accumulates at the Nuclear Rim and Is Required for Envelopment of Nucleocapsids

PubMed Central

Reynolds, Ashley E.; Ryckman, Brent J.; Baines, Joel D.; Zhou, Yuping; Liang, Li; Roller, Richard J.

2001-01-01

The herpes simplex virus type 1 (HSV-1) UL34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the UL31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with UL34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing UL31 protein fused to glutathione-S-transferase (UL31-GST) and UL34 protein fused to GST (UL34-GST) were demonstrated to specifically recognize the UL31 and UL34 proteins of approximately 34,000 and 30,000 Da, respectively. The UL31 and UL34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. UL34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of UL31 protein at the nuclear rim required the presence of UL34 protein, inasmuch as cells infected with a UL34 null mutant virus contained UL31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of UL34 protein exclusively at the nuclear rim required the presence of the UL31 gene product, inasmuch as UL34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a UL31 null virus. When transiently expressed in the absence of other viral factors, UL31 protein localized diffusely in the nucleoplasm, whereas UL34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the UL31 and UL34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the US3-encoded protein kinase, previously shown to phosphorylate the UL34 gene product, UL31 and UL34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, US3 kinase is required for even distribution of UL31 and UL34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the UL31 and UL34 deletion mutants, these data strongly suggest that the UL31 and UL34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane. PMID:11507225

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inagaki, Yuichi; Mitsutake, Susumu; Igarashi, Yasuyuki

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 NLSmore » 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.« less

RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheren, Jamie E.; Kassenbrock, C. Kenneth, E-mail: ken.kassenbrock@ucdenver.edu; Department of Biology, Colorado State University, Fort Collins, CO 80523-1878

2013-11-01

Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequencemore » (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.« less

Tracking STAT nuclear traffic.

PubMed

Reich, Nancy C; Liu, Ling

2006-08-01

Accurate cellular localization is crucial for the effective function of most signalling molecules and nuclear translocation is central to the function of transcription factors. The passage of large molecules between the cytoplasm and nucleus is restricted, and this restriction affords a mechanism to regulate transcription by controlling the access of transcription factors to the nucleus. In this Review, we focus on the signal transducer and activator of transcription (STAT) family of transcription factors. The regulation of the nuclear trafficking of STAT-family members is diverse. Some STAT proteins constitutively shuttle between the nucleus and cytoplasm, whereas others require tyrosine phosphorylation for nuclear localization. In either case, the regulation of nuclear trafficking can provide a target for therapeutic intervention.

In vivo delineation of subdivisions of the human amygdaloid complex in a high-resolution group template

PubMed Central

Tyszka, J. Michael; Pauli, Wolfgang M.

2016-01-01

The nuclei of the human amygdala remain difficult to distinguish in individual subject structural magnetic resonance images. However, interpretation of the amygdala’s role in whole brain networks requires accurate localization of functional activity to a particular nucleus or subgroup of nuclei. To address this, we constructed high spatial resolution, three-dimensional templates, using joint high accuracy diffeomorphic registration of T1- and T2-weighted structural images from 168 typical adults between 22 and 35 years old released by the Human Connectome Project. Several internuclear boundaries are clearly visible in these templates, which would otherwise be impossible to delineate in individual subject data. A probabilistic atlas of major nuclei and nuclear groups was constructed in this template space and mapped back to individual spaces by inversion of the individual diffeomorphisms. Group level analyses revealed a slight (approximately 2%) bias towards larger total amygdala and nuclear volumes in the right hemisphere. No substantial sex or age differences were found in amygdala volumes normalized to total intracranial volume, or subdivision volumes normalized to amygdala volume. The current delineation provides a finer parcellation of the amygdala with more accurate external boundary definition than current histology-based atlases when used in conjunction with high accuracy registration methods, such as diffeomorphic warping. These templates and delineation are intended to be an open and evolving resource for future functional and structural imaging studies of the human amygdala. PMID:27354150

The acromegaly--gigantism syndrome. Report of four cases treated surgically.

PubMed

Zampieri, P; Scanarini, M; Sicolo, N; Andrioli, G; Mingrino, S

1983-12-01

Four cases of growth-hormone-secreting pituitary adenoma, with associated aspects of acromegaly and gigantism, are reported in patients aged 12-26. All of the patients had macroadenomas and were treated surgically, three by the transsphenoidal approach and one with a transfrontal craniotomy. Histologic examination revealed eosinophilic adenomas in three of the cases and a mixed eosinophilic--chromophobe adenoma in one, all with cellular irregularities (mitosis and cellular and nuclear polymorphism), local invasivity, or both. Because surgical treatment did not produce complete normalization of growth hormone levels, radiotherapy followed the operations in all four cases. In our opinion, the treatment of acromegalic gigantism poses more therapeutic problems than that of simple acromegaly, with combined treatment (surgical, radiation, and medical) often being necessary.

Accelerated recovery of renal mitochondrial and tubule homeostasis with SIRT1/PGC-1α activation following ischemia-reperfusion injury.

PubMed

Funk, Jason A; Schnellmann, Rick G

2013-12-01

Kidney ischemia-reperfusion (I/R) injury elicits cellular injury in the proximal tubule, and mitochondrial dysfunction is a pathological consequence of I/R. Promoting mitochondrial biogenesis (MB) as a repair mechanism after injury may offer a unique strategy to restore both mitochondrial and organ function. Rats subjected to bilateral renal pedicle ligation for 22 min were treated once daily with the SIRT1 activator SRT1720 (5mg/kg) starting 24h after reperfusion until 72h-144 h. SIRT1 expression was elevated in the renal cortex of rats after I/R+vehicle treatment (IRV), but was associated with less nuclear localization. SIRT1 expression was even further augmented and nuclear localization was restored in the kidneys of rats after I/R+SRT1720 treatment (IRS). PGC-1α was elevated at 72 h-144 h in IRV and IRS kidneys; however, SRT1720 treatment induced deacetylation of PGC-1α, a marker of activation. Mitochondrial proteins ATP synthase β, COX I, and NDUFB8, as well as mitochondrial respiration, were diminished 24h-144 h in IRV rats, but were partially or fully restored in IRS rats. Urinary kidney injury molecule-1 (KIM-1) was persistently elevated in both IRV and IRS rats; however, KIM-1 tissue expression was attenuated in IRS rats. Additionally, sustained loss of Na(+),K(+)-ATPase expression and basolateral localization and elevated vimentin in IRV rats was normalized in IRS rats, suggesting restoration of a differentiated, polarized tubule epithelium. The results suggest that SRT1720 treatment expedited recovery of mitochondrial protein expression and function by enhancing MB, which was associated with faster proximal tubule repair. Targeting MB may offer unique therapeutic strategy following ischemic injury. © 2013. Published by Elsevier Inc. All rights reserved.

Androgen Receptor Content of the Normal and Hyperplastic Canine Prostate

PubMed Central

Shain, Sydney A.; Boesel, Robert W.

1978-01-01

A procedure was developed for measurement of androgen receptors in cytoplasmic extracts of prostates from intact dogs. The protocol utilized exchange saturation analysis at 15°C employing the synthetic androgen R1881 (17β-hydroxy-17α-methylestra-4,9,11-trien-3-one) as the ligand probe and quantitatively detected total cytoplasmic androgen receptor (Rc, androgen-free receptor, and RcA, androgen-occupied receptor) present at the initiation of the assay. This protocol was employed in conjunction with a tissue mince saturation analysis procedure (for quantitation of nuclear androgen receptor) to quantitate total androgen receptor content of normal and hyperplastic prostates obtained from young (2.5- or 4.6-yr old) and aged (12.5-yr old) purebred dogs of known birth date. The total cytoplasmic androgen receptor content (picomoles per prostate) of hyperplastic prostates was 4.6-fold greater than that of normal prostates. The total nuclear androgen receptor content of hyperplastic prostates (picomoles per prostate measured in crude nuclear preparations) was either 5.0- (4.6-yr-old dogs) or 7.8-fold (2.5-yr-old dogs) greater than that of normal prostates. However, androgen receptor content per cell was identical for hyperplastic and normal canine prostates, with the exception that nuclear androgen receptor was diminished in prostates from 2.5-yr-old dogs. The cell content per gram dry weight was identical for hyperplastic and normal canine prostates. We conclude that canine prostate hyperplasia is characterized by coordinate proliferation of androgen receptor-positive and androgen receptor-negative cells and is not a consequence of increased accumulation of 5α-dihydrotestosterone due to proliferation of androgen receptors per prostate cell. PMID:76635

Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function1

PubMed Central

Jaiswal, Aruna S; Armas, Melissa L; Izumi, Tadahide; Strauss, Phyllis R; Narayan, Satya

2012-01-01

In previous studies, we found that adenomatous polyposis coli (APC) blocks the base excision repair (BER) pathway by interacting with 5′-flap endonuclease 1 (Fen1). In this study, we identify the molecular features that contribute to the formation and/or stabilization of the APC/Fen1 complex that determines the extent of BER inhibition, and the subsequent accumulation of DNA damage creates mutagenic lesions leading to transformation susceptibility. We show here that APC binds to the nuclear localization sequence of Fen1 (Lys365Lys366Lys367), which prevents entry of Fen1 into the nucleus and participation in Pol-β-directed long-patch BER. We also show that levels of the APC/Fen1 complex are higher in breast tumors than in the surrounding normal tissues. These studies demonstrate a novel role for APC in the suppression of Fen1 activity in the BER pathway and a new biomarker profile to be explored to identify individuals who may be susceptible to the development of mammary and other tumors. PMID:22787431

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.

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

Progressive Disintegration of Brain Networking from Normal Aging to Alzheimer Disease: Analysis of Independent Components of 18F-FDG PET Data.

PubMed

Pagani, Marco; Giuliani, Alessandro; Öberg, Johanna; De Carli, Fabrizio; Morbelli, Silvia; Girtler, Nicola; Arnaldi, Dario; Accardo, Jennifer; Bauckneht, Matteo; Bongioanni, Francesca; Chincarini, Andrea; Sambuceti, Gianmario; Jonsson, Cathrine; Nobili, Flavio

2017-07-01

Brain connectivity has been assessed in several neurodegenerative disorders investigating the mutual correlations between predetermined regions or nodes. Selective breakdown of brain networks during progression from normal aging to Alzheimer disease dementia (AD) has also been observed. Methods: We implemented independent-component analysis of 18 F-FDG PET data in 5 groups of subjects with cognitive states ranging from normal aging to AD-including mild cognitive impairment (MCI) not converting or converting to AD-to disclose the spatial distribution of the independent components in each cognitive state and their accuracy in discriminating the groups. Results: We could identify spatially distinct independent components in each group, with generation of local circuits increasing proportionally to the severity of the disease. AD-specific independent components first appeared in the late-MCI stage and could discriminate converting MCI and AD from nonconverting MCI with an accuracy of 83.5%. Progressive disintegration of the intrinsic networks from normal aging to MCI to AD was inversely proportional to the conversion time. Conclusion: Independent-component analysis of 18 F-FDG PET data showed a gradual disruption of functional brain connectivity with progression of cognitive decline in AD. This information might be useful as a prognostic aid for individual patients and as a surrogate biomarker in intervention trials. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Relating normal vibrational modes to local vibrational modes with the help of an adiabatic connection scheme

NASA Astrophysics Data System (ADS)

Zou, Wenli; Kalescky, Robert; Kraka, Elfi; Cremer, Dieter

2012-08-01

Information on the electronic structure of a molecule and its chemical bonds is encoded in the molecular normal vibrational modes. However, normal vibrational modes result from a coupling of local vibrational modes, which means that only the latter can provide detailed insight into bonding and other structural features. In this work, it is proven that the adiabatic internal coordinate vibrational modes of Konkoli and Cremer [Int. J. Quantum Chem. 67, 29 (1998)], 10.1002/(SICI)1097-461X(1998)67:1<29::AID-QUA3>3.0.CO;2-0 represent a unique set of local modes that is directly related to the normal vibrational modes. The missing link between these two sets of modes are the compliance constants of Decius, which turn out to be the reciprocals of the local mode force constants of Konkoli and Cremer. Using the compliance constants matrix, the local mode frequencies of any molecule can be converted into its normal mode frequencies with the help of an adiabatic connection scheme that defines the coupling of the local modes in terms of coupling frequencies and reveals how avoided crossings between the local modes lead to changes in the character of the normal modes.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 foundmore » 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.« less

The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1

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 N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.

Functional relationship between CABIT, SAM and 14-3-3 binding domains of GAREM1 that play a role in its subcellular localization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki, E-mail: hkonishi@pu-hiroshima.ac.jp

2015-08-21

GAREM1 (Grb2-associated regulator of Erk/MAPK1) is an adaptor protein that is involved in the epidermal growth factor (EGF) pathway. The nuclear localization of GAREM1 depends on the nuclear localization sequence (NLS), which is located at the N-terminal CABIT (cysteine-containing, all in Themis) domain. Here, we identified 14-3-3ε as a GAREM-binding protein, and its binding site is closely located to the NLS. This 14-3-3 binding site was of the atypical type and independent of GAREM phosphorylation. Moreover, the binding of 14-3-3 had an effect on the nuclear localization of GAREM1. Unexpectedly, we observed that the CABIT domain had intramolecular association withmore » the C-terminal SAM (sterile alpha motif) domain. This association might be inhibited by binding of 14-3-3 at the CABIT domain. Our results demonstrate that the mechanism underlying the nuclear localization of GAREM1 depends on its NLS in the CABIT domain, which is controlled by the binding of 14-3-3 and the C-terminal SAM domain. We suggest that the interplay between 14-3-3, SAM domain and CABIT domain might be responsible for the distribution of GAREM1 in mammalian cells. - Highlights: • 14-3-3ε regulated the nuclear localization of GAREM1 as its binding partner. • The atypical 14-3-3 binding site of GAREM1 is located near the NLS in CABIT domain. • The CABIT domain had intramolecular association with the SAM domain in GAREM1. • Subcellular localization of GAREM1 is affected with its CABIT-SAM interaction.« less

Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner.

PubMed

Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

2012-01-01

Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

Lectin-binding histochemistry of non-decalcified growth plate cartilage: a postembedment method for light microscopy of epon-embedded tissue.

PubMed

Farnum, C E; Wilsman, N J

1984-06-01

A postembedment method for the localization of lectin-binding glycoconjugates was developed using Epon-embedded growth plate cartilage from Yucatan miniature swine. By testing a variety of etching, blocking, and incubation procedures, a standard protocol was developed for 1 micron thick sections that allowed visualization of both intracellular and extracellular glycoconjugates with affinity for wheat germ agglutinin and concanavalin A. Both fluorescent and peroxidase techniques were used, and comparisons were made between direct methods and indirect methods using the biotin-avidin bridging system. Differential extracellular lectin binding allowed visualization of interterritorial , territorial, and pericellular matrices. Double labeling experiments showed the precision with which intracellular binding could be localized to specific cytoplasmic compartments, with resolution of binding to the Golgi apparatus, endoplasmic reticulum, and nuclear membrane at the light microscopic level. This method allows the localization of both intracellular and extracellular lectin-binding glycoconjugates using fixation and embedment procedures that are compatible with simultaneous ultrastructural analysis. As such it should have applicability both to the morphological analysis of growth plate organization during normal endochondral ossification, as well as to the diagnostic pathology of matrix abnormalities in disease states of growing cartilage.

Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane.

PubMed

Taniyama, Toshiyuki; Tsuda, Natsumi; Sueda, Shinji

2018-06-15

The nuclear envelope (NE) is a double membrane that segregates nuclear components from the cytoplasm in eukaryotic cells. It is well-known that the NE undergoes a breakdown and reformation during mitosis in animal cells. However, the detailed mechanisms of the NE dynamics are not yet fully understood. Here, we propose a method for the fluorescent labeling of the NE in living cells, which enables the tracing of the NE dynamics during cell division under physiological conditions. In our method, labeling of the NE is accomplished by fixing green fluorescent protein carrying the nuclear localization signal on the inner nuclear membrane based on a unique biotinylation reaction from the archaeon Sulfolobus tokodaii. With this method, we observed HeLa cells during mitosis by confocal laser scanning microscopy and succeeded in clearly visualizing the difference in the timing of the formation of the NE and the nuclear lamina.

Confocal Microscopy and Molecular-Specific Optical Contrast Agents for the Detection of Oral Neoplasia

PubMed Central

Carlson, Alicia L.; Gillenwater, Ann M.; Williams, Michelle D.; El-Naggar, Adel K.; Richards-Kortum, R. R.

2009-01-01

Using current clinical diagnostic techniques, it is difficult to visualize tumor morphology and architecture at the cellular level, which is necessary for diagnostic localization of pathologic lesions. Optical imaging techniques have the potential to address this clinical need by providing real-time, sub-cellular resolution images. This paper describes the use of dual mode confocal microscopy and optical molecular-specific contrast agents to image tissue architecture, cellular morphology, and sub-cellular molecular features of normal and neoplastic oral tissues. Fresh tissue slices were prepared from 33 biopsies of clinically normal and abnormal oral mucosa obtained from 14 patients. Reflectance confocal images were acquired after the application of 6% acetic acid, and fluorescence confocal images were acquired after the application of a fluorescence contrast agent targeting the epidermal growth factor receptor (EGFR). The dual imaging modes provided images similar to light microscopy of hematoxylin and eosin and immunohistochemistry staining, but from thick fresh tissue slices. Reflectance images provided information on the architecture of the tissue and the cellular morphology. The nuclear-to-cytoplasmic (N/C) ratio from the reflectance images was at least 7.5 times greater for the carcinoma than the corresponding normal samples, except for one case of highly keratinized carcinoma. Separation of carcinoma from normal and mild dysplasia was achieved using this ratio (p<0.01). Fluorescence images of EGFR expression yielded a mean fluorescence labeling intensity (FLI) that was at least 2.7 times higher for severe dysplasia and carcinoma samples than for the corresponding normal sample, and could be used to distinguish carcinoma from normal and mild dysplasia (p<0.01). Analyzed together, the N/C ratio and the mean FLI may improve the ability to distinguish carcinoma from normal squamous epithelium. PMID:17877424

Morphometric computer-assisted image analysis of epithelial cells in different grades of oral squamous cell carcinoma.

PubMed

Ananjan, Chatterjee; Jyothi, Mahadesh; Laxmidevi, B L; Gopinathan, Pillai Arun; Nazir, Salroo Humaira; Pradeep, L

2018-01-01

Oral squamous cell carcinoma (OSCC) accounts 94% of all malignant lesions in the oral cavity. In the assessment of OSCC, nowadays the WHO grading system has been followed widely but due to its subjectivity, investigators applied the sophisticated technique of computer-assisted image analysis in the grading of carcinoma in larynx, lungs, esophagus, and cervix to make it more objective. Access, analyze, and compare the cellular area (CA); cytoplasmic area (Cyt A); nuclear area (NA); nuclear perimeter (NP); nuclear form factor (NF); and nuclear-cytoplasmic ratio (N/C) of the cells in different grades of OSCC. Fifty OSCC cases were obtained and stained with hematoxylin and eosin which were graded according to the WHO classification. The sections were subjected to morphometric analysis to analyze all the morphometric parameters in different grades of OSCC and subjected to one-way ANOVA statistical analysis. CA and Cyt A decreased from normal mucosa with dedifferentiation of OSCC. The NA and NP increased in carcinoma group when compared to normal mucosa but decreased with dedifferentiation of OSCC (P < 0.05). NF had no significance with normal mucosa and different grades of OSCC (P > 0.05), while N/C ratio increased from normal mucosa through increasing grades of OSCC, reaching the highest value in poorly differentiated squamous cell carcinoma (P < 0.05). Both cellular and nuclear variables provide a more accurate indication of tumor aggressiveness than any single parameter. Morphometric analysis can be a reliable tool to determine objectively the degree of malignancy at the invasive tumor front.

Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horie, Ryo; Yoneda, Misako, E-mail: yone@ims.u-tok

Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21–24 and 110–139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60–75 and 72–75 were importantmore » for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm. -- Highlights: •Nipah virus (NiV) infection resulted in high mortality, but effective treatment has not been established. •Several reports revealed that NiV nonstructural C protein (NiV-C) was essential for NiV pathogenicity, however, whole of NiV-C function is still unknown. •Although nonstructural C proteins of other Paramyxoviruses are expressed in similar mechanism and exert similar activity, subcellular localization and cellular targets are different. In this study, we evaluated the subcellular localization of NiV-C. •To our knowledge, this is the first report showing that NiV-C shuttles between the nucleus and cytoplasm. We also clarified that NiV-C has nuclear export signal and nuclear localization signal using NiV-C deleted, alanine substitution mutants and enhanced green fluorescent protein (EGFP) fused proteins. •And we also showed that interferon (IFN) antagonist activity of NiV-C related to its subcellular localization. Our results indicate that NiV-C exert IFN antagonist activity in the cytoplasm.« less

Crosstalk between ERK2 and RXR regulates nuclear import of transcription factor NGFI-B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacobs, Chris M.; Paulsen, Ragnhild E.

2005-10-21

Transcription factor NGFI-B initiates apoptosis when allowed to translocate to mitochondria. Retinoid-X receptor (RXR), another member of the nuclear receptor family, regulates NGFI-B signaling through heterodimerization and nuclear export. Growth factor EGF activates ERK2, which phosphorylates NGFI-B and determines if NGFI-B is allowed to translocate to mitochondria. In the present study, EGF treatment resulted in an increased nuclear import of NGFI-B. Likewise, active ERK2 resulted in a preferential nuclear localization of NGFI-B. When coexpressed with RXR the nuclear import and nuclear localization induced by active ERK2 were strongly reduced. In the presence of its ligand 9-cis-retinoic acid, RXR no longermore » inhibited ERK2-induced nuclear import. Thus, RXR serves a permissive role for ERK2-mediated nuclear accumulation of NGFI-B. This finding represents a novel crosstalk between ERK2 and RXR signaling pathways, and explains how two independent inhibitors of apoptosis (EGF and 9-cis-retinoic acid) may cooperate to regulate nuclear targeting of apoptosis inducer NGFI-B.« less

Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization.

PubMed

Pinarbasi, Emile S; Cağatay, Tolga; Fung, Ho Yee Joyce; Li, Ying C; Chook, Yuh Min; Thomas, Philip J

2018-05-04

ALS (Amyotrophic Lateral Sclerosis) is a neurodegenerative disease characterized by the redistribution of the RNA binding protein TDP-43 in affected neurons: from predominantly nuclear to aggregated in the cytosol. However, the determinants of TDP-43 localization and the cellular insults that promote redistribution are incompletely understood. Here, we show that the putative Nuclear Export Signal (NES) is not required for nuclear egress of TDP-43. Moreover, when the TDP-43 domain which contains the putative NES is fused to a reporter protein, YFP, the presence of the NES is not sufficient to mediate nuclear exclusion of the fusion protein. We find that the previously studied "∆NES" mutant, in which conserved hydrophobic residues are mutated to alanines, disrupts both solubility and splicing function. We further show that nuclear export of TDP-43 is independent of the exportin XPO1. Finally, we provide evidence that nuclear egress of TDP-43 is size dependent; nuclear export of dTomato TDP-43 is significantly impaired compared to Flag TDP-43. Together, these results suggest nuclear export of TDP-43 is predominantly driven by passive diffusion.

Mechanism for G2 phase-specific nuclear export of the kinetochore protein CENP-F.

PubMed

Loftus, Kyle M; Cui, Heying; Coutavas, Elias; King, David S; Ceravolo, Amanda; Pereiras, Dylan; Solmaz, Sozanne R

2017-08-03

Centromere protein F (CENP-F) is a component of the kinetochore and a regulator of cell cycle progression. CENP-F recruits the dynein transport machinery and orchestrates several cell cycle-specific transport events, including transport of the nucleus, mitochondria and chromosomes. A key regulatory step for several of these functions is likely the G2 phase-specific export of CENP-F from the nucleus to the cytosol, where the cytoplasmic dynein transport machinery resides; however, the molecular mechanism of this process is elusive. Here, we have identified 3 phosphorylation sites within the bipartite classical nuclear localization signal (cNLS) of CENP-F. These sites are specific for cyclin-dependent kinase 1 (Cdk1), which is active in G2 phase. Phosphomimetic mutations of these residues strongly diminish the interaction of the CENP-F cNLS with its nuclear transport receptor karyopherin α. These mutations also diminish nuclear localization of the CENP-F cNLS in cells. Notably, the cNLS is phosphorylated in the -1 position, which is important to orient the adjacent major motif for binding into its pocket on karyopherin α. We propose that localization of CENP-F is regulated by a cNLS, and a nuclear export pathway, resulting in nuclear localization during most of interphase. In G2 phase, the cNLS is weakened by phosphorylation through Cdk1, likely resulting in nuclear export of CENP-F via the still active nuclear export pathway. Once CENP-F resides in the cytosol, it can engage in pathways that are important for cell cycle progression, kinetochore assembly and the faithful segregation of chromosomes into daughter cells.

Nuclear translocation of glutathione S-transferase {pi} is mediated by a non-classical localization signal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawakatsu, Miho; Goto, Shinji, E-mail: sgoto@nagasaki-u.ac.jp; Yoshida, Takako

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 ofmore » 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

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 inmore » 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.« less

Absence of SUN-domain protein Slp1 blocks karyogamy and switches meiotic recombination and synapsis from homologs to sister chromatids

PubMed Central

Vasnier, Christelle; de Muyt, Arnaud; Zhang, Liangran; Tessé, Sophie; Kleckner, Nancy E.; Zickler, Denise; Espagne, Eric

2014-01-01

Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid–Sad1p, UNC-84–domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid “twin” nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly. PMID:25210014

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patterson, Edward I.; EH Graham Centre for Agricultural Innovation; Dombrovski, Andrew 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 mediatemore » 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.« less

Regulation of the Drosophila Hypoxia-Inducible Factor α Sima by CRM1-Dependent Nuclear Export ▿

PubMed Central

Romero, Nuria M.; Irisarri, Maximiliano; Roth, Peggy; Cauerhff, Ana; Samakovlis, Christos; Wappner, Pablo

2008-01-01

Hypoxia-inducible factor α (HIF-α) proteins are regulated by oxygen levels through several different mechanisms that include protein stability, transcriptional coactivator recruitment, and subcellular localization. It was previously reported that these transcription factors are mainly nuclear in hypoxia and cytoplasmic in normoxia, but so far the molecular basis of this regulation is unclear. We show here that the Drosophila melanogaster HIF-α protein Sima shuttles continuously between the nucleus and the cytoplasm. We identified the relevant nuclear localization signal and two functional nuclear export signals (NESs). These NESs are in the Sima basic helix-loop-helix (bHLH) domain and promote CRM1-dependent nuclear export. Site-directed mutagenesis of either NES provoked Sima nuclear retention and increased transcriptional activity, suggesting that nuclear export contributes to Sima regulation. The identified NESs are conserved and probably functional in the bHLH domains of several bHLH-PAS proteins. We propose that rapid nuclear export of Sima regulates the duration of cellular responses to hypoxia. PMID:18332128

Robust nuclear lamina-based cell classification of aging and senescent cells

PubMed Central

Righolt, Christiaan H.; van 't Hoff, Merel L.R.; Vermolen, Bart J.; Young, Ian T.; Raz, Vered

2011-01-01

Changes in the shape of the nuclear lamina are exhibited in senescent cells, as well as in cells expressing mutations in lamina genes. To identify cells with defects in the nuclear lamina we developed an imaging method that quantifies the intensity and curvature of the nuclear lamina. We show that this method accurately describes changes in the nuclear lamina. Spatial changes in nuclear lamina coincide with redistribution of lamin A proteins and local reduction in protein mobility in senescent cell. We suggest that local accumulation of lamin A in the nuclear envelope leads to bending of the structure. A quantitative distinction of the nuclear lamina shape in cell populations was found between fresh and senescent cells, and between primary myoblasts from young and old donors. Moreover, with this method mutations in lamina genes were significantly distinct from cells with wild-type genes. We suggest that this method can be applied to identify abnormal cells during aging, in in vitro propagation, and in lamina disorders. PMID:22199022

Robust nuclear lamina-based cell classification of aging and senescent cells.

PubMed

Righolt, Christiaan H; van 't Hoff, Merel L R; Vermolen, Bart J; Young, Ian T; Raz, Vered

2011-12-01

Changes in the shape of the nuclear lamina are exhibited in senescent cells, as well as in cells expressing mutations in lamina genes. To identify cells with defects in the nuclear lamina we developed an imaging method that quantifies the intensity and curvature of the nuclear lamina. We show that this method accurately describes changes in the nuclear lamina. Spatial changes in nuclear lamina coincide with redistribution of lamin A proteins and local reduction in protein mobility in senescent cell. We suggest that local accumulation of lamin A in the nuclear envelope leads to bending of the structure. A quantitative distinction of the nuclear lamina shape in cell populations was found between fresh and senescent cells, and between primary myoblasts from young and old donors. Moreover, with this method mutations in lamina genes were significantly distinct from cells with wild-type genes. We suggest that this method can be applied to identify abnormal cells during aging, in in vitro propagation, and in lamina disorders.

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates.

PubMed

Edelson, Benjamin S; Best, Timothy P; Olenyuk, Bogdan; Nickols, Nicholas G; Doss, Raymond M; Foister, Shane; Heckel, Alexander; Dervan, Peter B

2004-01-01

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.

Using an Authentic Radioisotope to Teach Half-Life

ERIC Educational Resources Information Center

Liddicoat, Scott; Sebranek, John

2005-01-01

Traditionally nuclear chemistry appears in the last few chapters of chemistry textbooks and is not normally considered a mainstream topic. In addition, some science teachers lack the training or equipment to teach nuclear chemistry. Yet nuclear chemistry is a very important topic that should be taught in all chemistry classrooms. Learning about…

Nuclear Transcription Factors in the Mitochondria: A New Paradigm in Fine-Tuning Mitochondrial Metabolism.

PubMed

Sepuri, Naresh Babu V; Tammineni, Prasad; Mohammed, Fareed; Paripati, Arunkumar

2017-01-01

Noncanonical functions of several nuclear transcription factors in the mitochondria have been gaining exceptional traction over the years. These transcription factors include nuclear hormone receptors like estrogen, glucocorticoid, and thyroid hormone receptors: p53, IRF3, STAT3, STAT5, CREB, NF-kB, and MEF-2D. Mitochondria-localized nuclear transcription factors regulate mitochondrial processes like apoptosis, respiration and mitochondrial transcription albeit being nuclear in origin and having nuclear functions. Hence, the cell permits these multi-stationed transcription factors to orchestrate and fine-tune cellular metabolism at various levels of operation. Despite their ubiquitous distribution in different subcompartments of mitochondria, their targeting mechanism is poorly understood. Here, we review the current status of mitochondria-localized transcription factors and discuss the possible targeting mechanism besides the functional interplay between these factors.

Fascin regulates nuclear actin during Drosophila oogenesis

PubMed Central

Kelpsch, Daniel J.; Groen, Christopher M.; Fagan, Tiffany N.; Sudhir, Sweta; Tootle, Tina L.

2016-01-01

Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5–9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved. PMID:27535426

Direct Estimation of Power Distribution in Reactors for Nuclear Thermal Space Propulsion

NASA Astrophysics Data System (ADS)

Aldemir, Tunc; Miller, Don W.; Burghelea, Andrei

2004-02-01

A recently proposed constant temperature power sensor (CTPS) has the capability to directly measure the local power deposition rate in nuclear reactor cores proposed for space thermal propulsion. Such a capability reduces the uncertainties in the estimated power peaking factors and hence increases the reliability of the nuclear engine. The CTPS operation is sensitive to the changes in the local thermal conditions. A procedure is described for the automatic on-line calibration of the sensor through estimation of changes in thermal .conditions.

Unfolded protein response regulation in keloid cells.

PubMed

Butler, Paris D; Wang, Zhen; Ly, Daphne P; Longaker, Michael T; Koong, Albert C; Yang, George P

2011-05-01

Keloids are a common form of pathologic wound healing characterized by excessive production of extracellular matrix. The unfolded protein response (UPR) is a cellular response to hypoxia, a component of the wound microenvironment, capable of protecting cells from the effects of over-accumulation of misfolded proteins. Since keloids have hypersecretion of extracellular matrix, we hypothesized that keloid fibroblasts (KFs) may have enhanced activation of the UPR compared with normal fibroblasts (NFs). KFs and NFs were placed in a hypoxia chamber for 0, 24, and 48h. We also used tunicamycin to specifically up-regulate the UPR. UPR activation was assayed by PCR for xbp-1 splicing and by immunoblotting with specific antibodies for the three UPR transducers. Nuclear localization of XBP-1 protein in KFs was confirmed by immunofluorescence. There is increased activation of XBP-1 protein in KFs compared with NFs following exposure to hypoxia. Pancreatic ER kinase (PERK) and ATF-6, two other pathways activated by the UPR, show comparable activation between KFs and NFs. We confirmed that there is enhanced activation of XBP-1 by demonstrating increased nuclear localization of XBP-1 using immunofluorescence. In contrast to our initial hypothesis that keloids would have broad activation of the UPR, we demonstrate here that there is a specific up-regulation of one facet of the UPR response. This may represent a specific molecular defect in KFs compared with NFs, and also suggests modulation of the UPR can be used in wound healing therapy. Published by Elsevier Inc.

The Effect of Non-Uniform Temperature and Velocity Fields on Long Range Ultrasonic Measurement Systems in MYRRHA

NASA Astrophysics Data System (ADS)

Van De Wyer, Nicolas; Schram, Christophe; Van Dyck, Dries; Dierckx, Marc

2017-02-01

SCK·CEN, the Belgian Nuclear Research Center, is developing MYRRHA, a generation IV liquid metal cooled nuclear research reactor. As the liquid metal coolant is opaque to light, normal visual feedback during fuel manipulations is not available and must therefore be replaced by a system that is not hindered by the opacity of the coolant. In this respect ultrasonic based instrumentation is under development at SCK·CEN to provide feedback during operations under liquid metal. One of the tasks that will be tackled using ultrasound is the detection and localization of a potentially lost fuel assembly. The development of this localization tool is detailed in this paper. In this application, the distance between ultrasonic sensor and target may be as large as 2.5m. At these distances, non uniform velocity and temperature fields in the liquid metal potentially influence the propagation of the ultrasonic signals, affecting the performance of the ultrasonic systems. In this paper, we investigate how relevant temperature and velocity gradients inside the liquid metal influence the propagation of ultrasonic waves. The effect of temperature and velocity gradients are simulated by means of a newly developed numerical raytracing model. The performance of the model is validated by dedicated water experiments. The setup is capable of creating velocity and temperature gradients representative for MYRRHA conditions. Once validated in water, the same model is used to make predictions for the effect of gradients in the MYRRHA liquid metal environment.

Prognostic significance of CCND1 (cyclin D1) overexpression in primary resected non-small-cell lung cancer.

PubMed Central

Betticher, D. C.; Heighway, J.; Hasleton, P. S.; Altermatt, H. J.; Ryder, W. D.; Cerny, T.; Thatcher, N.

1996-01-01

Amplification of the CCDN1 gene encoding cyclin D1 was examined by Southern blotting and multiplex polymerase chain reaction (PCR) and occurred in 8 of 53 patients (15%) with primary resected non-small-cell lung cancer (NSCLC). These tumours and 17 additional tumours with a normal gene copy number showed overexpression of cyclin D1 (25/53, 47%), as assessed by immunostaining using a monoclonal antibody. In 22/25 cases, cyclin D1 was localised in the cytoplasm, but some (7/25) had simultaneous nuclear staining. This result is in marked contrast to that reported in breast, hepatocellular and colorectal carcinoma studies where immunostaining was invariably nuclear. Examination of a restriction fragment length polymorphic (RFLP) site within the 3'untranslated region of the cDNA following reverse transcriptase (RT)-PCR (29/53 informative cases) showed a strong association between cytoplasmic staining and imbalance in allele-specific message levels. Cyclin D1 overexpression was associated with a poorly differentiated histology (P = 0.04), less lymphocytic infiltration of the tumour (P = 0.02) and a reduction in local relapse rate (P = 0.01). The relative risk of local relapse was 9.1 in tumours without cyclin D1 overexpression (P = 0.01, Cox regression analysis). We conclude that genetic alteration of cyclin D1 is a key abnormality in lung carcinogenesis and may have diagnostic and prognostic importance in the treatment of resectable NSCLC. Images Figure 1 Figure 2 Figure 3 PMID:8562333

Overexpression of an Arabidopsis heterogeneous nuclear ribonucleoprotein gene, AtRNP1, affects plant growth and reduces plant tolerance to drought and salt stresses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhenyu, E-mail: wzy72609@163.com; Zhao, Xiuyang, E-mail: xiuzh@psb.vib-ugent.be; Wang, Bing, E-mail: wangbing@ibcas.ac.cn

Heterogeneous nuclear ribonucleoproteins (hnRNPs) participate in diverse regulations of plant growth and environmental stress responses. In this work, an Arabidopsis hnRNP of unknown function, AtRNP1, was investigated. We found that AtRNP1 gene is highly expressed in rosette and cauline leaves, and slightly induced under drought, salt, osmotic and ABA stresses. AtRNP1 protein is localized to both the nucleus and cytoplasm. We performed homologous overexpression of AtRNP1 and found that the transgenic plants showed shortened root length and plant height, and accelerated flowering. In addition, the transgenic plants also showed reduced tolerance to drought, salt, osmotic and ABA stresses. Further studiesmore » revealed that under both normal and stress conditions, the proline contents in the transgenic plants are markedly decreased, associated with reduced expression levels of a proline synthase gene and several stress-responsive genes. These results suggested that the overexpression of AtRNP1 negatively affects plant growth and abiotic stress tolerance. - Highlights: • AtRNP1 is a widely expressed gene and its expression is slightly induced under abiotic stresses. • AtRNP1 protein is localized to both the nucleus and cytoplasm. • Overexpression of AtRNP1 affects plant growth. • Overexpression of AtRNP1 reduces plant tolerance to drought and salt stresses. • AtRNP1 overexpression plants show decreased proline accumulation and stress-responsive gene expressions.« less

Research on pressure control of pressurizer in pressurized water reactor nuclear power plant

NASA Astrophysics Data System (ADS)

Dai, Ling; Yang, Xuhong; Liu, Gang; Ye, Jianhua; Qian, Hong; Xue, Yang

2010-07-01

Pressurizer is one of the most important components in the nuclear reactor system. Its function is to keep the pressure of the primary circuit. It can prevent shutdown of the system from the reactor accident under the normal transient state while keeping the setting value in the normal run-time. This paper is mainly research on the pressure system which is running in the Daya Bay Nuclear Power Plant. A conventional PID controller and a fuzzy controller are designed through analyzing the dynamic characteristics and calculating the transfer function. Then a fuzzy PID controller is designed by analyzing the results of two controllers. The fuzzy PID controller achieves the optimal control system finally.

Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum

PubMed Central

Echevarría, Wihelma; Leite, M. Fatima; Guerra, Mateus T.; Zipfel, Warren R.; Nathanson, Michael H.

2013-01-01

Calcium is a second messenger in virtually all cells and tissues1. Calcium signals in the nucleus have effects on gene transcription and cell growth that are distinct from those of cytosolic calcium signals; however, it is unknown how nuclear calcium signals are regulated. Here we identify a reticular network of nuclear calcium stores that is continuous with the endoplasmic reticulum and the nuclear envelope. This network expresses inositol 1,4,5-trisphosphate (InsP3) receptors, and the nuclear component of InsP3-mediated calcium signals begins in its locality. Stimulation of these receptors with a little InsP3 results in small calcium signals that are initiated in this region of the nucleus. Localized release of calcium in the nucleus causes nuclear protein kinase C (PKC) to translocate to the region of the nuclear envelope, whereas release of calcium in the cytosol induces translocation of cytosolic PKC to the plasma membrane. Our findings show that the nucleus contains a nucleoplasmic reticulum with the capacity to regulate calcium signals in localized subnuclear regions. The presence of such machinery provides a potential mechanism by which calcium can simultaneously regulate many independent processes in the nucleus. PMID:12717445

Intracellular mediators of transforming growth factor beta superfamily signaling localize to endosomes in chicken embryo and mouse lenses in vivo.

PubMed

Rajagopal, Ramya; Ishii, Shunsuke; Beebe, David C

2007-06-25

Endocytosis is a key regulator of growth factor signaling pathways. Recent studies showed that the localization to endosomes of intracellular mediators of growth factor signaling may be required for their function. Although there is substantial evidence linking endocytosis and growth factor signaling in cultured cells, there has been little study of the endosomal localization of signaling components in intact tissues or organs. Proteins that are downstream of the transforming growth factor-beta superfamily signaling pathway were found on endosomes in chicken embryo and postnatal mouse lenses, which depend on signaling by members of the TGFbeta superfamily for their normal development. Phosphorylated Smad1 (pSmad1), pSmad2, Smad4, Smad7, the transcriptional repressors c-Ski and TGIF and the adapter molecules Smad anchor for receptor activation (SARA) and C184M, localized to EEA-1- and Rab5-positive vesicles in chicken embryo and/or postnatal mouse lenses. pSmad1 and pSmad2 also localized to Rab7-positive late endosomes. Smad7 was found associated with endosomes, but not caveolae. Bmpr1a conditional knock-out lenses showed decreased nuclear and endosomal localization of pSmad1. Many of the effectors in this pathway were distributed differently in vivo from their reported distribution in cultured cells. Based on the findings reported here and data from other signaling systems, we suggest that the localization of activated intracellular mediators of the transforming growth factor-beta superfamily to endosomes is important for the regulation of growth factor signaling.

Nuclear morphometry in histological specimens of canine prostate cancer: Correlation with histological subtypes, Gleason score, methods of collection and survival time.

PubMed

Di Donato, Guido; Laufer-Amorim, Renée; Palmieri, Chiara

2017-10-01

Ten normal prostates, 22 benign prostatic hyperplasia (BPH) and 29 prostate cancer (PC) were morphometrically analyzed with regard to mean nuclear area (MNA), mean nuclear perimeter (MNP), mean nuclear diameter (MND), coefficient of variation of the nuclear area (NACV), mean nuclear diameter maximum (MDx), mean nuclear diameter minimum (MDm), mean nuclear form ellipse (MNFe) and form factor (FF). The relationship between nuclear morphometric parameters and histological type, Gleason score, methods of sample collection, presence of metastases and survival time of canine PC were also investigated. Overall, nuclei from neoplastic cells were larger, with greater variation in nuclear size and shape compared to normal and hyperplastic cells. Significant differences were found between more (small acinar/ductal) and less (cribriform, solid) differentiated PCs with regard to FF (p<0.05). MNA, MNP, MND, MDx, and MDm were significantly correlated with the Gleason score of PC (p<0.05). MNA, MNP, MDx and MNFe may also have important prognostic implications in canine prostatic cancer since negatively correlated with the survival time. Biopsy specimens contained nuclei that were smaller and more irregular in comparison to those in prostatectomy and necropsy specimens and therefore factors associated with tissue sampling and processing may influence the overall morphometric evaluation. The results indicate that nuclear morphometric analysis in combination with Gleason score can help in canine prostate cancer grading, thus contributing to the establishment of a more precise prognosis and patient's management. Copyright © 2017 Elsevier Ltd. All rights reserved.

Contractile properties of skinned muscle fibres from young and adult normal and dystrophic (mdx) mice.

PubMed Central

Williams, D A; Head, S I; Lynch, G S; Stephenson, D G

1993-01-01

1. Single muscle fibres were enzymatically isolated from the soleus and extensor digitorum longus (EDL) muscles of genetically dystrophic mdx and normal (C57BL/10) mice aged 3-6 or 17-23 weeks. 2. Fibres of both muscles were chemically skinned with the non-ionic detergent Triton X-100 (2% v/v). Ca(2+)- and Sr(2+)-activated contractile responses were recorded and comparisons were made between several contractile parameters of various fibre types of normal and dystrophic mice of similar age. 3. There were no significant differences in the following contractile parameters of skinned fibres of normal and mdx mice of the same age: sensitivity to activating Ca2+ (pCa50) or Sr2+ (pSr50) and differential sensitivity to the activating ions (pCa50-pSr50). However the maximum isometric tension (Po) and the frequency of myofibrillar force oscillations in EDL fast-twitch fibres of young mdx mice were significantly lower than those of soleus fast-twitch fibres of the same animals, or fast-twitch fibres (EDL or soleus) of normal mice. 4. Age-related differences were apparent in some contractile parameters of both normal and mdx mice. In particular the steepness of force-pCa and force-pSr curves increased with age in normal mice, yet decreased with age in fibres of mdx mice. 5. A fluorescent probe, ethidium bromide, which interchelates with DNA, was used with laser-scanning confocal microscopy to determine the distribution of myonuclei in fibres. Fibres isolated from either muscle type of normal animals displayed a characteristic peripheral spiral of myonuclei. Fibres from muscles of mdx mice displayed three major patterns of nuclear distribution; the normal peripheral spiral, long central strands of nuclei, and a mixture of these two patterns. 6. The contractile characteristics of mdx fibres were not markedly influenced by the nuclear distribution pattern in that there were no discernible differences in the major contractile parameters (the Hill coefficients nCa and nSr, which are associated with the steepness of the Ca2+ and Sr2+ activation curves, pCa50, pSr50, pCa50-pSr50) of skinned fibres possessing peripheral or central nuclei. However, except for nSr, these values were all lower in individual fibres which displayed similar proportions of central and peripheral nuclei. The presence of mixed nucleation and absence of fibres with embryonic contractile characteristics in mdx mice suggest that the dystrophin-negative fibres can repair locally occurring muscle damage. Images Fig. 1 Fig. 1(Contd.) Fig. 4 Fig. 5 PMID:8487206

Xenon monitoring and the Comprehensive Nuclear-Test-Ban Treaty

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bowyer, Theodore W.

How do you monitor (verify) a CTBT? It is a difficult challenge to monitor the entire world for nuclear tests, regardless of size. Nuclear tests 'normally' occur underground, above ground or underwater. Setting aside very small tests (let's limit our thinking to 1 kiloton or more), nuclear tests shake the ground, emit large amounts of radioactivity, and make loud noises if in the atmosphere (or hydroacoustic waves if underwater)

Compensation as Means for Local Acceptance The Case of the Final Disposal of Spent Nuclear Fuel in Eurajoki, Finland

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kojo, M.

The paper sheds light on the local negotiations on compensation as a part of the site selection for the spent nuclear fuel repository in Finland. The negotiation took place between the representatives of the Municipality of Eurajoki, the nuclear power company Teollisuuden Voima Ltd (TVO) and the nuclear waste management company Posiva Ltd in the late 1990's. The compensation negotiation process and the development of the requirements are elucidated in detail on the basis of the analysis of the minutes of the meetings of the Vuojoki working party. The paper helps to understand the smooth site selection process in Finland.more » The context of the local decision-making is viewed from the policy, institutional and economic aspect. It is concluded in the paper that when trying to understand the progress of the Finnish site selection process more emphasis should be put on the role of TVO, the economic dependency of the Municipality of Eurajoki on TVO and the partnership between TVO and the leading local politicians. (authors)« less

SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity.

PubMed

Yao, Fan; Zhou, Zhicheng; Kim, Jongchan; Hang, Qinglei; Xiao, Zhenna; Ton, Baochau N; Chang, Liang; Liu, Na; Zeng, Liyong; Wang, Wenqi; Wang, Yumeng; Zhang, Peijing; Hu, Xiaoyu; Su, Xiaohua; Liang, Han; Sun, Yutong; Ma, Li

2018-06-11

Dysregulation of YAP localization and activity is associated with pathological conditions such as cancer. Although activation of the Hippo phosphorylation cascade is known to cause cytoplasmic retention and inactivation of YAP, emerging evidence suggests that YAP can be regulated in a Hippo-independent manner. Here, we report that YAP is subject to non-proteolytic, K63-linked polyubiquitination by the SCF SKP2 E3 ligase complex (SKP2), which is reversed by the deubiquitinase OTUD1. The non-proteolytic ubiquitination of YAP enhances its interaction with its nuclear binding partner TEAD, thereby inducing YAP's nuclear localization, transcriptional activity, and growth-promoting function. Independently of Hippo signaling, mutation of YAP's K63-linkage specific ubiquitination sites K321 and K497, depletion of SKP2, or overexpression of OTUD1 retains YAP in the cytoplasm and inhibits its activity. Conversely, overexpression of SKP2 or loss of OTUD1 leads to nuclear localization and activation of YAP. Altogether, our study sheds light on the ubiquitination-mediated, Hippo-independent regulation of YAP.

Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus.

PubMed

Horie, Ryo; Yoneda, Misako; Uchida, Shotaro; Sato, Hiroki; Kai, Chieko

2016-10-01

Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21-24 and 110-139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60-75 and 72-75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm. Copyright © 2016 Elsevier Inc. All rights reserved.

Importance of nuclear localization for the apoptosis-induced activity of a fungal galectin AAL (Agrocybe aegerita lectin)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Yi; Feng, Lei; Tong, Xin

2009-08-28

Agrocybe aegerita lectin (AAL) was identified previously in our group as a novel galectin from medicinal fungi Agrocybe aegerita, and has been shown to effectively induce cancer cell cycle arrest and apoptosis in vitro and tumor regression in vivo. Here, AAL was observed to translocate into the HeLa cell nucleus and induce cell apoptosis when it was predominantly in the nucleus. The N-terminus and C-terminus of AAL were required for nuclear localization. Site mutated proteins were generated based on AAL structure. Dimer interface mutant I25G, carbohydrate recognition domain (CRD) mutant R63H, and loop region mutant L33A could not enter themore » nucleus and lost the ability to induce apoptosis. CRD mutant H59Q and loop region mutant I144G maintained nuclear localization activity, and H59Q retained residual bioability but I144G had no activity, indicating that nuclear localization is important but not sufficient for AAL to become apoptotically active. Our findings provide a novel antitumor mechanism of fungal galectin.« less

Intracellular distribution of a speech/language disorder associated FOXP2 mutant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mizutani, Akifumi; Department of Pediatrics, Jichi Medical University, Yakushiji 3311-1, Shimotsukeshi, Tochigi 329-0498; Matsuzaki, Ayumi

Although a mutation (R553H) in the forkhead box (FOX)P2 gene is associated with speech/language disorder, little is known about the function of FOXP2 or its relevance to this disorder. In the present study, we identify the forkhead nuclear localization domains that contribute to the cellular distribution of FOXP2. Nuclear localization of FOXP2 depended on two distally separated nuclear localization signals in the forkhead domain. A truncated version of FOXP2 lacking the leu-zip, Zn{sup 2+} finger, and forkhead domains that was observed in another patient with speech abnormalities demonstrated an aggregated cytoplasmic localization. Furthermore, FOXP2 (R553H) mainly exhibited a cytoplasmic localizationmore » despite retaining interactions with nuclear transport proteins (importin {alpha} and {beta}). Interestingly, wild type FOXP2 promoted the transport of FOXP2 (R553H) into the nucleus. Mutant and wild type FOXP2 heterodimers in the nucleus or FOXP2 R553H in the cytoplasm may underlie the pathogenesis of the autosomal dominant speech/language disorder.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.

Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associatedmore » proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.« less

Nuclear Architecture of Mouse Spermatocytes: Chromosome Topology, Heterochromatin, and Nucleolus.

PubMed

Berrios, Soledad

2017-01-01

The nuclear organization of spermatocytes in meiotic prophase I is primarily determined by the synaptic organization of the bivalents that are bound by their telomeres to the nuclear envelope and described as arc-shaped trajectories through the 3D nuclear space. However, over this basic meiotic organization, a spermatocyte nuclear architecture arises that is based on higher-ordered patterns of spatial associations among chromosomal domains from different bivalents that are conditioned by the individual characteristics of chromosomes and the opportunity for interactions between their domains. Consequently, the nuclear architecture is species-specific and prone to modification by chromosomal rearrangements. This model is valid for the localization of any chromosomal domain in the meiotic prophase nucleus. However, constitutive heterochromatin plays a leading role in shaping nuclear territories. Thus, the nuclear localization of nucleoli depends on the position of NORs in nucleolar bivalents, but the association among nucleolar chromosomes mainly depends on the presence of constitutive heterochromatin that does not affect the expression of the ribosomal genes. Constitutive heterochromatin and nucleoli form complex nuclear territories whose distribution in the nuclear space is nonrandom, supporting the hypothesis regarding the existence of a species-specific nuclear architecture in first meiotic prophase spermatocytes. © 2017 S. Karger AG, Basel.

Role of Nuclear Pools of Aminoacyl-tRNA Synthetases in tRNA Nuclear Export

PubMed Central

Azad, Abul K.; Stanford, David R.; Sarkar, Srimonti; Hopper, Anita K.

2001-01-01

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 2000a) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export. PMID:11359929

Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export.

PubMed

Azad, A K; Stanford, D R; Sarkar, S; Hopper, A K

2001-05-01

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.

Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

PubMed

Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

2016-03-10

In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

A calreticulin-dependent nuclear export signal is involved in the regulation of liver receptor homologue-1 protein folding.

PubMed

Yang, Feng-Ming; Feng, Shan-Jung; Lai, Tsai-Chun; Hu, Meng-Chun

2015-10-15

As an orphan member of the nuclear receptor family, liver receptor homologue-1 (LRH-1) controls a tremendous range of transcriptional programmes that are essential for metabolism and hormone synthesis. Our previous studies have shown that nuclear localization of the LRH-1 protein is mediated by two nuclear localization signals (NLSs) that are karyopherin/importin-dependent. It is unclear whether LRH-1 can be actively exported from the nucleus to the cytoplasm. In the present study, we describe a nuclear export domain containing two leucine-rich motifs [named nuclear export signal (NES)1 and NES2] within the ligand-binding domain (LBD). Mutation of leucine residues in NES1 or NES2 abolished nuclear export, indicating that both NES1 and NES2 motifs are essential for full nuclear export activity. This NES-mediated nuclear export was insensitive to the chromosomal region maintenance 1 (CRM1) inhibitor leptomycin B (LMB) or to CRM1 knockdown. However, knockdown of calreticulin (CRT) prevented NES-mediated nuclear export. Furthermore, our data show that CRT interacts with LRH-1 and is involved in the nuclear export of LRH-1. With full-length LRH-1, mutation of NES1 led to perinuclear accumulation of the mutant protein. Immunofluorescence analysis showed that these perinuclear aggregates were co-localized with the centrosome marker, microtubule-associated protein 1 light chain 3 (LC3), ubiquitin and heat shock protein 70 (Hsp70), indicating that the mutant was misfolded and sequestered into aggresome-like structures via the autophagic clearance pathway. Our study demonstrates for the first time that LRH-1 has a CRT-dependent NES which is not only required for cytoplasmic trafficking, but also essential for correct protein folding to avoid misfolding-induced aggregation. © 2015 Authors; published by Portland Press Limited.

MNDA binds NPM/B23 and the NPM-MLF1 chimera generated by the t(3;5) associated with myelodysplastic syndrome and acute myeloid leukemia.

PubMed

Xie, J; Briggs, J A; Morris, S W; Olson, M O; Kinney, M C; Briggs, R C

1997-10-01

The myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein expressed specifically in developing cells of the human myelomonocytic lineage, including the end-stage monocytes/macrophages and granulocytes. Nuclear localization, lineage- and stage-specific expression, association with chromatin, and regulation by interferon alpha indicate that this protein is involved in regulating gene expression uniquely associated with the differentiation process and/or function of the monocyte/macrophage. MNDA does not bind specific DNA sequences, but rather a set of nuclear proteins that includes nucleolin (C23). Both in vitro binding assays and co-immunoprecipitation were used to demonstrate that MNDA also binds protein B23 (nucleophosmin/NPM). Three reciprocal chromosome translocations found in certain cases of leukemia/lymphoma involve fusions with the NPM/B23 gene, t(5;17) NPM-RARalpha, t(2;5) NPM-ALK, and the t(3;5) NPM-MLF1. In the current study, MNDA was not able to bind the NPM-ALK chimera originating from the t(2;5) and containing residues 1-117 of NPM. However, MNDA did bind the NPM-MLF1 product of the t(3;5) that contains the N-terminal 175 residues of NPM. The additional 58 amino acids (amino acids 117-175) of the NPM sequence that are contained in the product of the NPM-MLF1 fusion gene relative to the product of the NPM-ALK fusion appear responsible for MNDA binding. This additional NPM sequence contains a nuclear localization signal and clusters of acidic residues believed to bind nuclear localization signals of other proteins. Whereas NPM and nucleolin are primarily localized within the nucleolus, MNDA is distributed throughout the nucleus including the nucleolus, suggesting that additional interactions define overall MNDA localization.

FOXP2 promotes the nuclear translocation of POT1, but FOXP2(R553H), mutation related to speech-language disorder, partially prevents it

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanabe, Yuko; Fujita, Eriko; Department of Pediatrics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498

Highlights: {yields} We isolated protection of telomeres 1 (POT1) as a FOXP2-associated protein by a yeast two-hybrid. {yields} FOXP2 associated and co-localized with POT1 in the nuclei. {yields} FOXP2(R553H) also co-localized with POT1 in both the cytoplasm and nuclei. {yields} FOXP2(R553H) partially prevented the nuclear translocation of POT1. {yields} FOXP2(R553H) mutation may be associated with the pathogenesis of speech-language disorder. -- Abstract: FOXP2 is a forkhead box-containing transcription factor with several recognizable sequence motifs. However, little is known about the FOXP2-associated proteins except for C-terminal binding protein (CtBP). In the present study, we attempted to isolate the FOXP2-associated protein withmore » a yeast two-hybrid system using the C-terminal region, including the forkhead domain, as a bait probe, and identified protection of telomeres 1 (POT1) as a FOXP2-associated protein. Immunoprecipitation assay confirmed the association with FOXP2 and POT1. POT1 alone localized in the cytoplasm but co-localized with FOXP2 and the forkhead domain of FOXP2 in nuclei. However, both FOXP2 with mutated nuclear localization signals and (R553H) mutated forkhead, which is associated with speech-language disorder, prevented the nuclear translocation of POT1. These results suggest that FOXP2 is a binding partner for the nuclear translocation of POT1. As loss of POT1 function induces the cell arrest, the impaired nuclear translocation of POT1 in the developing neuronal cells may be associated with the pathogenesis of speech-language disorder with FOXP2(R553H) mutation.« less

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. © 2014 Wiley Periodicals, Inc.

Exact solution of equations for proton localization in neutron star matter

NASA Astrophysics Data System (ADS)

Kubis, Sebastian; Wójcik, Włodzimierz

2015-11-01

The rigorous treatment of proton localization phenomenon in asymmetric nuclear matter is presented. The solution of proton wave function and neutron background distribution is found by the use of the extended Thomas-Fermi approach. The minimum of energy is obtained in the Wigner-Seitz approximation of a spherically symmetric cell. The analysis of four different nuclear models suggests that the proton localization is likely to take place in the interior of a neutron star.

Activation of peroxisome proliferator-activated receptor-gamma reverses squamous metaplasia and induces transitional differentiation in normal human urothelial cells.

PubMed

Varley, Claire Lucy; Stahlschmidt, Jens; Smith, Barbara; Stower, Michael; Southgate, Jennifer

2004-05-01

We observed that in urothelium, both cornifying and noncornifying forms of squamous metaplasia are accompanied by changes in the localization of the nuclear hormone receptors, peroxisome proliferator activated receptor gamma (PPAR-gamma) and retinoid X receptor (RXR-alpha). To obtain objective evidence for a role for PPAR-gamma-mediated signaling in urothelial differentiation, we examined expression of the cytokeratin isotypes CK13, CK20, and CK14 as indicators of transitional, terminal transitional, and squamous differentiation, respectively, in cultures of normal human urothelial cells. In control culture conditions, normal human urothelial cells showed evidence of squamous differentiation (CK14+, CK13-, CK20-). Treatment with the high-affinity PPAR-gamma agonist, troglitazone (TZ), resulted in gain of CK13 and loss of CK14 protein expression. The effect of TZ was significantly augmented when the autocrine-stimulated epidermal growth factor receptor pathway was inhibited and this resulted in induction of CK20 expression. The RXR-specific inhibitors PA452, HX531, and HX603 inhibited the TZ-induced CK13 expression, supporting a role for RXR in the induction of CK13 expression. Thus, signaling through PPAR-gamma can mediate transitional differentiation of urothelial cells and this is modulated by growth regulatory programs.

Towards hyperpolarized 13C-succinate imaging of brain cancer

PubMed Central

Bhattacharya, Pratip; Chekmenev, Eduard Y.; Perman, William H.; Harris, Kent C.; Lin, Alexander P.; Norton, Valerie A.; Tan, Chou T.; Ross, Brian D.; Weitekamp, Daniel P.

2009-01-01

We describe a novel 13C enriched precursor molecule, sodium 1-13C acetylenedicarboxylate, which after hydrogenation by PASADE-NA (Parahydrogen and Synthesis Allows Dramatically Enhanced Nuclear Alignment) under controlled experimental conditions, becomes hyperpolarized 13C sodium succinate. Fast in vivo 3D FIESTA MR imaging demonstrated that, following carotid arterial injection, the hyperpolarized 13C-succinate appeared in the head and cerebral circulation of normal and tumor-bearing rats. At this time, no in vivo hyperpolarized signal has been localized to normal brain or brain tumor. On the other hand, ex vivo samples of brain harvested from rats bearing a 9L brain tumor, 1 h or more following in vivo carotid injection of hyperpolarized 13C sodium succinate, contained significant concentrations of the injected substrate, 13C sodium succinate, together with 13C maleate and succinate metabolites 1-13C-glutamate, 5-13C-glutamate, 1-13C-glutamine and 5-13C-glutamine. The 13C substrates and products were below the limits of NMR detection in ex vivo samples of normal brain consistent with an intact blood–brain barrier. These ex vivo results indicate that hyperpolarized 13C sodium succinate may become a useful tool for rapid in vivo identification of brain tumors, providing novel biomarkers in 13C MR spectral-spatial images. PMID:17303454

Towards hyperpolarized 13C-succinate imaging of brain cancer

NASA Astrophysics Data System (ADS)

Bhattacharya, Pratip; Chekmenev, Eduard Y.; Perman, William H.; Harris, Kent C.; Lin, Alexander P.; Norton, Valerie A.; Tan, Chou T.; Ross, Brian D.; Weitekamp, Daniel P.

2007-05-01

We describe a novel 13C enriched precursor molecule, sodium 1- 13C acetylenedicarboxylate, which after hydrogenation by PASADENA (Parahydrogen and Synthesis Allows Dramatically Enhanced Nuclear Alignment) under controlled experimental conditions, becomes hyperpolarized 13C sodium succinate. Fast in vivo 3D FIESTA MR imaging demonstrated that, following carotid arterial injection, the hyperpolarized 13C-succinate appeared in the head and cerebral circulation of normal and tumor-bearing rats. At this time, no in vivo hyperpolarized signal has been localized to normal brain or brain tumor. On the other hand, ex vivo samples of brain harvested from rats bearing a 9L brain tumor, 1 h or more following in vivo carotid injection of hyperpolarized 13C sodium succinate, contained significant concentrations of the injected substrate, 13C sodium succinate, together with 13C maleate and succinate metabolites 1- 13C-glutamate, 5- 13C-glutamate, 1- 13C-glutamine and 5- 13C-glutamine. The 13C substrates and products were below the limits of NMR detection in ex vivo samples of normal brain consistent with an intact blood-brain barrier. These ex vivo results indicate that hyperpolarized 13C sodium succinate may become a useful tool for rapid in vivo identification of brain tumors, providing novel biomarkers in 13C MR spectral-spatial images.

Effects of sulfite on the uptake and binding of benzo[a]pyrene diol epoxide in cultured murine respiratory epithelial cells.

PubMed Central

Green, J L; Jones, B C; Reed, G A

1994-01-01

Sulfur dioxide (SO2) may act as a cocarcinogen with benzo[a]pyrene (BaP) in the respiratory tract. We have modeled this effect by examining the interactions of 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with sulfite, the physiological form of SO2, in a murine respiratory epithelial cell line (C10). We exposed C10 cells to [3H]-anti-BPDE and determined the effects of 1 and 10 mM sulfite on the uptake and subcellular localization of labeled products. Autoradiographic analysis showed that sulfite doubled the nuclear localization of anti-BPDE-derived materials after a 4-hr incubation period. The net nuclear localization of anti-BPDE-derived materials was not affected by sulfite during the first 60 min, but nuclear localization continued to increase in the sulfite-containing incubations throughout the 4-hr incubation period. Little increase in nuclear localization of anti-BPDE-derived material was noted in the incubations without sulfite after 60 min. Subcellular fractionation was performed to determine the amount of label associated with cytosolic and nuclear fractions and to determine covalent binding to protein and DNA. Sulfite produced a modest increase in the amount of [3H]-anti-BPDE-derived products bound to protein; however, binding to nuclear DNA increased by more than 200% with 10 mM sulfite. Analysis of the supernatants from the cytosolic and nuclear fractions of cells exposed to anti-BPDE and sulfite demonstrated the presence of 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10c-su lfonate (BPT-10-sulfonate). [3H]-BPT-10-sulfonate was unable to enter C10 cells, suggesting that it is formed intracellularly.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1. Figure 2. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 4. PMID:8033853

PROTEINS IN NUCLEOCYTOPLASMIC INTERACTIONS

PubMed Central

Prescott, David; Goldstein, Lester

1968-01-01

The behavior of nuclear proteins in Amoeba proteus was studied by tritiated amino acid labeling, nuclear transplantation, and cytoplasmic amputation. During prophase at least 77% (but probably over 95%) of the nuclear proteins is released to the cytoplasm. These same proteins return to the nucleus within the first 3 hr of interphase. When cytoplasm is amputated from an ameba in mitosis (shen the nuclear proteins are in the cytoplasm), the resultant daughter nuclei are depleted in the labeled nuclear proteins. The degree of depletion is less than proportional to the amount of cytoplasm removed because a portion of rapidly migrating protein (a nuclear protein that is normally shuttling between nucleus and cytoplasm and is thus also present in the cytoplasm) which would normally remain in the cytoplasm is taken up by the reconstituting daughter nuclei. Cytoplasmic fragments cut from mitotic cells are enriched in both major classes of nuclear proteins, i.e. rapidly migrating protein and slow turn-over protein. An interphase nucleus implanted into such an enucleated cell acquires from the cytoplasm essentially all of the excess nuclear proteins of both classes. The data indicate that there is a lack of binding sites in the cytoplasm for the rapidly migrating nuclear protein. The quantitative aspects of the distribution of rapidly migrating protein between the nucleus and the cytoplasm indicate that the distribution is governed primarily by factors within the nucleus. PMID:5677972

Close but Distinct Regions of Human Herpesvirus 8 Latency-Associated Nuclear Antigen 1 Are Responsible for Nuclear Targeting and Binding to Human Mitotic Chromosomes

PubMed Central

Piolot, Tristan; Tramier, Marc; Coppey, Maité; Nicolas, Jean-Claude; Marechal, Vincent

2001-01-01

Human herpesvirus 8 is associated with all forms of Kaposi's sarcoma, AIDS-associated body cavity-based lymphomas, and some forms of multicentric Castleman's disease. Herpesvirus 8, like other gammaherpesviruses, can establish a latent infection in which viral genomes are stably maintained as multiple episomes. The latent nuclear antigen (LANA or LNAI) may play an essential role in the stable maintenance of latent episomes, notably by interacting concomitantly with the viral genomes and the metaphase chromosomes, thus ensuring an efficient transmission of the neoduplicated episomes to the daughter cells. To identify the regions responsible for its nuclear and subnuclear localization in interphase and mitotic cells, LNAI and various truncated forms were fused to a variant of green fluorescent protein. This enabled their localization and chromosome binding activity to be studied by low-light-level fluorescence microscopy in living HeLa cells. The results demonstrate that nuclear localization of LNAI is due to a unique signal, which maps between amino acids 24 and 30. Interestingly, this nuclear localization signal closely resembles those identified in EBNA1 from Epstein-Barr virus and herpesvirus papio. A region encompassing amino acids 5 to 22 was further proved to mediate the specific interaction of LNA1 with chromatin during interphase and the chromosomes during mitosis. The presence of putative phosphorylation sites in the chromosome binding sites of LNA1 and EBNA1 suggests that their activity may be regulated by specific cellular kinases. PMID:11264383

The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses.

PubMed

Takada, Saeko; Collins, Eric R; Kurahashi, Kayo

2015-05-15

DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage. © 2015 Takada et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

CGGBP1 is a nuclear and midbody protein regulating abscission

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Umashankar, E-mail: umashankar.singh@genpat.uu.se; Westermark, Bengt

2011-01-15

Abscission marks the completion of cell division and its failure is associated with delayed cytokinesis and even tetraploidization. Aberrant abscission and consequential ploidy changes can underlie various diseases including cancer. Midbody, a transient structure formed in the intercellular bridge during telophase, contains several proteins including Aurora kinase B (AURKB), which participate in abscission. We report here an unexpected expression pattern and function of the transcription repressor protein CGG triplet repeat-binding protein 1 (CGGBP1), in normal human fibroblasts. We show that CGGBP1, a chromatin-associated protein, trans-localizes to spindle midzone and midbodies in a manner similar to that of AURKB. CGGBP1 depletionmore » resulted in a cell cycle block at G2, characterized by failure of cells to undergo mitosis and also reduced entry into S phase. Consistent with its presence in the midbodies, live microscopy showed that CGGBP1 deficiency caused mitotic failure at abscission resulting in tetraploidy, which could be rescued by CGGBP1 overexpression. These results show that CGGBP1 is a bona fide midbody protein required for normal abscission and mitosis in general.« less

Meiosis completion and various sperm responses lead to unisexual and sexual reproduction modes in one clone of polyploid Carassius gibelio.

PubMed

Zhang, Jun; Sun, Min; Zhou, Li; Li, Zhi; Liu, Zhen; Li, Xi-Yin; Liu, Xiao-Li; Liu, Wei; Gui, Jian-Fang

2015-06-04

Unisexual polyploid vertebrates are commonly known to reproduce by gynogenesis, parthenogenesis, or hybridogenesis. One clone of polyploid Carassius gibelio has been revealed to possess multiple modes of unisexual gynogenesis and sexual reproduction, but the cytological and developmental mechanisms have remained unknown. In this study, normal meiosis completion was firstly confirmed by spindle co-localization of β-tubulin and Spindlin. Moreover, three types of various nuclear events and development behaviors were revealed by DAPI staining and BrdU-incorporated immunofluorescence detection during the first mitosis in the fertilized eggs by three kinds of different sperms. They include normal sexual reproduction in response to sperm from the same clone male, typical unisexual gynogenesis in response to sperm from the male of another species Cyprinus carpio, and an unusual hybrid-similar development mode in response to sperm from another different clone male. Based on these findings, we have discussed cytological and developmental mechanisms on multiple reproduction modes in the polyploid fish, and highlighted evolutionary significance of meiosis completion and evolutionary consequences of reproduction mode diversity in polyploid vertebrates.

Meiosis completion and various sperm responses lead to unisexual and sexual reproduction modes in one clone of polyploid Carassius gibelio

PubMed Central

Zhang, Jun; Sun, Min; Zhou, Li; Li, Zhi; Liu, Zhen; Li, Xi-Yin; Liu, Xiao-Li; Liu, Wei; Gui, Jian-Fang

2015-01-01

Unisexual polyploid vertebrates are commonly known to reproduce by gynogenesis, parthenogenesis, or hybridogenesis. One clone of polyploid Carassius gibelio has been revealed to possess multiple modes of unisexual gynogenesis and sexual reproduction, but the cytological and developmental mechanisms have remained unknown. In this study, normal meiosis completion was firstly confirmed by spindle co-localization of β-tubulin and Spindlin. Moreover, three types of various nuclear events and development behaviors were revealed by DAPI staining and BrdU-incorporated immunofluorescence detection during the first mitosis in the fertilized eggs by three kinds of different sperms. They include normal sexual reproduction in response to sperm from the same clone male, typical unisexual gynogenesis in response to sperm from the male of another species Cyprinus carpio, and an unusual hybrid-similar development mode in response to sperm from another different clone male. Based on these findings, we have discussed cytological and developmental mechanisms on multiple reproduction modes in the polyploid fish, and highlighted evolutionary significance of meiosis completion and evolutionary consequences of reproduction mode diversity in polyploid vertebrates. PMID:26042995

Natural History of the Central Structural Abnormalities in Choroideremia: A Prospective Cross-Sectional Study.

PubMed

Aleman, Tomas S; Han, Grace; Serrano, Leona W; Fuerst, Nicole M; Charlson, Emily S; Pearson, Denise J; Chung, Daniel C; Traband, Anastasia; Pan, Wei; Ying, Gui-Shuang; Bennett, Jean; Maguire, Albert M; Morgan, Jessica I W

2017-03-01

To describe in detail the central retinal structure of a large group of patients with choroideremia (CHM). A prospective, cross-sectional, descriptive study. Patients (n = 97, age 6-71 years) with CHM and subjects with normal vision (n = 44; ages 10-50 years) were included. Subjects were examined with spectral-domain optical coherence tomography (SD OCT) and near-infrared reflectance imaging. Visual acuity (VA) was measured during their encounter or obtained from recent ophthalmic examinations. Visual thresholds were measured in a subset of patients (n = 24) with automated static perimetry within the central regions (±15°) examined with SD OCT. Visual acuity and visual thresholds; total nuclear layer, inner nuclear layer (INL), and outer nuclear layer (ONL) thicknesses; and horizontal extent of the ONL and the photoreceptor outer segment (POS) interdigitation zone (IZ). Earliest abnormalities in regions with normally appearing retinal pigment epithelium (RPE) were the loss of the POS and ellipsoid zone associated with rod dysfunction. Transition zones (TZs) from relatively preserved retina to severe ONL thinning and inner retinal thickening moved centripetally with age. Most patients (88%) retained VAs better than 20/40 until their fifth decade of life. The VA decline coincided with migration of the TZ near the foveal center. There were outer retinal tubulations in degenerated, nonatrophic retina in the majority (69%) of patients. In general, RPE abnormalities paralleled photoreceptor degeneration, although there were regions with detectable but abnormally thin ONL co-localizing with severe RPE depigmentation and choroidal thinning. Abnormalities of the POS and rod dysfunction are the earliest central abnormalities observed in CHM. Foveal function is relatively preserved until the fifth decade of life. Migration of the TZs to the foveal center with foveal thinning and structural disorganization heralded central VA loss. The relationships established may help outline the eligibility criteria and outcome measures for clinical trials for CHM. Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Environmental assessment proposed license renewal of Nuclear Metals, Inc. Concord, Massachusetts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, R.L.; Easterly, C.E.; Lombardi, C.E.

1997-02-01

The US Nuclear Regulatory Commission (NRC) has prepared this Environmental Assessment (EA) to evaluate environmental issues associated with the renewal of licenses issued by NRC for facilities operated by Nuclear Metals, Inc. (NMI) in Concord, Massachusetts. By renewing the licenses, NRC proposes to allow the continuation of ongoing operations involving radioactive materials at NMI`s facilities. This EA focuses on the potential impacts related to air emissions at NMI during normal (incident-free) operations and accidental releases. Findings indicate that there are only two areas of potential concern. First, modeling results for sulfur dioxide (SO{sub 2}) emissions from the boilers during normalmore » operations indicate that the potential exists for exceeding the short-term National Ambient Air Quality Standards (NAAQS). NMI is prepared to undertake mitigative action to prevent potential exceedances of the short-term SO{sub 2} NAAQS, and the Massachusetts Department of Environmental Protection is prepared to resolve the issue via a permit/approval change or through a Consent Order. Second, in the unlikely event of a severe fire, predicted sulfuric acid (H{sub 2}SO{sub 4}) concentrations based on conservative (upper bound) modeling exceed the Emergency Response Planning Guideline (ERPG) levels. NMI has committed to NRC to give a briefing for local emergency response officials regarding the potential for an accidental H{sub 2}SO{sub 4} release.« less

Propagation Distance of the α-Particle-Induced Bystander Effect: The Role of Nuclear Traversal and Gap Junction Communication

PubMed Central

Gaillard, Sylvain; Pusset, David; de Toledo, Sonia M.; Fromm, Michel; Azzam, Edouard I.

2009-01-01

When cell populations are exposed to low-dose α-particle radiation, a significant fraction of the cells will not be traversed by a radiation track. However, stressful effects occur in both irradiated and bystander cells in the population. Characterizing these effects, and investigating their underlying mechanism(s), is critical to understanding human health risks associated with exposure to α particles. To this end, confluent normal human fibroblast cultures were grown on polyethylene terephthalate foil grafted to an ultrathin solid-state nuclear track detector and exposed under non-perturbing conditions to low-fluence α particles from a broadbeam irradiator. Irradiated and affected bystander cells were localized with micrometer precision. The stress-responsive protein p21Waf1 (also known as CDKN1A) was induced in bystander cells within a 100-µm radius from an irradiated cell. The mean propagation distance ranged from 20 to 40 µm around the intranuclear α-particle impact point, which corresponds to a set of ∼30 cells. Nuclear traversal, induced DNA damage, and gap junction communication were critical contributors to propagation of this stressful effect The strategy described here may be ideal to investigate the size of radiation-affected target and the relative contribution of different cellular organelles to bystander effects induced by energetic particles, which is relevant to radioprotection and cancer radiotherapy. PMID:19580486

The nuclear import factor importin α4 can protect against oxidative stress.

PubMed

Young, Julia C; Ly-Huynh, Jennifer D; Lescesen, Helen; Miyamoto, Yoichi; Browne, Cate; Yoneda, Yoshihiro; Koopman, Peter; Loveland, Kate L; Jans, David A

2013-10-01

The importin (IMP) superfamily of nuclear transport proteins is essential to key developmental pathways, including in the murine testis where expression of the 6 distinct IMPα proteins is highly dynamic. Present predominantly from the spermatocyte stage onwards, IMPα4 is unique in showing a striking nuclear localization, a property we previously found to be linked to maintenance of pluripotency in embryonic stem cells and to the cellular stress response in cultured cells. Here we examine the role of IMPα4 in vivo for the first time using a novel transgenic mouse model in which we overexpress an IMPα4-EGFP fusion protein from the protamine 1 promoter to recapitulate endogenous testicular germ cell IMPα4 expression in spermatids. IMPα4 overexpression did not affect overall fertility, testis morphology/weight or spermatogenic progression under normal conditions, but conferred significantly (>30%) increased resistance to oxidative stress specifically in the spermatid subpopulation expressing the transgene. Consistent with a cell-specific role for IMPα4 in protecting against oxidative stress, haploid germ cells from IMPα4 null mice were significantly (c. 30%) less resistant to oxidative stress than wild type controls. These results from two unique and complementary mouse models demonstrate a novel protective role for IMPα4 in stress responses specifically within haploid male germline cells, with implications for male fertility and genetic integrity. Copyright © 2013 Elsevier B.V. All rights reserved.

Intracellular translocation of calmodulin and Ca{sup 2+}/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gangopadhyay, Jaya Pal, E-mail: jaya@bbri.org; Ikemoto, Noriaki; Department of Neurology, Harvard Medical School, Boston, MA 02115

We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca{sup 2+} leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 ). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in themore » nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca{sup 2+}/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca{sup 2+} transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca{sup 2+} transients results in the appearance of trains of Ca{sup 2+} spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca{sup 2+} events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional sites to activate HT program.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kojo, Matti

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 Finnishmore » 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)« less

Nuclear reprogramming: the strategy used in normal development is also used in somatic cell nuclear transfer and parthenogenesis.

PubMed

Gao, Tianlong; Zheng, Junke; Xing, Fengying; Fang, Haiyan; Sun, Feng; Yan, Ayong; Gong, Xun; Ding, Hui; Tang, Fan; Sheng, Hui Z

2007-02-01

Somatic cell nuclear transfer (SCNT) and parthenogenesis are alternative forms of reproduction and development, building new life cycles on differentiated somatic cell nuclei and duplicated maternal chromatin, respectively. In the preceding paper (Sun F, et al., Cell Res 2007; 17:117-134.), we showed that an "erase-and-rebuild" strategy is used in normal development to transform the maternal gene expression profile to a zygotic one. Here, we investigate if the same strategy also applies to SCNT and parthenogenesis. The relationship between chromatin and chromatin factors (CFs) during SCNT and parthenogenesis was examined using immunochemical and GFP-fusion protein assays. Results from these studies indicated that soon after nuclear transfer, a majority of CFs dissociated from somatic nuclei and were redistributed to the cytoplasm of the egg. The erasure process in oogenesis is recaptured during the initial phase in SCNT. Most CFs entered pseudo-pronuclei shortly after their formation. In parthenogenesis, all parthenogenotes underwent normal oogenesis, and thus had removed most CFs from chromosomes before the initiation of development. The CFs were subsequently re-associated with female pronuclei in time and sequence similar to that in fertilized embryos. Based on these data, we conclude that the "erase-and-rebuild" process observed in normal development also occurs in SCNT and in parthenogenesis, albeit in altered fashions. The process is responsible for transcription reprogramming in these procedures. The "erase" process in SCNT is compressed and the efficiency is compromised, which likely contribute to the developmental defects often observed in nuclear transfer (nt) embryos. Furthermore, results from this study indicated that the cytoplasm of an egg contains most, if not all, essential components for assembling the zygotic program and can assemble them onto appropriate diploid chromatin of distinct origins.

Microscopically based energy density functionals for nuclei using the density matrix expansion. II. Full optimization and validation

NASA Astrophysics Data System (ADS)

Navarro Pérez, R.; Schunck, N.; Dyhdalo, A.; Furnstahl, R. J.; Bogner, S. K.

2018-05-01

Background: Energy density functional methods provide a generic framework to compute properties of atomic nuclei starting from models of nuclear potentials and the rules of quantum mechanics. Until now, the overwhelming majority of functionals have been constructed either from empirical nuclear potentials such as the Skyrme or Gogny forces, or from systematic gradient-like expansions in the spirit of the density functional theory for atoms. Purpose: We seek to obtain a usable form of the nuclear energy density functional that is rooted in the modern theory of nuclear forces. We thus consider a functional obtained from the density matrix expansion of local nuclear potentials from chiral effective field theory. We propose a parametrization of this functional carefully calibrated and validated on selected ground-state properties that is suitable for large-scale calculations of nuclear properties. Methods: Our energy functional comprises two main components. The first component is a non-local functional of the density and corresponds to the direct part (Hartree term) of the expectation value of local chiral potentials on a Slater determinant. Contributions to the mean field and the energy of this term are computed by expanding the spatial, finite-range components of the chiral potential onto Gaussian functions. The second component is a local functional of the density and is obtained by applying the density matrix expansion to the exchange part (Fock term) of the expectation value of the local chiral potential. We apply the UNEDF2 optimization protocol to determine the coupling constants of this energy functional. Results: We obtain a set of microscopically constrained functionals for local chiral potentials from leading order up to next-to-next-to-leading order with and without three-body forces and contributions from Δ excitations. These functionals are validated on the calculation of nuclear and neutron matter, nuclear mass tables, single-particle shell structure in closed-shell nuclei, and the fission barrier of 240Pu. Quantitatively, they perform noticeably better than the more phenomenological Skyrme functionals. Conclusions: The inclusion of higher-order terms in the chiral perturbation expansion seems to produce a systematic improvement in predicting nuclear binding energies while the impact on other observables is not really significant. This result is especially promising since all the fits have been performed at the single-reference level of the energy density functional approach, where important collective correlations such as center-of-mass correction, rotational correction, or zero-point vibrational energies have not been taken into account yet.

Nucleolar localization of cirhin, the protein mutated in North American Indian childhood cirrhosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Bin; Mitchell, Grant A.; Richter, Andrea

2005-12-10

Cirhin (NP{sub 1}16219), the product of the CIRH1A gene is mutated in North American Indian childhood cirrhosis (NAIC/CIRH1A, OMIM 604901), a severe autosomal recessive intrahepatic cholestasis. It is a 686-amino-acid WD40-repeat containing protein of unknown function that is predicted to contain multiple targeting signals, including an N-terminal mitochondrial targeting signal, a C-terminal monopartite nuclear localization signal (NLS) and a bipartite nuclear localization signal (BNLS). We performed the direct determination of subcellular localization of cirhin as a crucial first step in unraveling its biological function. Using EGFP and His-tagged cirhin fusion proteins expressed in HeLa and HepG2, cells we show thatmore » cirhin is a nucleolar protein and that the R565W mutation, for which all NAIC patients are homozygous, has no effect on subcellular localization. Cirhin has an active C-terminal monopartite nuclear localization signal (NLS) and a unique nucleolar localization signal (NrLS) between residues 315 and 432. The nucleolus is not known to be important specifically for intrahepatic cholestasis. These observations provide a new dimension in the study of hereditary cholestasis.« less

Medical aspects of power generation, present and future.

PubMed

Linnemann, R E

1979-01-01

It can be seen that the radiation emissions of nuclear power plants are small indeed, compared to natural background radiation and other man-made sources of radiation. For example, the poulation is exposed to 100 times more radiation from television sets than from nuclear power reactors. The assumed risks to the people in this country from nuclear power reactors are also small compared to the normal risks which are tolerated in this society. The complete elimination of all hazards is a most difficult if not impossible task. If we need and desire a certain level of electrical energy, if we must choose between alternative sourves of the energy, then it is apparent that the total impact on our health from nuclear power generation of electricity, under normal operations and in consideration of catastrophic accident probabilities, is significantly less than that of continuing or increasing use of fossil fuels to generate electricity.

Stalled RNAP-II molecules bound to non-coding rDNA spacers are required for normal nucleolus architecture.

PubMed

Freire-Picos, M A; Landeira-Ameijeiras, V; Mayán, María D

2013-07-01

The correct distribution of nuclear domains is critical for the maintenance of normal cellular processes such as transcription and replication, which are regulated depending on their location and surroundings. The most well-characterized nuclear domain, the nucleolus, is essential for cell survival and metabolism. Alterations in nucleolar structure affect nuclear dynamics; however, how the nucleolus and the rest of the nuclear domains are interconnected is largely unknown. In this report, we demonstrate that RNAP-II is vital for the maintenance of the typical crescent-shaped structure of the nucleolar rDNA repeats and rRNA transcription. When stalled RNAP-II molecules are not bound to the chromatin, the nucleolus loses its typical crescent-shaped structure. However, the RNAP-II interaction with Seh1p, or cryptic transcription by RNAP-II, is not critical for morphological changes. Copyright © 2013 John Wiley & Sons, Ltd.

Effects of the nuclear localization of the N(pro) protein of classical swine fever virus on its virulence in pigs.

PubMed

Li, Yongfeng; Shen, Liang; Sun, Yuan; Wang, Xiao; Li, Chao; Huang, Junhua; Chen, Jianing; Li, Lianfeng; Zhao, Bibo; Luo, Yuzi; Li, Su; Qiu, Hua-Ji

2014-12-05

The N(pro) protein of classical swine fever virus (CSFV) is localized in the cytoplasm and nucleus. However, it is unknown whether the nuclear localization of N(pro) correlates with the virulence of CSFV in the host. Previously, we showed that the N(pro) protein fused with interferon regulatory factor 3 (IRF3) was present only in the cytoplasm. Here, we generated and evaluated a recombinant CSFV vSM-IRF3 harboring the IRF3 gene inserted into the N(pro) gene of the highly virulent CSFV Shimen strain. Compared to the even nuclear and cytoplasmic distribution of the enhanced green fluorescent protein (EGFP)-N(pro) fusion expressed by the recombinant CSFV EGFP-CSFV, vSM-IRF3 expressed an IRF3-N(pro) fusion protein that only was localized in the cytoplasm. vSM-IRF3 was markedly attenuated in vitro and in vivo, and the inoculated pigs were completely protected from lethal CSFV challenge, whereas the parental virus as well as EGFP-CSFV exhibited a typical virulent phenotype. Taken together, the nuclear localization of N(pro) plays a significant role in the CSFV replication and virulence. Copyright © 2014 Elsevier B.V. All rights reserved.

Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix.

PubMed

Yun, Jing-Ping; Chew, Eng Ching; Liew, Choong-Tsek; Chan, John Y H; Jin, Mei-Lin; Ding, Ming-Xiao; Fai, Yam Hin; Li, H K Richard; Liang, Xiao-Man; Wu, Qiu-Liang

2003-12-15

It has become obvious that a better understanding and potential elucidation of the nucleolar phosphoprotein B23 involving in functional interrelationship between nuclear organization and gene expression. In present study, protein B23 expression were investigated in the regenerative hepatocytes at different periods (at days 0, 1, 2, 3, 4, 7) during liver regeneration after partial hepatectomy on the rats with immunohistochemistry and Western blot analysis. Another experiment was done with immunolabeling methods and two-dimensional (2-D) gel electrophoresis for identification of B23 in the regenerating hepatocytes and HepG2 cells (hepatoblastoma cell line) after sequential extraction with detergents, nuclease, and salt. The results showed that its expression in the hepatocytes had a locative move and quantitative change during the process of liver regeneration post-operation. Its immunochemical localization in the hepatocytes during the process showed that it moved from nucleoli of the hepatocytes in the stationary stage to nucleoplasm, cytoplasm, mitotic spindles, and mitotic chromosomes of the hepatocytes in the regenerating livers. It was quantitatively increased progressively to peak level at day 3 post-operation and declined gradually to normal level at day 7. It was detected in nuclear matrix protein (NMP) composition extracted from the regenerating hepatocytes and HepG2 cells and identified with isoelectric point (pI) value of 5.1 and molecular weight of 40 kDa. These results indicated that B23 was a proliferate shuttle protein involving in cell cycle and cell proliferation associated with nuclear matrix. Copyright 2003 Wiley-Liss, Inc.

BAG3 affects the nucleocytoplasmic shuttling of HSF1 upon heat stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Young-Hee; Ahn, Sang-Gun; Kim, Soo-A., E-mail: ksooa@dongguk.ac.kr

2015-08-21

Bcl2-associated athoanogene (BAG) 3 is a member of the co-chaperone BAG family. It is induced by stressful stimuli such as heat shock and heavy metals, and it regulates cellular adaptive responses against stressful conditions. In this study, we identified a novel role for BAG3 in regulating the nuclear shuttling of HSF1 during heat stress. The expression level of BAG3 was induced by heat stress in HeLa cells. Interestingly, BAG3 rapidly translocalized to the nucleus upon heat stress. Immunoprecipitation assay showed that BAG3 interacts with HSF1 under normal and stressed conditions and co-translocalizes to the nucleus upon heat stress. We alsomore » demonstrated that BAG3 interacts with HSF1 via its BAG domain. Over-expression of BAG3 down-regulates the level of nuclear HSF1 by exporting it to the cytoplasm during the recovery period. Depletion of BAG3 using siRNA results in reduced nuclear HSF1 and decreased Hsp70 promoter activity. BAG3 in MEF(hsf1{sup −/−}) cells actively translocalizes to the nucleus upon heat stress suggesting that BAG3 plays a key role in the processing of the nucleocytoplasmic shuttling of HSF1 upon heat stress. - Highlights: • The expression level of BAG3 is induced by heat stress. • BAG3 translocates to the nucleus upon heat stress. • BAG3 interacts with HSF1 and co-localizes to the nucleus. • BAG3 is a key regulator for HSF1 nuclear shuttling.« less

The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme

PubMed Central

Wang, Yi-Ping; Du, Wen-Juan; Huang, Li-Ping; Wei, Yan-Wu; Wu, Hong-Li; Feng, Li; Liu, Chang-Ming

2016-01-01

Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354–370 and that K354, R355, and K367 are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system. PMID:26913023

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Tadanobu; Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, CREST, JST, and COE Program in the 21st Century, Shizuoka 422-8526; Moriyama, Yusuke

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.

14 CFR 1216.306 - Actions normally requiring an EIS.

Code of Federal Regulations, 2013 CFR

2013-01-01

... normally requiring an EIS. (a) NASA will prepare an EIS for actions with the potential to significantly... action or mitigation of its potentially significant impacts. (b) Typical NASA actions normally requiring... material greater than the quantity for which the NASA Nuclear Flight Safety Assurance Manager may grant...

Radioactive fallout projections and arms control agreements: INF (Intermediate-range Nuclear Forces) and START (Strategic Arms Reduction Treaty)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shapiro, C.S.

1988-02-01

Projections of levels of radioactive fallout from a nuclear war are sensitive to assumptions about the structure of the nuclear stockpiles as well as the assumed scenarios for a nuclear war. Recent arms control proposals would change these parameters. This paper examines the implications of the proposed (Intermediate-range Nuclear Forces) INF treaty and (Strategic Arms Reduction Treaty) START on fallout projections from a major nuclear war. We conclude that the INF reductions are likely to have negligible effects on estimates of global and local fallout, whereas the START reductions could result in reductions in estimates of local fallout that rangemore » from significant to dramatic, depending upon the nature of the reduced strategic forces. Should a major war occur, projections of total fatalities from direct effects of blast, thermal radiation, a nd fallout, and the phenomenon known as nuclear winter, would not be significantly affected by INF and START initiatives as now drafted. 14 refs.« less

Opposite nuclear level and binding activity of STAT5B and STAT3 proteins with rat haptoglobin gene under normal and turpentine induced acute phase conditions.

PubMed

Grigorov, I; Lazić, T; Cvetković, I; Milosavljević, T; Petrović, M

2001-01-01

Transcription of the rat gene encoding haptoglobin (Hp) is highly induced during acute phase (AP) response which has been previously shown to be mediated by inducible STAT3 member of the Signal Transducer and Activators of Transcription (STATs) family proteins. In this study, we observed that under normal but not in the turpentine induced AP conditions, another member of the STAT family proteins, STAT5b is expressed and binds to the hormone regulatory element (HRE) of the rat Hp gene. We found that the nuclear amounts of constitutively active STAT5b in rat liver decreased significantly with time of turpentine treatment as opposed to that of cytosol STAT5b, suggesting possible export of constitutive STAT5b from the nucleus. Nuclear accumulation and binding of inducible STAT3 proteins to the rat Hp gene HRE following turpentine treatment implicated that STAT5b negatively regulates Hp gene expression during normal conditions.

STATs get their move on.

PubMed

Reich, Nancy C

2013-10-01

Understanding the mechanisms that regulate dynamic localization of a protein within a cell can provide critical insight to its functional molecular interactions. Signal transducers and activators of transcription (STATs) play essential roles in development, proliferation, and immune defense. However the consequences of STAT hyperactivity can predispose to diseases including autoimmunity and cancer. To function as transcription factors STATs must gain access to the nucleus, and knowledge of the mechanisms that regulate STAT nuclear trafficking can provide a means to control STAT action. This review presents a synopsis of some of the studies that address the nuclear dynamics of the STAT proteins. Evidence suggests that not all STATs are the same. Nuclear import of STAT1 and STAT4 appears linked to their tyrosine phosphorylation and the formation of parallel dimers via reciprocal phosphotyrosine and Src homology 2 domain interactions. This dimer arrangement generates a conformational nuclear localization signal. STAT2 is imported continually to the nucleus in an unphosphorylated state due to its association with IRF9, but the dominant nuclear export signal of STAT2 shuttles the complex back to the cytoplasm. Following STAT2 tyrosine phosphorylation, it can form dimers with STAT1 to affect nuclear import as the trimeric complex (ISGF3). Distinctly, STAT3, STAT5, and STAT6 are continually imported to the nucleus independent of tyrosine phosphorylation. Mutational studies indicate the nuclear localization signals in these STATs require the conformational structure of their coiled-coil domains. Increases in STAT nuclear accumulation following cytokine stimulation appear coordinate with their ability to bind DNA.

CHANGES IN FERRITIN H- AND L-CHAINS IN CANINE LENSES WITH AGE-RELATED NUCLEAR CATARACT

PubMed Central

Goralska, Małgorzata; Nagar, Steven; Colitz, Carmen M.H.; Fleisher, Lloyd N.; McGahan, M. Christine

2014-01-01

PURPOSE To determine potential differences in the characteristics of the iron storage protein, ferritin and its heavy (H) and light (L) subunits in fiber cells from cataractous and normal lenses of older dogs. METHODS Lens fiber cell homogenates were analyzed by SDS-PAGE and ferritin chains were immunodetected with ferritin chain-specific antibodies. Ferritin concentration was measured by ELISA. Immunohistochemistry was used to localize ferritin chains in lens sections. RESULTS The concentration of assembled ferritin was comparable in normal and cataractous lenses of similarly aged dogs. The ferritin L-chain detected in both lens types was modified and was about 11 kDa larger (30 kDa) than standard L-chain (19 kDa) purified from canine liver. The H-chain identified in cataractous fiber cells (29 kDa) differed from 21 kDa standard canine H-chain and from 12 kDa modified H-chain present in fiber cells of normal lenses. Histologic analysis revealed that the H-chain was distributed differently throughout cataractous lenses when compared to normal lenses. There was also a difference in subunit makeup of assembled ferritin between the two lens types. Ferritin from cataractous lenses contained more H-chain and bound 11-fold more iron than ferritin from normal lenses. CONCLUSIONS There are significant differences in the characteristics of ferritin H-chain and its distribution in canine cataractous lenses as compared to normal lenses. The higher content of H-chain in assembled ferritin allows this molecule to sequester more iron. In addition the accumulation of H-chain in deeper fiber layers of the lens may be part of a defense mechanism by which the cataractous lens limits iron-catalyzed oxidative damage. PMID:18708625

Prolonged exposure to particulate chromate inhibits RAD51 nuclear import mediator proteins.

PubMed

Browning, Cynthia L; Wise, John Pierce

2017-09-15

Particulate hexavalent chromium (Cr(VI)) is a human lung carcinogen and a human health concern. The induction of structural chromosome instability is considered to be a driving mechanism of Cr(VI)-induced carcinogenesis. Homologous recombination repair protects against Cr(VI)-induced chromosome damage, due to its highly accurate repair of Cr(VI)-induced DNA double strand breaks. However, recent studies demonstrate Cr(VI) inhibits homologous recombination repair through the misregulation of RAD51. RAD51 is an essential protein in HR repair that facilitates the search for a homologous sequence. Recent studies show prolonged Cr(VI) exposure prevents proper RAD51 subcellular localization, causing it to accumulate in the cytoplasm. Since nuclear import of RAD51 is crucial to its function, this study investigated the effect of Cr(VI) on the RAD51 nuclear import mediators, RAD51C and BRCA2. We show acute (24h) Cr(VI) exposure induces the proper localization of RAD51C and BRCA2. In contrast, prolonged (120h) exposure increased the cytoplasmic localization of both proteins, although RAD51C localization was more severely impaired. These results correlate temporally with the previously reported Cr(VI)-induced RAD51 cytoplasmic accumulation. In addition, we found Cr(VI) does not inhibit interaction between RAD51 and its nuclear import mediators. Altogether, our results suggest prolonged Cr(VI) exposure inhibits the nuclear import of RAD51C, and to a lesser extent, BRCA2, which results in the cytoplasmic accumulation of RAD51. Cr(VI)-induced inhibition of nuclear import may play a key role in its carcinogenic mechanism since the nuclear import of many tumor suppressor proteins and DNA repair proteins is crucial to their function. Copyright © 2017 Elsevier Inc. All rights reserved.

Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals.

PubMed

Zlopasa, Livija; Brachner, Andreas; Foisner, Roland

2016-06-01

Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle1 is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. It contains a GIY-YIG-type nuclease domain, but its function is unknown. The mammalian genome encodes only one other GIY-YIG domain protein, termed Slx1. Slx1 has been described as a resolvase that processes Holliday junctions during homologous recombination-mediated DNA double strand break repair. Resolvase activity is regulated in a spatial and temporal manner during the cell cycle. We hypothesized that Ankle1 may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of Ankle1 activity. Hence, we aimed at identifying the domains mediating Ankle1 shuttling and investigating whether cellular localization is affected during DNA damage response. Sequence analysis predicts the presence of two canonical nuclear import and export signals in Ankle1. Immunofluorescence microscopy of cells expressing wild-type and various mutated Ankle1-fusion proteins revealed a C-terminally located classical monopartite nuclear localization signal and a centrally located CRM1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of Ankle1. These sequences are also functional in heterologous proteins. The predominant localization of Ankle1 in the cytoplasm, however, does not change upon induction of several DNA damage response pathways throughout the cell cycle. We identified the domains mediating nuclear import and export of Ankle1. Ankle1's cellular localization was not affected following DNA damage.

Polyad breaking phenomenon associated with a local-to-normal mode transition and suitability to estimate force constants

NASA Astrophysics Data System (ADS)

Bermúdez-Montaña, M.; Lemus, R.; Castaños, O.

2017-12-01

In a system of two interacting harmonic oscillators a local-to-normal mode transition is manifested as a polyad breaking phenomenon. This phenomenon is associated with the suitability to estimate zeroth-order force constants in the framework of a local mode description. This transition is also exhibited in two interacting Morse oscillators. To study this case, an appropriate parameterisation going from a molecule with local mode behaviour (H2O) to a molecule presenting a normal mode behaviour (CO2) is introduced. Concepts from quantum mechanics like fidelity, entropy and probability density, as well from nonlinear classical mechanics like Poincaré sections are used to detect the transition region. It is found that fidelity and entropy are sensitive complementary properties to detect the local-to-normal transition. Poincaré sections allow the local-to-normal transition to be detected through the appearance of chaos as a consequence of the polyad breaking phenomenon. In addition, two kinds of avoided energy crossings are identified in accordance with the different regions of the spectrum.

Roles of the Nuclear Lamina in Stable Nuclear Association and Assembly of a Herpesviral Transactivator Complex on Viral Immediate-Early Genes

PubMed Central

Silva, Lindsey; Oh, Hyung Suk; Chang, Lynne; Yan, Zhipeng; Triezenberg, Steven J.; Knipe, David M.

2012-01-01

ABSTRACT Little is known about the mechanisms of gene targeting within the nucleus and its effect on gene expression, but most studies have concluded that genes located near the nuclear periphery are silenced by heterochromatin. In contrast, we found that early herpes simplex virus (HSV) genome complexes localize near the nuclear lamina and that this localization is associated with reduced heterochromatin on the viral genome and increased viral immediate-early (IE) gene transcription. In this study, we examined the mechanism of this effect and found that input virion transactivator protein, virion protein 16 (VP16), targets sites adjacent to the nuclear lamina and is required for targeting of the HSV genome to the nuclear lamina, exclusion of heterochromatin from viral replication compartments, and reduction of heterochromatin on the viral genome. Because cells infected with the VP16 mutant virus in1814 showed a phenotype similar to that of lamin A/C−/− cells infected with wild-type virus, we hypothesized that the nuclear lamina is required for VP16 activator complex formation. In lamin A/C−/− mouse embryo fibroblasts, VP16 and Oct-1 showed reduced association with the viral IE gene promoters, the levels of VP16 and HCF-1 stably associated with the nucleus were lower than in wild-type cells, and the association of VP16 with HCF-1 was also greatly reduced. These results show that the nuclear lamina is required for stable nuclear localization and formation of the VP16 activator complex and provide evidence for the nuclear lamina being the site of assembly of the VP16 activator complex. PMID:22251972

Nuclear accumulation of SHIP1 mutants derived from AML patients leads to increased proliferation of leukemic cells.

PubMed

Nalaskowski, Marcus M; Ehm, Patrick; Rehbach, Christoph; Nelson, Nina; Täger, Maike; Modest, Kathrin; Jücker, Manfred

2018-05-28

The inositol 5-phosphatase SHIP1 acts as negative regulator of intracellular signaling in myeloid cells and is a tumor suppressor in myeloid leukemogenesis. After relocalization from the cytoplasm to the plasma membrane SHIP1 terminates PI3-kinase mediated signaling processes. Furthermore, SHIP1 is also found in distinct puncta in the cell nucleus and nuclear SHIP1 has a pro-proliferative function. Here we report the identification of five nuclear export signals (NESs) which regulate together with the two known nuclear localization signals (NLSs) the nucleocytoplasmic shuttling of SHIP1. Mutation of NLSs reduced the nuclear import and mutation of NESs decreased the nuclear export of SHIP1 in the acute myeloid leukemia (AML) cell line UKE-1. Interestingly, four SHIP1 mutants (K210R, N508D, V684E, Q1153L) derived from AML patients showed a nuclear accumulation after expression in UKE-1 cells. In addition, overexpression of the AML patient-derived mutation N508D caused an increased proliferation rate of UKE-1 cells in comparison to wild type SHIP1. Furthermore, we identified serine and tyrosine phosphorylation as a molecular mechanism for the regulation of nucleocytoplasmic shuttling of SHIP1 where tyrosine phosphorylation of distinct residues i.e. Y864, Y914, Y1021 reduces nuclear localization, whereas serine phosphorylation at S933 enhances nuclear localization of SHIP1. In summary, our data further implicate nuclear SHIP1 in cellular signaling and suggest that enhanced accumulation of SHIP1 mutants in the nucleus may be a contributory factor of abnormally high proliferation of AML cells. Copyright © 2018 Elsevier Inc. All rights reserved.

The pathomechanism of cytochrome c oxidase deficiency includes nuclear DNA damage.

PubMed

Douiev, Liza; Saada, Ann

2018-06-07

Mitochondrial cytochrome c oxidase (COX, respiratory chain complex IV), contributes to ATP production via oxidative phosphorylation (OXPHOS). Clinical presentation of COX deficiency is heterogeneous ranging from mild to severe neuromuscular diseases. Anemia is among the symptoms and we have previously reported Fanconi anemia like features in COX4-1 deficiency, suggesting genomic instability and our preliminary results detected nuclear double stranded DNA breaks (DSB). We now quantified the DSB by phospho histone H2AX Ser139 staining of COX4-1 and COX6B1 deficient fibroblasts (225% and 215% of normal, respectively) and confirmed their occurrence by neutral comet assay. We further explored the mechanism of DNA damage by studying normal fibroblasts treated with micromolar concentrations of cyanide (KCN). Present results demonstrate elevated nuclear DSB in cells treated with 50 μM KCN for 24 h (170% of normal) in high-glucose medium conditions where ROS and ATP remain normal, although Glutathione content was partially decreased. In glucose-free and serum-free medium, where growth is hampered, DSB were not elevated. Additionally we demonstrate the benefit of nicotinamide riboside (NR) which ameliorated DSB in COX4-1, COX6B1 and KCN treated cells (130%, 154% and 87% of normal cells, respectively). Conversely a negative effect of a poly[ADP-ribose] polymerase (PARP) inhibitor was found. Although additional investigation is needed, our findings raise the possibility that the pathomechanism of COX deficiency and possibly also in other OXPHOS defects, include nuclear DNA damage resulting from nicotinamide adenine dinucleotide (NAD + ) deficit combined with a replicative state, rather than oxidative stress and energy depletion. Copyright © 2018 Elsevier B.V. All rights reserved.

Fluctuations and symmetry energy in nuclear fragmentation dynamics.

PubMed

Colonna, M

2013-01-25

Within a dynamical description of nuclear fragmentation, based on the liquid-gas phase transition scenario, we explore the relation between neutron-proton density fluctuations and nuclear symmetry energy. We show that, along the fragmentation path, isovector fluctuations follow the evolution of the local density and approach an equilibrium value connected to the local symmetry energy. Higher-density regions are characterized by smaller average asymmetry and narrower isotopic distributions. This dynamical analysis points out that fragment final state isospin fluctuations can probe the symmetry energy of the density domains from which fragments originate.

Role of ANC-1 in tethering nuclei to the actin cytoskeleton.

PubMed

Starr, Daniel A; Han, Min

2002-10-11

Mutations in anc-1 (nuclear anchorage defective) disrupt the positioning of nuclei and mitochondria in Caenorhabditis elegans. ANC-1 is shown to consist of mostly coiled regions with a nuclear envelope localization domain (called the KASH domain) and an actin-binding domain; this structure was conserved with the Drosophila protein Msp-300 and the mammalian Syne proteins. Antibodies against ANC-1 localized cytoplasmically and were enriched at the nuclear periphery in an UNC-84-dependent manner. Overexpression of the KASH domain or the actin-binding domain caused a dominant negative anchorage defect. Thus, ANC-1 may connect nuclei to the cytoskeleton by interacting with UNC-84 at the nuclear envelope and with actin in the cytoplasm.

Alert status of nuclear weapons

NASA Astrophysics Data System (ADS)

Kristensen, Hans M.

2017-11-01

Nuclear Alert Forces. Four nuclear-armed states deploy nuclear warheads on alert, ready to be used on relatively short notice: United States, Russia, France and Britain. Combined, the four countries deploy an estimated 1,869 nuclear alert warheads. Russia and the United States deploy 1,749 alert warheads combined, or 94% of all alert warheads. Despite some debate about possible need to increase readiness of nuclear forces (China, Pakistan), the five other nuclear-armed states (China, Pakistan, India, Israel and North Korea) are thought to store their warheads separate from launchers under normal circumstances. The overall number of alert warheads has remained relatively stable during the past five years.

Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase

PubMed Central

Schawalder, James; Paric, Enesa; Neff, Norma F

2003-01-01

Background Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci. Results Chromatin immunoprecipitation of BLM complexes recovered telomere and ribosomal DNA repeats. The N-terminus of BLM, required for NB localization, is the same as the telomere association domain of BLM. The C-terminus is required for ribosomal DNA localization. BLM localizes primarily to the non-transcribed spacer region of the ribosomal DNA repeat where replication forks initiate. Bloom syndrome cells expressing the deletion alleles lacking the ribosomal DNA and telomere association domains have altered cell cycle populations with increased S or G2/M cells relative to normal. Conclusion These results identify telomere and ribosomal DNA repeated sequence elements as chromosomal targets for the BLM DNA helicase during the S/G2 phase of the cell cycle. BLM is localized in nuclear bodies when it associates with telomeric repeats in both telomerase positive and negative cells. The BLM DNA helicase participates in genomic stability at ribosomal DNA repeats and telomeres. PMID:14577841

Fire resistant nuclear fuel cask

DOEpatents

Heckman, Richard C.; Moss, Marvin

1979-01-01

The disclosure is directed to a fire resistant nuclear fuel cask employing reversibly thermally expansible bands between adjacent cooling fins such that normal outward flow of heat is not interfered with, but abnormal inward flow of heat is impeded or blocked.

Altered Expression of Retinal Molecular Markers in the Canine RPE65 Model of Leber Congenital Amaurosis

PubMed Central

Hernández, Maria; Pearce-Kelling, Susan E.; Rodriguez, F. David; Aguirre, Gustavo D.; Vecino, Elena

2010-01-01

Purpose. Leber congenital amaurosis (LCA) is a group of childhood-onset retinal diseases characterized by severe visual impairment or blindness. One form is caused by mutations in the RPE65 gene, which encodes the retinal pigment epithelium (RPE) isomerase. In this study, the retinal structure and expression of molecular markers for different retinal cell types were characterized, and differences between control and RPE65 mutant dogs during the temporal evolution of the disease were analyzed. Methods. Retinas from normal and mutant dogs of different ages were examined by immunofluorescence with a panel of 16 different antibodies. Results. Cones and rods were preserved in the mutant retinas, and the number of cones was normal. However, there was altered expression of cone arrestin and delocalization of rod opsin. The ON bipolar cells showed sprouting of the dendritic arbors toward the outer nuclear layer (ONL) and retraction of their axons in the inner nuclear layer (INL). A decreased expression of GABA, and an increased expression of intermediate filament glial markers was also found in the mutant retinas. These changes were more evident in the adult than the young mutant retinas. Conclusions. The structure of the retina is well preserved in the mutant retina, but several molecular changes take place in photoreceptors and in bipolar and amacrine cells. Some of these changes are structural, whereas others reflect a change in localization of the examined proteins. This study provides new information that can be applied to the interpretation of outcomes of retinal gene therapy in animal models and humans. PMID:20671290

Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment.

PubMed

Chen, Xinhua; James Swanson, R; Kolb, Juergen F; Nuccitelli, Richard; Schoenbach, Karl H

2009-12-01

Nanosecond pulsed electric fields (nsPEFs) can affect the intracellular structures of cells in vitro. This study shows the direct effects of nsPEFs on tumor growth, tumor volume, and histological characteristics of normal skin and B16-F10 melanoma in SKH-1 mice. A melanoma model was set up by injecting B16-F10 into female SKH-1 mice. After a 100-pulse treatment with an nsPEF (40-kV/cm field strength; 300-ns duration; 30-ns rise time; 2-Hz repetition rate), tumor growth and histology were studied using transillumination, light microscopy with hematoxylin and eosin stain and transmission electron microscopy. Melanin and iron within the melanoma tumor were also detected with specific stains. After nsPEF treatment, tumor development was inhibited with decreased volumes post-nsPEF treatment compared with control tumors (P<0.05). The nsPEF-treated tumor volume was reduced significantly compared with the control group (P<0.01). Hematoxylin and eosin stain and transmission electron microscopy showed morphological changes and nuclear shrinkage in the tumor. Fontana-Masson stain indicates that nsPEF can externalize the melanin. Iron stain suggested nsPEF caused slight hemorrhage in the treated tissue. Histology confirmed that repeated applications of nsPEF disrupted the vascular network. nsPEF treatment can significantly disrupt the vasculature, reduce subcutaneous murine melanoma development, and produce tumor cell contraction and nuclear shrinkage while concurrently, but not permanently, damaging peripheral healthy skin tissue in the treated area, which we attribute to the highly localized electric fields surrounding the needle electrodes.

p54nrb is a new regulator of progression of malignant melanoma.

PubMed

Schiffner, Susanne; Zimara, Nicole; Schmid, Rainer; Bosserhoff, Anja-Katrin

2011-08-01

Nuclear RNA-binding protein p54(nrb) and its murine homolog NonO are known to be involved in a variety of nuclear processes including transcription and RNA processing. Melanoma inhibitory activity (MIA) has been shown to play an essential role in the progression of malignant melanoma and to influence melanoma-associated molecules and pathways in the early tumor formation steps. Interestingly, recent studies suggest that MIA is a regulator of p54(nrb). Here, we show that p54(nrb) is strongly expressed and localized in the nucleus of both melanoma cell lines and melanoma tissue samples compared with normal human melanocytes or normal skin, respectively. Furthermore, all tested melanoma cell lines revealed strong p54(nrb) promoter activity. Treatment with MIA-specific small interfering RNAs showed an influence of MIA on p54(nrb) expression on both messenger RNA (mRNA) and protein level. Knockdown of p54(nrb) protein in melanoma cell lines led to reduced proliferation rates and to a strong decrease in their migratory potential. In addition, attachment to laminin and poly-l-lysine was significantly increased. We could identify Connexin-43 (Cx-43) as a downstream target molecule of p54(nrb) as knockdown of p54(nrb) resulted in enhanced Cx-43 mRNA and protein levels. As a confirmation of these findings, melanoma cell lines showed very low Cx-43 expression levels compared with melanocytes. Our results demonstrate that p54(nrb) is highly expressed in malignant melanoma and, as a MIA target molecule, it seems to be involved in the development and progression of malignant melanoma.

Interplay Between Cytoplasmic and Nuclear Androgen Receptor Splice Variants Mediate Castration Resistance

PubMed Central

Zhan, Yang; Zhang, Guanyi; Wang, Xiaojie; Qi, Yanfeng; Bai, Shanshan; Li, Dongying; Ma, Tianfang; Sartor, Oliver; Flemington, Erik K.; Zhang, Haitao; Lee, Peng; Dong, Yan

2016-01-01

Androgen receptor splice variants (AR-Vs) are implicated in resistance of prostate cancer to androgen-directed therapies. When expressed alone in cells, some AR-Vs (e.g., AR-V7) localize primarily to the nucleus, whereas others (e.g., AR-V1, AR-V4, and AR-V6) localize mainly to the cytoplasm. Significantly, the latter are often co-expressed with the nucleus-predominant AR-Vs and the full-length AR (AR-FL). An important question to be addressed is whether the cytoplasmic-localized AR-Vs play a role in castration-resistant prostate cancer (CRPC) through interaction with the nucleus-predominant AR-Vs and AR-FL. Here, it is demonstrated that AR-V1, -V4, and -V6 can dimerize with both AR-V7 and AR-FL. Consequently, AR-V7 and androgen-bound AR-FL induced nuclear localization of AR-V1, -V4, and -V6, and these variants, in turn, mitigated the ability of the anti-androgen enzalutamide to inhibit androgen-induced AR-FL nuclear localization. Interestingly, the impact of nuclear localization of AR-V4 and -V6 on AR transactivation differs from that of AR-V1. Nuclear localization leads to an increased ability of AR-V4 and -V6 to transactivate both canonical AR targets and AR-V-specific targets and to confer castration-resistant cell growth. However, while AR-V1, which lacks inherent transcriptional activity, appears to activate AR-FL in an androgen-independent manner, it significantly antagonizes AR-V7 transactivation. Together, these data demonstrate that the complex interactions among different AR-Vs and AR-FL play a significant role in castration resistant disease. Implications This study suggests important consequences for clinical castration resistance due to simultaneous expression of AR-FL and AR-Vs in patient tumors and suggests that dissecting these interactions should help develop effective strategies to disrupt AR-V signaling. PMID:27671337

Proton pump inhibitors suppress iNOS-dependent DNA damage in Barrett's esophagus by increasing Mn-SOD expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thanan, Raynoo; Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507; Ma, Ning

2012-05-04

Highlights: Black-Right-Pointing-Pointer Inflammation by Barrett's esophagus (BE) is a risk factor of its adenocarcinoma (BEA). Black-Right-Pointing-Pointer 8-Nitroguanine and 8-oxodG are inflammation-related DNA lesions. Black-Right-Pointing-Pointer DNA lesions and iNOS expression were higher in the order, BEA > BE > normal tissues. Black-Right-Pointing-Pointer Proton pump inhibitors suppress DNA damage by increasing Mn-SOD via Nrf2 activation. Black-Right-Pointing-Pointer DNA lesions can be useful biomarkers to predict risk of BEA in BE patients. -- Abstract: Barrett's esophagus (BE), an inflammatory disease, is a risk factor for Barrett's esophageal adenocarcinoma (BEA). Treatment of BE patients with proton pump inhibitors (PPIs) is expected to reduce the riskmore » of BEA. We performed an immunohistochemical study to examine the formation of nitrative and oxidative DNA lesions, 8-nitroguanine and 8-oxo-7,8-dihydro-2 Prime -deoxygaunosine (8-oxodG), in normal esophageal, BE with pre- and post-treatment by PPIs and BEA tissues. We also observed the expression of an oxidant-generating enzyme (iNOS) and its transcription factor NF-{kappa}B, an antioxidant enzyme (Mn-SOD), its transcription factor (Nrf2) and an Nrf2 inhibitor (Keap1). The immunoreactivity of DNA lesions was significantly higher in the order of BEA > BE > normal tissues. iNOS expression was significantly higher in the order of BEA > BE > normal tissues, while Mn-SOD expression was significantly lower in the order of BEA < BE < normal tissues. Interestingly, Mn-SOD expression and the nuclear localization of Nrf2 were significantly increased, and the formation of DNA lesions was significantly decreased in BE tissues after PPIs treatment for 3-6 months. Keap1 and iNOS expression was not significantly changed by the PPIs treatment in BE tissues. These results indicate that 8-nitroguanine and 8-oxodG play a role in BE-derived BEA. Additionally, PPIs treatment may trigger the activation and nuclear translocation of Nrf2 resulting in the expression of antioxidant genes, leading to DNA damage suppression. These DNA lesions can be useful biomarkers to predict both the risk of BEA and the efficacy of PPIs treatment to prevent BEA in BE patients.« less

Nuclear ferritin: A new role for ferritin in cell biology.

PubMed

Alkhateeb, Ahmed A; Connor, James R

2010-08-01

Ferritin has been traditionally considered a cytoplasmic iron storage protein. However, several studies over the last two decades have reported the nuclear localization of ferritin, specifically H-ferritin, in developing neurons, hepatocytes, corneal epithelial cells, and some cancer cells. These observations encouraged a new perspective on ferritin beyond iron storage, such as a role in the regulation of iron accessibility to nuclear components, DNA protection from iron-induced oxidative damage, and transcriptional regulation. This review will address the translocation and functional significance of nuclear ferritin in the context of human development and disease. The nuclear translocation of ferritin is a selective energy-dependent process that does not seem to require a consensus nuclear localization signal. It is still unclear what regulates the nuclear import/export of ferritin. Some reports have implicated the phosphorylation and O-glycosylation of the ferritin protein in nuclear transport; others suggested the existence of a specific nuclear chaperone for ferritin. The data argue strongly for nuclear ferritin as a factor in human development and disease. Ferritin can bind and protect DNA from oxidative damage. It also has the potential of playing a regulatory role in transcription. Nuclear ferritin represents a novel new outlook on ferritin functionality beyond its classical role as an iron storage molecule. Copyright 2010 Elsevier B.V. All rights reserved.

77 FR 8904 - Entergy Nuclear Indian Point 3, LLC.; Entergy Nuclear Operations, Inc., Indian Point Nuclear...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-15

... that the fire loading in this area is low and that fixed combustibles consist of cable insulation... reactor coolant system decay heat removal when the normal feedwater system is not available, such as... coolant system heat removal function and that the III.G.2 exemption for Fire Zone 23 remains valid. 3.2...

Evaluation of Radiation Impacts of Spent Nuclear Fuel Storage (SNFS-2) of Chernobyl NPP - 13495

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paskevych, Sergiy; Batiy, Valiriy; Sizov, Andriy

2013-07-01

Radiation effects are estimated for the operation of a new dry storage facility for spent nuclear fuel (SNFS-2) of Chernobyl NPP RBMK reactors. It is shown that radiation exposure during normal operation, design and beyond design basis accidents are minor and meet the criteria for safe use of radiation and nuclear facilities in Ukraine. (authors)

NIMBY, CLAMP, and the location of new nuclear-related facilities: U.S. national and 11 site-specific surveys.

PubMed

Greenberg, Michael R

2009-09-01

Public and political opposition have made finding locations for new nuclear power plants, waste management, and nuclear research and development facilities a challenge for the U.S. government and the nuclear industry. U.S. government-owned properties that already have nuclear-related activities and commercial nuclear power generating stations are logical locations. Several studies and utility applications to the Nuclear Regulatory Commission suggest that concentrating locations at major plants (CLAMP) has become an implicit siting policy. We surveyed 2,101 people who lived within 50 miles of 11 existing major nuclear sites and 600 who lived elsewhere in the United States. Thirty-four percent favored CLAMP for new nuclear power plants, 52% for waste management facilities, and 50% for new nuclear laboratories. College educated, relatively affluent male whites were the strongest CLAMP supporters. They disproportionately trusted those responsible for the facilities and were not worried about existing nuclear facilities or other local environmental issues. Notably, they were concerned about continuing coal use. Not surprisingly, CLAMP proponents tended to be familiar with their existing local nuclear site. In short, likely CLAMP sites have a large and politically powerful core group to support a CLAMP policy. The challenge to proponents of nuclear technologies will be to sustain this support and expand the base among those who clearly are less connected and receptive to new nearby sites.

Experimental and Theoretical Understanding of Neutron Capture on Uranium Isotopes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ullmann, John Leonard

2017-09-21

Neutron capture cross sections on uranium isotopes are important quantities needed to model nuclear explosion performance, nuclear reactor design, nuclear test diagnostics, and nuclear forensics. It has been difficult to calculate capture accurately, and factors of 2 or more be- tween calculation and measurements are not uncommon, although normalization to measurements of the average capture width and nuclear level density can improve the result. The calculations of capture for 233,235,237,239U are further complicated by the need to accurately include the fission channel.

Cytoplasmic and nuclear localization of cadherin in honey bee (Apis mellifera L.) gonads.

PubMed

Florecki, Mônica M; Hartfelder, Klaus

2011-01-01

Cadherins are crucial molecules mediating cell-cell interactions between somatic and germline cells in insect and mammalian male and female gonads. We analysed the presence and localization of cadherins in ovaries of honeybee queens and in testes of drones. Transcripts representing two classical cadherins, E-cadherin (shotgun) and N-cadherin, as well as three protocadherins (Starry night, Fat and Fat-like) were detected in gonads of both sexes. Pan-cadherin antibodies, which most probably detect a honeybee N-cadherin, were used in immunolocalization analyses. In the germarium of ovarioles, cadherin-IR (cadherin immunoreactivity) was evidenced as homogeneously distributed in the cytoplasm and as nuclear foci, in both germline and somatic cells. It was also detected in polyfusomes and ring canals. In testiolar tubules, cadherin-IR showed a cytoplasmic and nuclear distributon alike in ovaries. The unexpected nuclear localization and cytoplasmic distribution in ovaries and testes were corroborated by immunogold electron microscopy, which revealed cadherin aggregates associated with electron-dense nuclear structures. With respect to cadherin localization, the honeybee differs from Drosophila, the model for gametogenesis in insects, raising the question as to how differences among solitary and social species may be built into and generated from the general architecture of polytrophic meroistic ovaries. It also indicates the possibility of divergent roles for cadherin in the functional architecture of insect gonads, in general, especially in taxa with high reproductive output.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andrews, Joel F.; Sykora, Landon J.; Barik Letostak, Tiasha

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 implymore » 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).« less

Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji, E-mail: tanaka-kj@igakuken.or.jp

2013-07-05

Highlights: •We succeeded to control the proteasome localization by the anchor-away technique. •Nuclear proteasome-depleted cells showed a lethal phenotype. •Cytoplasmic proteasomes are not indispensable for cell growth in dividing cells. -- Abstract: The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specificmore » compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.« less

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

Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms.

PubMed Central

Zhao, Xiangshan; Gan, Lixia; Pan, Haiyun; Kan, Donghui; Majeski, Michael; Adam, Stephen A; Unterman, Terry G

2004-01-01

FOXO1, a Forkhead transcription factor, is an important target of insulin and growth factor action. Phosphorylation of Thr-24, Ser-256 and Ser-319 promotes nuclear exclusion of FOXO1, yet the mechanisms regulating nuclear/cytoplasmic shuttling of FOXO1 are poorly understood. Previous studies have identified an NLS (nuclear localization signal) in the C-terminal basic region of the DBD (DNA-binding domain), and a leucine-rich, leptomycin-B sensitive NES (nuclear export signal) located further downstream. Here, we find that other elements in the DBD also contribute to nuclear localization, and that multiple mechanisms contribute to nuclear exclusion of FOXO1. Phosphorylation of Ser-319 and a cluster of nearby residues (Ser-322, Ser-325 and Ser-329) functions co-operatively with the nearby NES to promote nuclear exclusion. The N-terminal region of FOXO1 (amino acids 1-149) also is sufficient to promote nuclear exclusion, and does so through multiple mechanisms. Amino acids 1-50 are sufficient to promote nuclear exclusion of green fluorescent protein fusion proteins, and the phosphorylation of Thr-24 is required for this effect. A leucine-rich, leptomycin B-sensitive export signal is also present nearby. Phosphorylated FOXO1 binds 14-3-3 proteins, and co-precipitation studies with tagged proteins indicate that 14-3-3 binding involves co-operative interactions with both Thr-24 and Ser-256. Ser-256 is located in the C-terminal region of the DBD, where 14-3-3 proteins may interfere both with DNA-binding and with nuclear-localization functions. Together, these studies demonstrate that multiple elements contribute to nuclear/cytoplasmic shuttling of FOXO1, and that phosphorylation and 14-3-3 binding regulate the cellular distribution and function of FOXO1 through multiple mechanisms. The presence of these redundant mechanisms supports the concept that the regulation of FOXO1 function plays a critical role in insulin and growth factor action. PMID:14664696

Effects of pituitary dwarfism in the mouse on fast and slow skeletal muscles.

PubMed

Stickland, N C; Crook, A R; Sutton, C M

1994-01-01

The Snell dwarf mouse exhibits impaired growth of the anterior pituitary resulting in reduced levels of growth hormone and thyroid stimulating hormone. Ten dwarf mice and 10 phenotypically normal littermates were killed at 33 days of age. M. biceps brachii (a predominantly fast muscle) and m. soleus (a relatively slow muscle) were removed from each animal and complete frozen transverse sections obtained. Serial sections were reacted for various enzyme activities in order to identify muscle fibre types. There was no difference in the total number of muscle fibres in m. biceps brachii but a small difference in m. soleus between normal and dwarf mice. There were marked differences in the size of all fibre types between normal and dwarf mice with the largest differences in m. soleus. The percentage of slow oxidative fibres was similar (about 32%) in both groups of mice for m. soleus but there was a marked difference for this fibre type in m. biceps brachii being about 1.5% in normal mice and 8.0% in dwarf mice. This may be related to a difference in levels of thyroid hormone. Nuclear density was very significantly greater in dwarf muscles although total nuclear numbers were less than in normal muscles. These differences are most likely due to growth hormone levels. Differences in nuclear content were much greater in m. soleus than in m. biceps brachii.

Identifying biomarkers of papillary renal cell carcinoma associated with pathological stage by weighted gene co-expression network analysis.

PubMed

He, Zhongshi; Sun, Min; Ke, Yuan; Lin, Rongjie; Xiao, Youde; Zhou, Shuliang; Zhao, Hong; Wang, Yan; Zhou, Fuxiang; Zhou, Yunfeng

2017-04-25

Although papillary renal cell carcinoma (PRCC) accounts for 10%-15% of renal cell carcinoma (RCC), no predictive molecular biomarker is currently applicable to guiding disease stage of PRCC patients. The mRNASeq data of PRCC and adjacent normal tissue in The Cancer Genome Atlas was analyzed to identify 1148 differentially expressed genes, on which weighted gene co-expression network analysis was performed. Then 11 co-expressed gene modules were identified. The highest association was found between blue module and pathological stage (r = 0.45) by Pearson's correlation analysis. Functional enrichment analysis revealed that biological processes of blue module focused on nuclear division, cell cycle phase, and spindle (all P < 1e-10). All 40 hub genes in blue module can distinguish localized (pathological stage I, II) from non-localized (pathological stage III, IV) PRCC (P < 0.01). A good molecular biomarker for pathological stage of RCC must be a prognostic gene in clinical practice. Survival analysis was performed to reversely validate if hub genes were associated with pathological stage. Survival analysis unveiled that all hub genes were associated with patient prognosis (P < 0.01).The validation cohort GSE2748 verified that 30 hub genes can differentiate localized from non-localized PRCC (P < 0.01), and 18 hub genes are prognosis-associated (P < 0.01).ROC curve indicated that the 17 hub genes exhibited excellent diagnostic efficiency for localized and non-localized PRCC (AUC > 0.7). These hub genes may serve as a biomarker and help to distinguish different pathological stages for PRCC patients.

Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eto, Masumi, E-mail: masumi.eto@jefferson.edu; Kirkbride, Jason A.; Chugh, Rishika

2013-04-26

Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentratedmore » in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.« less

Genetic variability in captive populations of the stingless bee Tetragonisca angustula.

PubMed

Santiago, Leandro R; Francisco, Flávio O; Jaffé, Rodolfo; Arias, Maria C

2016-08-01

Low genetic variability has normally been considered a consequence of animal husbandry and a major contributing factor to declining bee populations. Here, we performed a molecular analysis of captive and wild populations of the stingless bee Tetragonisca angustula, one of the most commonly kept species across South America. Microsatellite analyses showed similar genetic variability between wild and captive populations However, captive populations showed lower mitochondrial genetic variability. Male-mediated gene flow, transport and division of nests are suggested as the most probable explanations for the observed patterns of genetic structure. We conclude that increasing the number of colonies kept through nest divisions does not negatively affect nuclear genetic variability, which seems to be maintained by small-scale male dispersal and human-mediated nest transport. However, the transport of nests from distant localities should be practiced with caution given the high genetic differentiation observed between samples from western and eastern areas. The high genetic structure verified is the result of a long-term evolutionary process, and bees from distant localities may represent unique evolutionary lineages.

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…

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP)

PubMed Central

Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R. Max; Tu, Benjamin P.; MacMillan, John B.; De Brabander, Jef K.; Veech, Richard L.; Uyeda, Kosaku

2016-01-01

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. PMID:26984404

Nuclear transporters in a multinucleated organism: functional and localization analyses in Aspergillus nidulans

PubMed Central

Markina-Iñarrairaegui, Ane; Etxebeste, Oier; Herrero-García, Erika; Araújo-Bazán, Lidia; Fernández-Martínez, Javier; Flores, Jairo A.; Osmani, Stephen A.; Espeso, Eduardo A.

2011-01-01

Nuclear transporters mediate bidirectional macromolecule traffic through the nuclear pore complex (NPC), thus participating in vital processes of eukaryotic cells. A systematic functional analysis in Aspergillus nidulans permitted the identification of 4 essential nuclear transport pathways of a hypothetical number of 14. The absence of phenotypes for most deletants indicates redundant roles for these nuclear receptors. Subcellular distribution studies of these carriers show three main distributions: nuclear, nucleocytoplasmic, and in association with the nuclear envelope. These locations are not specific to predicted roles as exportins or importins but indicate that bidirectional transport may occur coordinately in all nuclei of a syncytium. Coinciding with mitotic NPC rearrangements, transporters dynamically modified their localizations, suggesting supplementary roles to nucleocytoplasmic transport specifically during mitosis. Loss of transportin-SR and Mex/TAP from the nuclear envelope indicates absence of RNA transport during the partially open mitosis of Aspergillus, whereas nucleolar accumulation of Kap121 and Kap123 homologues suggests a role in nucleolar disassembly. This work provides new insight into the roles of nuclear transporters and opens an avenue for future studies of the molecular mechanisms of transport among nuclei within a common cytoplasm, using A. nidulans as a model organism. PMID:21880896

NLSdb-major update for database of nuclear localization signals and nuclear export signals.

PubMed

Bernhofer, Michael; Goldberg, Tatyana; Wolf, Silvana; Ahmed, Mohamed; Zaugg, Julian; Boden, Mikael; Rost, Burkhard

2018-01-04

NLSdb is a database collecting nuclear export signals (NES) and nuclear localization signals (NLS) along with experimentally annotated nuclear and non-nuclear proteins. NES and NLS are short sequence motifs related to protein transport out of and into the nucleus. The updated NLSdb now contains 2253 NLS and introduces 398 NES. The potential sets of novel NES and NLS have been generated by a simple 'in silico mutagenesis' protocol. We started with motifs annotated by experiments. In step 1, we increased specificity such that no known non-nuclear protein matched the refined motif. In step 2, we increased the sensitivity trying to match several different families with a motif. We then iterated over steps 1 and 2. The final set of 2253 NLS motifs matched 35% of 8421 experimentally verified nuclear proteins (up from 21% for the previous version) and none of 18 278 non-nuclear proteins. We updated the web interface providing multiple options to search protein sequences for NES and NLS motifs, and to evaluate your own signal sequences. NLSdb can be accessed via Rostlab services at: https://rostlab.org/services/nlsdb/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulation of subcellular localization of the Aryl Hydrocarbon Receptor (AhR)

USGS Publications Warehouse

Richter, Catherine A.; Tillitt, Donald E.; Hannink, Mark

2001-01-01

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxin and other xenobiotics. In the absence of exogenous ligand, AhR is cytosolic. We investigated how AhR is retained in the cytosol and how dioxin induces AhR to move to the nucleus. Disruption of nuclear export of AhR by the nuclear export inhibitor leptomycin B (LMB) or by mutation of the AhR nuclear export signal resulted in nuclear accumulation of AhR in the absence of exogenous ligand. Mutation of the AhR nuclear localization signal resulted in defects in nuclear import of AhR in both the presence and the absence of exogenous ligand. Dioxin treatment caused a more rapid accumulation of AhR in the nucleus than LMB treatment. In the presence of both dioxin and LMB, nuclear accumulation of AhR was more rapid than in the presence of dioxin alone. Our results show that AhR shuttles between the nucleus and the cytosol in the absence of exogenous ligand. Binding of ligand induces an increase in the rate of nuclear import of AhR but does not eliminate nuclear export of AhR.

Nuclear NF-kappaB p65 phosphorylation at serine 276 by protein kinase A contributes to the malignant phenotype of head and neck cancer.

PubMed

Arun, Pattatheyil; Brown, Matthew S; Ehsanian, Reza; Chen, Zhong; Van Waes, Carter

2009-10-01

Aberrant nuclear activation and phosphorylation of the canonical NF-kappaB subunit RELA/p65 at Serine-536 by inhibitor kappaB kinase is prevalent in head and neck squamous cell carcinoma (HNSCC), but the role of other kinases in NF-kappaB activation has not been well defined. Here, we investigated the prevalence and function of p65-Ser276 phosphorylation by protein kinase A (PKA) in the malignant phenotype and gene transactivation, and studied p65-Ser276 as a potential target for therapy. Phospho and total p65 protein expression and localization were determined in HNSCC tissue array and in cell lines. The effects of the PKA inhibitor H-89 on NF-kappaB activation, downstream gene expression, cell proliferation and cell cycle were examined. Knockdown of PKA by specific siRNA confirmed the specificity. NF-kappaB p65 phosphorylated at Ser276 was prevalent in HNSCC and adjacent dysplastic mucosa, but localized to the cytoplasm in normal mucosa. In HNSCC lines, tumor necrosis factor-alpha (TNF-alpha) significantly increased, whereas H-89 inhibited constitutive and TNF-alpha-induced nuclear p65 (Ser276) phosphorylation, and significantly suppressed NF-kappaB and target gene IL-8 reporter activity. Knockdown of PKA by small interfering RNA inhibited NF-kappaB, IL-8, and BCL-XL reporter gene activities. H-89 suppressed cell proliferation, induced cell death, and blocked the cell cycle in G(1)-S phase. Consistent with its biological effects, H-89 down-modulated expression of NF-kappaB-related genes Cyclin D1, BCL2, BCL-XL, COX2, IL-8, and VEGF, as well as induced cell cycle inhibitor p21(CIP1/WAF1), while suppressing proliferative marker Ki67. NF-kappaB p65 (Ser276) phosphorylation by PKA promotes the malignant phenotype and holds potential as a therapeutic target in HNSCC.

14 CFR § 1216.306 - Actions normally requiring an EIS.

Code of Federal Regulations, 2014 CFR

2014-01-01

...) § 1216.306 Actions normally requiring an EIS. (a) NASA will prepare an EIS for actions with the potential...) Typical NASA actions normally requiring an EIS include: (1) Development and operation of new launch... using a total quantity of radioactive material greater than the quantity for which the NASA Nuclear...

Function of Nup98 subtypes and their fusion proteins, Nup98-TopIIβ and Nup98-SETBP1 in nuclear-cytoplasmic transport.

PubMed

Saito, Shoko; Yokokawa, Takafumi; Iizuka, Gemmei; Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

2017-05-20

Nup98 is a component of the nuclear pore complex. The nup98-fusion genes derived by chromosome translocations are involved in hematopoietic malignancies. Here, we investigated the functions of Nup98 isoforms and two unexamined Nup98-fusion proteins, Nup98-TopIIβ and Nup98-SETBP1. We first demonstrated that two Nup98 isoforms are expressed in various mouse tissues and similarly localized in the nucleus and the nuclear envelope. We also showed that Nup98-TopIIβ and Nup98-SETBP1 are localized in the nucleus and partially co-localized with full-length Nup98 and a nuclear export receptor XPO1. We demonstrated that Nup98-TopIIβ and Nup98-SETBP1 negatively regulate the XPO1-mediated protein export. Our results will contribute to the understanding of the molecular mechanism by which the Nup98-fusion proteins induce tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Cyclophilin B enhances HIV-1 Infection

PubMed Central

DeBoer, Jason; Madson, Christian J.; Belshan, Michael

2016-01-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. PMID:26774171

Cyclophilin B enhances HIV-1 infection.

PubMed

DeBoer, Jason; Madson, Christian J; Belshan, Michael

2016-02-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. Copyright © 2015 Elsevier Inc. All rights reserved.

Nucleocytoplasmic shuttling of hexokinase II in a cancer cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neary, Catherine L., E-mail: nearycl@umdnj.edu; Pastorino, John G.

2010-04-16

In yeast, the hexokinase type II enzyme (HXKII) translocates to the nucleus in the presence of excess glucose, and participates in glucose repression. However, no evidence has suggested a nuclear function for HXKII in mammalian cells. Herein, we present data showing nuclear localization of HXKII in HeLa cells, both by immunocytochemistry and subcellular fractionation. HXKII is extruded from the nucleus, at least in part, by the activity of the exportin 1/CrmA system, as demonstrated by increased nuclear expression and decreased cytoplasmic expression after incubation with leptomycin B, a bacterially-derived exportin inhibitor. Furthermore, cytoplasmic localization of HXKII is dependent on itsmore » enzymatic activity, as inhibiting HXKII activity using 2-deoxy-D-glucose (2DG) increased nuclear localization. This effect was more significant in cells incubated in the absence of glucose for 24 h prior to addition of 2DG. Regulated translocation of HXKII to the nucleus of mammalian cells could represent a previously unknown glucose-sensing mechanism.« less

Nonlocal Polarization Feedback in a Fractional Quantum Hall Ferromagnet.

PubMed

Hennel, Szymon; Braem, Beat A; Baer, Stephan; Tiemann, Lars; Sohi, Pirouz; Wehrli, Dominik; Hofmann, Andrea; Reichl, Christian; Wegscheider, Werner; Rössler, Clemens; Ihn, Thomas; Ensslin, Klaus; Rudner, Mark S; Rosenow, Bernd

2016-04-01

In a quantum Hall ferromagnet, the spin polarization of the two-dimensional electron system can be dynamically transferred to nuclear spins in its vicinity through the hyperfine interaction. The resulting nuclear field typically acts back locally, modifying the local electronic Zeeman energy. Here we report a nonlocal effect arising from the interplay between nuclear polarization and the spatial structure of electronic domains in a ν=2/3 fractional quantum Hall state. In our experiments, we use a quantum point contact to locally control and probe the domain structure of different spin configurations emerging at the spin phase transition. Feedback between nuclear and electronic degrees of freedom gives rise to memristive behavior, where electronic transport through the quantum point contact depends on the history of current flow. We propose a model for this effect which suggests a novel route to studying edge states in fractional quantum Hall systems and may account for so-far unexplained oscillatory electronic-transport features observed in previous studies.

Glutathione S-transferase (placental) as a marker of transformation in the human cervix uteri: an immunohistochemical study.

PubMed Central

Randall, B. J.; Angus, B.; Akiba, R.; Hall, A.; Cattan, A. R.; Proctor, S. J.; Jones, R. A.; Horne, C. H.

1990-01-01

Using an indirect immunohistochemical technique on paraffin sections, employing a polyclonal antibody to the acidic (placental) form of glutathione-S-transferase (GST), we have evaluated cytoplasmic and nuclear staining in a series of 67 cervical biopsies including normal non neoplastic tissue, immature squamous metaplasia, all grades of cervical intraepithelial neoplasia (CIN) and invasive carcinomas of keratinising and non-keratinising types. No differences in cytoplasmic staining between the varied lesions studied were seen. However, there were marked differences in nuclear staining. While normal non-neoplastic stratified squamous epithelium showed weak staining of the lower one-third of the epithelium only, in immature squamous metaplasia and in all grades of CIN there was intense nuclear staining in all layers of the epithelium. Invasive carcinomas showed generally less intense nuclear staining than CIN lesions. Endocervical cell nuclei also showed intense nuclear staining. These findings indicate that GST is of limited use as a marker of transformation in the human cervix uteri. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2223578

48 CFR 926.7103 - Requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... preference in hiring to an eligible employee of Department of Energy Defense Nuclear Facilities. This right... and subcontractors employed at Department of Energy Defense Nuclear Facilities, to the extent... implementation of Section 3161 at the Department of Energy Defense Nuclear Facility and local counsel, should...

48 CFR 926.7103 - Requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... preference in hiring to an eligible employee of Department of Energy Defense Nuclear Facilities. This right... and subcontractors employed at Department of Energy Defense Nuclear Facilities, to the extent... implementation of Section 3161 at the Department of Energy Defense Nuclear Facility and local counsel, should...

48 CFR 926.7103 - Requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... preference in hiring to an eligible employee of Department of Energy Defense Nuclear Facilities. This right... and subcontractors employed at Department of Energy Defense Nuclear Facilities, to the extent... implementation of Section 3161 at the Department of Energy Defense Nuclear Facility and local counsel, should...

48 CFR 926.7103 - Requirements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... preference in hiring to an eligible employee of Department of Energy Defense Nuclear Facilities. This right... and subcontractors employed at Department of Energy Defense Nuclear Facilities, to the extent... implementation of Section 3161 at the Department of Energy Defense Nuclear Facility and local counsel, should...

48 CFR 926.7103 - Requirements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... preference in hiring to an eligible employee of Department of Energy Defense Nuclear Facilities. This right... and subcontractors employed at Department of Energy Defense Nuclear Facilities, to the extent... implementation of Section 3161 at the Department of Energy Defense Nuclear Facility and local counsel, should...

Do nuclear collisions create a locally equilibrated quark–gluon plasma?

DOE PAGES

Romatschke, P.

2017-01-10

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.

A Role for Myosin Va in Human Cytomegalovirus Nuclear Egress.

PubMed

Wilkie, Adrian R; Sharma, Mayuri; Pesola, Jean M; Ericsson, Maria; Fernandez, Rosio; Coen, Donald M

2018-03-15

Herpesviruses replicate and package their genomes into capsids in replication compartments within the nuclear interior. Capsids then move to the inner nuclear membrane for envelopment and release into the cytoplasm in a process called nuclear egress. We previously found that nuclear F-actin is induced upon infection with the betaherpesvirus human cytomegalovirus (HCMV) and is important for nuclear egress and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Despite these and related findings, it has not been shown that any specific motor protein is involved in herpesvirus nuclear egress. In this study, we have investigated whether the host motor protein, myosin Va, could be fulfilling this role. Using immunofluorescence microscopy and coimmunoprecipitation, we observed associations between a nuclear population of myosin Va and the viral major capsid protein, with both concentrating at the periphery of replication compartments. Immunoelectron microscopy showed that nearly 40% of assembled nuclear capsids associate with myosin Va. We also found that myosin Va and major capsid protein colocalize with nuclear F-actin. Importantly, antagonism of myosin Va with RNA interference or a dominant negative mutant revealed that myosin Va is important for the efficient production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Our results lead us to suggest a working model whereby human cytomegalovirus capsids associate with myosin Va for movement from replication compartments to the nuclear periphery during nuclear egress. IMPORTANCE Little is known regarding how newly assembled and packaged herpesvirus capsids move from the nuclear interior to the periphery during nuclear egress. While it has been proposed that an actomyosin-based mechanism facilitates intranuclear movement of alphaherpesvirus capsids, a functional role for any specific myosin in nuclear egress has not been reported. Furthermore, the notion that an actomyosin-based mechanism facilitates intranuclear capsid movement is controversial. Here we show that human cytomegalovirus capsids associate with nuclear myosin Va and F-actin and that antagonism of myosin Va impairs capsid localization toward the nuclear rim and nuclear egress. Together with our previous results showing that nuclear F-actin is induced upon HCMV infection and is also important for these processes, our results lend support to the hypothesis that nascent human cytomegalovirus capsids migrate to the nuclear periphery via actomyosin-based movement. These results shed light on a poorly understood viral process and the cellular machinery involved. Copyright © 2018 American Society for Microbiology.

Notch1 is asymmetrically distributed from the beginning of embryogenesis and controls the ventral center.

PubMed

Castro Colabianchi, Aitana M; Revinski, Diego R; Encinas, Paula I; Baez, María Verónica; Monti, Renato J; Abinal, Mateo Rodríguez; Kodjabachian, Laurent; Franchini, Lucía F; López, Silvia L

2018-06-04

Based on functional evidence, we have previously demonstrated that an early ventral Notch1 activity restricts dorsoanterior development in Xenopus We found that Notch1 has ventralizing properties and abolishes the dorsalizing activity of β-catenin by reducing its steady state levels, in a process that does not require β-catenin phosphorylation by glycogen synthase kinase-3β. In the present work, we demonstrate that Notch1 mRNA and protein are enriched in the ventral region from the beginning of the embryogenesis in Xenopus This is the earliest sign of ventral development, preceding the localized expression of wnt8a , bmp4 and ventxs genes in the ventral center and the dorsal accumulation of nuclear β-catenin. Knock-down experiments indicate that Notch1 is necessary for the normal expression of genes essential for ventral-posterior development. These results indicate that during early embryogenesis, ventrally located Notch1 promotes the development of the ventral center. Together with our previous evidence, these results suggest that ventral enrichment of Notch1 underlies the process by which Notch1 participates in restricting nuclear accumulation of β-catenin to the dorsal side. © 2018. Published by The Company of Biologists Ltd.

A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

NASA Technical Reports Server (NTRS)

Lu, C.; Fedoroff, N.

2000-01-01

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia

PubMed Central

Foster, K. Wade; Liu, Zhaoli; Nail, Clinton D.; Li, Xingnan; Fitzgerald, Thomas J.; Bailey, Sarah K.; Frost, Andra R.; Louro, Iuri D.; Townes, Tim M.; Paterson, Andrew J.; Kudlow, Jeffrey E.; Lobo-Ruppert, Susan M.; Ruppert, J. Michael

2006-01-01

KLF4/GKLF normally functions in differentiating epithelial cells, but also acts as a transforming oncogene in vitro. To examine the role of this zinc finger protein in skin, we expressed the wild-type human allele from inducible and constitutive promoters. When induced in basal keratinocytes KLF4 rapidly abolished the distinctive properties of basal and parabasal epithelial cells. KLF4 caused a transitory apoptotic response and the skin progressed through phases of hyperplasia and dysplasia. By 6 weeks, lesions exhibited nuclear KLF4 and other morphologic and molecular similarities to squamous cell carcinoma in situ. p53 determined the patch size sufficient to establish lesions, as induction in a mosaic pattern produced skin lesions only when p53 was deficient. Compared with p53 wild-type animals, p53 hemizygous animals had early onset of lesions and a pronounced fibrovascular response that included outgrowth of subcutaneous sarcoma. A KLF4-estrogen receptor fusion protein showed tamoxifen-dependent nuclear localization and conditional transformation in vitro. The results suggest that KLF4 can function in the nucleus to induce squamous epithelial dysplasia, and indicate roles for p53 and epithelial-mesenchymal signaling in these early neoplastic lesions. PMID:15674344

Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia.

PubMed

Foster, K Wade; Liu, Zhaoli; Nail, Clinton D; Li, Xingnan; Fitzgerald, Thomas J; Bailey, Sarah K; Frost, Andra R; Louro, Iuri D; Townes, Tim M; Paterson, Andrew J; Kudlow, Jeffrey E; Lobo-Ruppert, Susan M; Ruppert, J Michael

2005-02-24

KLF4/GKLF normally functions in differentiating epithelial cells, but also acts as a transforming oncogene in vitro. To examine the role of this zinc finger protein in skin, we expressed the wild-type human allele from inducible and constitutive promoters. When induced in basal keratinocytes, KLF4 rapidly abolished the distinctive properties of basal and parabasal epithelial cells. KLF4 caused a transitory apoptotic response and the skin progressed through phases of hyperplasia and dysplasia. By 6 weeks, lesions exhibited nuclear KLF4 and other morphologic and molecular similarities to squamous cell carcinoma in situ. p53 determined the patch size sufficient to establish lesions, as induction in a mosaic pattern produced skin lesions only when p53 was deficient. Compared with p53 wild-type animals, p53 hemizygous animals had early onset of lesions and a pronounced fibrovascular response that included outgrowth of subcutaneous sarcoma. A KLF4-estrogen receptor fusion protein showed tamoxifen-dependent nuclear localization and conditional transformation in vitro. The results suggest that KLF4 can function in the nucleus to induce squamous epithelial dysplasia, and indicate roles for p53 and epithelial-mesenchymal signaling in these early neoplastic lesions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shiheido, Hirokazu, E-mail: shiheido@ak.med.kyoto-u.ac.jp; Shimizu, Jun

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,more » 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.« less

The Role of Ect2 Nuclear RhoGEF Activity in Ovarian Cancer Cell Transformation

PubMed Central

Huff, Lauren P.; DeCristo, Molly J.; Trembath, Dimitri; Kuan, Pei Fen; Yim, Margaret; Liu, Jinsong; Cook, Danielle R.; Miller, C. Ryan; Der, Channing J.

2013-01-01

Ect2, a Rho guanine nucleotide exchange factor (RhoGEF), is atypical among RhoGEFs in its predominantly nuclear localization in interphase cells. One current model suggests that Ect2 mislocalization drives cellular transformation by promoting aberrant activation of cytoplasmic Rho family GTPase substrates. However, in ovarian cancers, where Ect2 is both amplified and overexpressed at the mRNA level, we observed that the protein is highly expressed and predominantly nuclear and that nuclear but not cytoplasmic Ect2 increases with advanced disease. Knockdown of Ect2 in ovarian cancer cell lines impaired their anchorage-independent growth without affecting their growth on plastic. Restoration of Ect2 expression rescued the anchorage-independent growth defect, but not if either the DH catalytic domain or the nuclear localization sequences of Ect2 were mutated. These results suggested a novel mechanism whereby Ect2 could drive transformation in ovarian cancer cells by acting as a RhoGEF specifically within the nucleus. Interestingly, Ect2 had an intrinsically distinct GTPase specificity profile in the nucleus versus the cytoplasm. Nuclear Ect2 bound preferentially to Rac1, while cytoplasmic Ect2 bound to RhoA but not Rac. Consistent with nuclear activation of endogenous Rac, Ect2 overexpression was sufficient to recruit Rac effectors to the nucleus, a process that required a functional Ect2 catalytic domain. Furthermore, expression of active nuclearly targeted Rac1 rescued the defect in transformed growth caused by Ect2 knockdown. Our work suggests a novel mechanism of Ect2-driven transformation, identifies subcellular localization as a regulator of GEF specificity, and implicates activation of nuclear Rac1 in cellular transformation. PMID:24386507

Light Water Reactor Sustainability Program: Evaluation of Localized Cable Test Methods for Nuclear Power Plant Cable Aging Management Programs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glass, Samuel W.; Fifield, Leonard S.; Hartman, Trenton S.

This Pacific Northwest National Laboratory (PNNL) milestone report describes progress to date on the investigation of nondestructive test (NDE) methods focusing particularly on local measurements that provide key indicators of cable aging and damage. The work includes a review of relevant literature as well as hands-on experimental verification of inspection capabilities. As NPPs consider applying for second, or subsequent, license renewal (SLR) to extend their operating period from 60 years to 80 years, it important to understand how the materials installed in plant systems and components will age during that time and develop aging management programs (AMPs) to assure continuedmore » safe operation under normal and design basis events (DBE). Normal component and system tests typically confirm the cables can perform their normal operational function. The focus of the cable test program is directed toward the more demanding challenge of assuring the cable function under accident or DBE. Most utilities already have a program associated with their first life extension from 40 to 60 years. Regrettably, there is neither a clear guideline nor a single NDE that can assure cable function and integrity for all cables. Thankfully, however, practical implementation of a broad range of tests allows utilities to develop a practical program that assures cable function to a high degree. The industry has adopted 50% elongation at break (EAB) relative to the un-aged cable condition as the acceptability standard. All tests are benchmarked against the cable EAB test. EAB is a destructive test so the test programs must apply an array of other NDE tests to assure or infer the overall set of cable’s system integrity. These cable NDE programs vary in rigor and methodology. As the industry gains experience with the efficacy of these programs, it is expected that implementation practice will converge to a more common approach. This report addresses the range of local NDE cable tests that are or could be practically implemented in a field test situation. These tests include: visual, infrared thermography, interdigital capacitance, indenter, relaxation time indenter, dynamic mechanical analyzer, infrared/near-infrared spectrometry, ultrasound, and distributed fiber optic temperature measurement.« less

STATs get their move on

PubMed Central

Reich, Nancy C

2013-01-01

Understanding the mechanisms that regulate dynamic localization of a protein within a cell can provide critical insight to its functional molecular interactions. Signal transducers and activators of transcription (STATs) play essential roles in development, proliferation, and immune defense. However the consequences of STAT hyperactivity can predispose to diseases including autoimmunity and cancer. To function as transcription factors STATs must gain access to the nucleus, and knowledge of the mechanisms that regulate STAT nuclear trafficking can provide a means to control STAT action. This review presents a synopsis of some of the studies that address the nuclear dynamics of the STAT proteins. Evidence suggests that not all STATs are the same. Nuclear import of STAT1 and STAT4 appears linked to their tyrosine phosphorylation and the formation of parallel dimers via reciprocal phosphotyrosine and Src homology 2 domain interactions. This dimer arrangement generates a conformational nuclear localization signal. STAT2 is imported continually to the nucleus in an unphosphorylated state due to its association with IRF9, but the dominant nuclear export signal of STAT2 shuttles the complex back to the cytoplasm. Following STAT2 tyrosine phosphorylation, it can form dimers with STAT1 to affect nuclear import as the trimeric complex (ISGF3). Distinctly, STAT3, STAT5, and STAT6 are continually imported to the nucleus independent of tyrosine phosphorylation. Mutational studies indicate the nuclear localization signals in these STATs require the conformational structure of their coiled-coil domains. Increases in STAT nuclear accumulation following cytokine stimulation appear coordinate with their ability to bind DNA. PMID:24470978

A Role for the NF-kb/Rel Transcription Factors in Human Breast Cancer

DTIC Science & Technology

1998-07-01

binding proteins present in a series of nuclear extracts from cell lines and from breast tumor tissues as well as normal mammary epithelium. Finally, we...RelA is nuclear in several examples. Our recent data on nuclear extracts of breast tumors shows that there is a significant increase in NF-KB binding...Figure 2 in the appendix). Additionally, immunoblotting of nuclear extracts versus adjacent tissue controls showed that NF-KB p50, p52 and c-Rel were

Identification of candidates for interacting partners of the tail domain of DcNMCP1, a major component of the Daucus carota nuclear lamina-like structure.

PubMed

Mochizuki, Ryota; Tsugama, Daisuke; Yamazaki, Michihiro; Fujino, Kaien; Masuda, Kiyoshi

2017-05-04

NMCP/CRWN (NUCLEAR MATRIX CONSTITUENT PROTEIN/CROWDED NUCLEI) is a major component of a protein fibrous meshwork (lamina-like structure) on the plant inner nuclear membrane. NMCP/CRWN contributes to regulating nuclear shape and nuclear functions. An NMCP/CRWN protein in Daucus carota (DcNMCP1) is localized to the nuclear periphery in interphase cells, and surrounds chromosomes in cells in metaphase and anaphase. The N-terminal region and the C-terminal region of DcNMCP1 are both necessary for localizing DcNMCP1 to the nuclear periphery. Here candidate interacting partners of the amino acid position 975-1053 of DcNMCP1 (T975-1053), which is present in the C-terminal region and contains a conserved sequence that plays a role in localizing DcNMCP1 to the nuclear periphery, are screened for. Arabidopsis thaliana nuclear proteins were subjected to far-Western blotting with GST-fused T975-1053 as a probe, and signals were detected at the positions corresponding to ∼70, ∼40, and ∼18 kDa. These ∼70, ∼40, and ∼18 kDa nuclear proteins were identified by mass spectrometry, and subjected to a yeast 2-hybrid (Y2H) analysis with T975-1053 as bait. In this analysis, the ∼40 kDa protein ARP7, which is a nuclear actin-related protein possibly involved in regulating chromatin structures, was confirmed to interact with T975-1053. Independently of the far-Western blotting, a Y2H screen was performed using T975-1053 as bait. Targeted Y2H assays confirmed that 3 proteins identified in the screen, MYB3, SINAT1, and BIM1, interact with T975-1053. These proteins might have roles in NMCP/CRWN protein-mediated biologic processes.

Specific Nuclear Localizing Sequence Directs Two Myosin Isoforms to the Cell Nucleus in Calmodulin-Sensitive Manner

PubMed Central

Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

2012-01-01

Background Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the “cytoplasmic” myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. Methodology/Principal Findings We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. Conclusions/Significance We have shown that the novel specific NLS brings to the cell nucleus not only the “nuclear” isoform of myosin I (NM1 protein) but also its “cytoplasmic” isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus. PMID:22295092

Local bifurcations in differential equations with state-dependent delay.

PubMed

Sieber, Jan

2017-11-01

A common task when analysing dynamical systems is the determination of normal forms near local bifurcations of equilibria. As most of these normal forms have been classified and analysed, finding which particular class of normal form one encounters in a numerical bifurcation study guides follow-up computations. This paper builds on normal form algorithms for equilibria of delay differential equations with constant delay that were developed and implemented in DDE-Biftool recently. We show how one can extend these methods to delay-differential equations with state-dependent delay (sd-DDEs). Since higher degrees of regularity of local center manifolds are still open for sd-DDEs, we give an independent (still only partial) argument which phenomena from the truncated normal must persist in the full sd-DDE. In particular, we show that all invariant manifolds with a sufficient degree of normal hyperbolicity predicted by the normal form exist also in the full sd-DDE.

5 CFR 5801.102 - Prohibited securities.

Code of Federal Regulations, 2014 CFR

2014-01-01

... licenses for facilities which generate electric energy by means of a nuclear reactor; (2) State or local... reactor or a low-level waste facility; (3) Entities manufacturing or selling nuclear power or test reactors; (4) Architectural-engineering companies providing services relating to a nuclear power reactor...

5 CFR 5801.102 - Prohibited securities.

Code of Federal Regulations, 2010 CFR

2010-01-01

... licenses for facilities which generate electric energy by means of a nuclear reactor; (2) State or local... reactor or a low-level waste facility; (3) Entities manufacturing or selling nuclear power or test reactors; (4) Architectural-engineering companies providing services relating to a nuclear power reactor...

Generation of a transgenic medaka (Oryzias latipes) strain for visualization of nuclear dynamics in early developmental stages.

PubMed

Inoue, Takanobu; Iida, Atsuo; Maegawa, Shingo; Sehara-Fujisawa, Atsuko; Kinoshita, Masato

2016-12-01

In this study, we verified nuclear transport activity of an artificial nuclear localization signal (aNLS) in medaka fish (Oryzias latipes). We generated a transgenic medaka strain expresses the aNLS tagged enhanced green fluorescent protein (EGFP) driven by a medaka beta-actin promoter. The aNLS-EGFP was accumulated in the nuclei of somatic tissues and yolk nuclei of oocytes, but undetectable in the spermatozoa. The fluorescent signal was observed from immediately after fertilization by a maternal contribution. Furthermore, male and female pronuclei were visualized in fertilized eggs, and nuclear dynamics of pronuclear fusion and subsequent cleavage were captured by time-lapse imaging. In contrast, SV40NLS exhibited no activity of nuclear transport in early embryos. In conclusion, the aNLS possesses a strong nuclear localization activity and is a useful probe for fluorescent observation of the pronuclei and nuclei in early developmental stage of medaka. © 2016 Japanese Society of Developmental Biologists.

Demonstration of nuclear compartmentalization of glutathione in hepatocytes.

PubMed Central

Bellomo, G; Vairetti, M; Stivala, L; Mirabelli, F; Richelmi, P; Orrenius, S

1992-01-01

The intracellular distribution of glutathione (GSH) in cultured hepatocytes has been investigated by using the compound monochlorobimane (BmCl), which interacts specifically with GSH to form a highly fluorescent adduct. Image analysis of BmCl-labeled hepatocytes predominantly localized the fluorescence in the nucleus; the nuclear/cytoplasmic concentration gradient was approximately three. This concentration gradient was collapsed by treatment of the cells with ATP-depleting agents. The uneven distribution of BmCl fluorescence was not attributable to (i) nonspecific interaction of BmCl with protein sulfhydryl groups, (ii) any selective nuclear localization of the GSH transferase(s) catalyzing formation of the GSH-BmCl conjugate, or (iii) any apparent alterations in cell morphology from culture conditions, suggesting that this distribution did, indeed, reflect a nuclear compartmentalization of GSH. That the nuclear pool of GSH was found more resistant to depletion by several agents than the cytoplasmic pool supports the assumption that GSH is essential in protecting DNA and other nuclear structures from chemical injury. Images PMID:1584774

Distinct molecular cues ensure a robust microtubule-dependent nuclear positioning in the Drosophila oocyte

PubMed Central

Tissot, Nicolas; Lepesant, Jean-Antoine; Bernard, Fred; Legent, Kevin; Bosveld, Floris; Martin, Charlotte; Faklaris, Orestis; Bellaïche, Yohanns; Coppey, Maïté; Guichet, Antoine

2017-01-01

Controlling nucleus localization is crucial for a variety of cellular functions. In the Drosophila oocyte, nuclear asymmetric positioning is essential for the reorganization of the microtubule (MT) network that controls the polarized transport of axis determinants. A combination of quantitative three-dimensional live imaging and laser ablation-mediated force analysis reveal that nuclear positioning is ensured with an unexpected level of robustness. We show that the nucleus is pushed to the oocyte antero-dorsal cortex by MTs and that its migration can proceed through distinct tracks. Centrosome-associated MTs favour one migratory route. In addition, the MT-associated protein Mud/NuMA that is asymmetrically localized in an Asp-dependent manner at the nuclear envelope hemisphere where MT nucleation is higher promotes a separate route. Our results demonstrate that centrosomes do not provide an obligatory driving force for nuclear movement, but together with Mud, contribute to the mechanisms that ensure the robustness of asymmetric nuclear positioning. PMID:28447612

Four nucleocytoplasmic-shuttling proteins and p53 interact specifically with the YB-NLS and are involved in anticancer reagent-induced nuclear localization of YB-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanaka, Toru; Ohashi, Sachiyo; Kobayashi, Shunsuke

In cancer cells, anticancer reagents often trigger nuclear accumulation of YB-1, which participates in the progression of cancer malignancy. YB-1 has a non-canonical nuclear localization signal (YB-NLS). Here we found that four nucleocytoplasmic-shuttling RNA-binding proteins and p53 interact specifically with the YB-NLS and co-accumulate with YB-1 in the nucleus of actinomycin D-treated cells. To elucidate the roles of these YB-NLS-binding proteins, we performed a dominant-negative experiment in which a large excess of YB-NLS interacts with the YB-NLS-binding proteins, and showed inhibitory effects on actinomycin D-induced nuclear transport of endogenous YB-1 and subsequent MDR1 gene expression. Furthermore, the YB-NLS-expressing cells weremore » also found to show increased drug sensitivity. Our results suggest that these YB-NLS-associating proteins are key factors for nuclear translocation/accumulation of YB-1 in cancer cells. - Highlights: • Four nucleocytoplasmic-shuttling proteins and p53 associate with YB-NLS. • They showed nuclear co-accumulation with YB-1 in actinomycin D-treated cells. • Overexpression of YB-NLS was carried out to take YB-NLS-binding proteins from YB-1. • YB-NLS inhibited actinomycin D-induced nuclear localization of endogenous YB-1. • YB-NLS suppressed actinomycin D-induced expression of MDR1.« less

Off-site Emergency Planning at UK Nuclear Licensed Sites.

PubMed

Leonard, Paul; Thomas, Gareth

2017-04-01

Nuclear emergency planning arrangements in the UK are continually kept under review. This work proposes to outline how experience from nuclear exercises and undertaking emergency response duties can be based on radiological knowledge of specific sites and utilised in the future. In 2014, the UK regulator, the Office for Nuclear Regulation (ONR) revised their principles for the determination of off-site emergency planning areas around nuclear sites where predetermined countermeasures and other protection measures are applied to protect those people who may be affected by a radiation emergency. The revised principles also enhanced communication from the nuclear site operators and local authorities to the public. This updated ONR's application of the UK Radiation (Emergency Preparedness and Public Information) Regulations 2001 (REPPIR) http://www.hse.gov.uk/radiation/ionising/reppir.htm, which includes details of minimising potential doses to the public, as well as assessment and reassurance, linked to other concurrent risks such as flooding. ONR undertakes site-specific assessments of each operators' hazard identification and risk evaluation, which include consideration of whether the public might receive a significant radiation dose in the year following the emergency (excluding countermeasures in the first 24 hours). In defining the areas for off-site emergency planning, practical and strategic factors are then considered, which include other local non-nuclear emergency planning arrangements and experience, and whether local geographic and demographic aspects could aid public credibility and confidence. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Regulatory T cells facilitate the nuclear accumulation of inducible cAMP early repressor (ICER) and suppress nuclear factor of activated T cell c1 (NFATc1)

PubMed Central

Vaeth, Martin; Gogishvili, Tea; Bopp, Tobias; Klein, Matthias; Berberich-Siebelt, Friederike; Gattenloehner, Stefan; Avots, Andris; Sparwasser, Tim; Grebe, Nadine; Schmitt, Edgar; Hünig, Thomas; Serfling, Edgar; Bodor, Josef

2011-01-01

Inducible cAMP early repressor (ICER) is a transcriptional repressor, which, because of alternate promoter use, is generated from the 3′ region of the cAMP response modulator (Crem) gene. Its expression and nuclear occurrence are elevated by high cAMP levels in naturally occurring regulatory T cells (nTregs). Using two mouse models, we demonstrate that nTregs control the cellular localization of ICER/CREM, and thereby inhibit IL-2 synthesis in conventional CD4+ T cells. Ablation of nTregs in depletion of regulatory T-cell (DEREG) mice resulted in cytosolic localization of ICER/CREM and increased IL-2 synthesis upon stimulation. Direct contacts between nTregs and conventional CD4+ T cells led to nuclear accumulation of ICER/CREM and suppression of IL-2 synthesis on administration of CD28 superagonistic (CD28SA) Ab. In a similar way, nTregs communicated with B cells and induced the cAMP-driven nuclear localization of ICER/CREM. High levels of ICER suppressed the induction of nuclear factor of activated T cell c1 (Nfatc1) gene in T cells whose inducible Nfatc1 P1 promoter bears two highly conserved cAMP-responsive elements to which ICER/CREM can bind. These findings suggest that nTregs suppress T-cell responses by the cAMP-dependent nuclear accumulation of ICER/CREM and inhibition of NFATc1 and IL-2 induction. PMID:21262800

Vitamin D Receptor Expression in Normal, Premalignant, and Malignant Human Lung Tissue

PubMed Central

Menezes, Ravi J.; Cheney, Richard T.; Husain, Aliya; Tretiakova, Maria; Loewen, Gregory; Johnson, Candace S.; Jayaprakash, Vijay; Moysich, Kirsten B.; Salgia, Ravi; Reid, Mary E.

2009-01-01

Background There is a strong interest in identifying chemopreventive agents that might help decrease the burden of lung cancer. The active metabolite of vitamin D, 1,25-dihydroxycholecalciferol (calcitriol), has been shown to have antiproliferative effects in several tumor types, mediated by the vitamin D receptor (VDR). This is the first comprehensive survey of VDR expression in a series of human lung tissues, including normal and premalignant central airway biopsies and lung tumors. Methods Immunohistochemical expression of nuclear and cytoplasmic VDR was examined in 180 premalignant or malignant bronchial biopsies from bronchoscopy of 78 high-risk individuals at the Roswell Park Cancer Institute and also in 63 tumor samples from 35 lung cancer patients from the University of Chicago Hospitals. Associations between clinicopathologic data and VDR expression were examined. Results VDR expression was present in many samples. In biopsies, VDR was commonly detected throughout the full epithelial layer. Most histologically normal (60%, 53 of 88) and metaplastic (61%, 39 of 64) samples had moderate to high nuclear intensity; dysplastic samples mostly had low nuclear intensity (10 of 18, 55%). In tumor samples, 62% (38 of 61) were lacking cytoplasmic VDR, with nuclear expression present in 79%(49 of 62). Analysis of all samples revealed a positive linear trend between proportion of samples with greater nuclear than cytoplasmic intensity and increasing histologic grade (P < 0.01). Conclusions VDR expression spanned the lung carcinogenesis spectrum. Nuclear expression was similar across various histologies, whereas cytoplasmic expression decreased with increasing histologic grade. These results indicate that there is potential for the use of calcitriol as a chemopreventive agent against the development of lung cancer. (Cancer Epidemiol Bio-markers Prev 2008;17(5):1104–10) PMID:18483332

Nuclear-localized AtHSPR links abscisic acid-dependent salt tolerance and antioxidant defense in Arabidopsis.

PubMed

Yang, Tao; Zhang, Liang; Hao, Hongyan; Zhang, Peng; Zhu, Haowei; Cheng, Wei; Wang, Yongli; Wang, Xinyu; Wang, Chongying

2015-12-01

Salt stress from soil or irrigation water limits plant growth. A T-DNA insertion mutant in C24, named athspr (Arabidopsis thaliana heat shock protein-related), showed several phenotypes, including reduced organ size and enhanced sensitivity to environmental cues. The athspr mutant is severely impaired under salinity levels at which wild-type (WT) plants grow normally. AtHSPR encodes a nuclear-localized protein with ATPase activity, and its expression was enhanced by high salinity and abscisic acid (ABA). Overexpression (OE) of AtHSPR significantly enhanced tolerance to salt stress by increasing the activities of the antioxidant system and by maintaining K(+) /Na(+) homeostasis. Quantitative RT-PCR analyses showed that OE of AtHSPR increased the expression of ABA/stress-responsive, salt overly sensitive (SOS)-related and antioxidant-related genes. In addition, ABA content was reduced in athspr plants with or without salt stress, and exogenous ABA restored WT-like salt tolerance to athspr plants. athspr exhibited increased leaf stomatal density and stomatal index, slower ABA-induced stomatal closure and reduced drought tolerance relative to the WT. AtHSPR OE enhanced drought tolerance by reducing leaf water loss and stomatal aperture. Transcript profiling in athspr showed a differential salt-stress response for genes involved in accumulation of reactive oxygen species (ROS), ABA signaling, cell death, stress response and photosynthesis. Taken together, our results suggested that AtHSPR is involved in salt tolerance in Arabidopsis through modulation of ROS levels, ABA-dependent stomatal closure, photosynthesis and K(+) /Na(+) homeostasis. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Recovery of deficient homologous recombination in Brca2-depleted mouse cells by wild-type Rad51 expression.

PubMed

Lee, Shauna A; Roques, Céline; Magwood, Alissa C; Masson, Jean-Yves; Baker, Mark D

2009-02-01

The BRCA2 tumor suppressor is important in maintaining genomic stability. BRCA2 is proposed to control the availability, cellular localization and DNA binding activity of the central homologous recombination protein, RAD51, with loss of BRCA2 resulting in defective homologous recombination. Nevertheless, the roles of BRCA2 in regulating RAD51 and how other proteins implicated in RAD51 regulation, such as RAD52 and RAD54 function relative to BRCA2 is not known. In this study, we tested whether defective homologous recombination in Brca2-depleted mouse hybridoma cells could be rectified by expression of mouse Rad51 or the Rad51-interacting mouse proteins, Rad52 and Rad54. In the Brca2-depleted cells, defective homologous recombination can be restored by over-expression of wild-type mouse Rad51, but not mouse Rad52 or Rad54. Correction of the homologous recombination defect requires Rad51 ATPase activity. A sizeable fraction ( approximately 50%) of over-expressed wild-type Rad51 is nuclear localized. The restoration of homologous recombination in the presence of a low (i.e., non-functional) level of Brca2 by wild-type Rad51 over-expression is unexpected. We suggest that Rad51 may access the nuclear compartment in a Brca2-independent manner and when Rad51 is over-expressed, the normal requirement for Brca2 control over Rad51 function in homologous recombination is dispensable. Our studies support loss of Rad51 function as a critical underlying factor in the homologous recombination defect in the Brca2-depleted cells.

IE1 and hr facilitate the localization of Bombyx mori nucleopolyhedrovirus ORF8 to specific nuclear sites.

PubMed

Kang, WonKyung; Imai, Noriko; Kawasaki, Yu; Nagamine, Toshihiro; Matsumoto, Shogo

2005-11-01

The Bombyx mori nucleopolyhedrovirus (BmNPV) ORF8 protein has previously been reported to colocalize with IE1 to specific nuclear sites during infection. Transient expression of green fluorescent protein (GFP)-fused ORF8 showed the protein to have cytoplasmic localization, but following BmNPV infection the protein formed foci, suggesting that ORF8 requires some other viral factor(s) for this. Therefore, interacting factors were looked for using the yeast two-hybrid system and IE1 was identified. We mapped the interacting region of ORF8 using a yeast two-hybrid assay. An N-terminal region (residues 1-110) containing a predicted coiled-coil domain interacted with IE1, while a truncated N-terminal region (residues 1-78) that lacks this domain did not. In addition, a protein with a complete deletion of the N-terminal region failed to interact with IE1. These results suggest that the ORF8 N-terminal region containing the coiled-coil domain is required for the interaction with IE1. Next, whether IE1 plays a role in ORF8 localization was investigated. In the presence of IE1, GFP-ORF8 localized to the nucleus. In addition, cotransfection with a plasmid expressing IE1 and a plasmid containing the hr3 element resulted in nuclear foci formation. A GFP-fused ORF8 mutant protein containing the coiled-coil domain, previously shown to interact with IE1, also formed nuclear foci in the presence of IE1 and hr3. However, ORF8 mutant proteins that did not interact with IE1 failed to form nuclear foci. In contrast to wild-type IE1, focus formation was not observed for an IE1 mutant protein that was deficient in hr binding. These results suggest that IE1 and hr facilitate the localization of BmNPV ORF8 to specific nuclear sites.

Nuclear uptake and dosimetry of 64Cu-labeled chelator somatostatin conjugates in an SSTr2-transfected human tumor cell line.

PubMed

Eiblmaier, Martin; Andrews, Rebecca; Laforest, Richard; Rogers, Buck E; Anderson, Carolyn J

2007-08-01

64Cu radiopharmaceuticals have shown tumor growth inhibition in tumor-bearing animal models with a relatively low radiation dose that may be related to nuclear localization of the 64Cu in tumor cells. Here we address whether the nuclear localization of 64Cu from a 64Cu-labeled chelator-somatostatin conjugate is related to the dissociation of the radio-copper from its chelator. The 64Cu complex of 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) has demonstrated instability in vivo, whereas 64Cu-CB-TE2A (CB-TE2A is 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane) was highly stable. Receptor binding, nuclear uptake, internalization, and efflux assays were performed to characterize the interaction with the somatostatin receptor and the intracellular fate of 64Cu-labeled chelator-peptide conjugates in A427-7 cells. From these data, the absorbed dose to cells was calculated. 64Cu-TETA-Y3-TATE (64Cu-[1]) and 64Cu-CB-TE2A-Y3-TATE (64Cu-[2]) had high affinity for somatostatin receptor subtype 2 (SSTr2) in A427-7 cells. After 3 h, 64Cu-[2] showed greater internalization (>30%) compared with 64Cu-[1] (approximately 15%). There was uptake of 64Cu-[1] in nuclei of 427-7 cells (9.4% +/- 1.7% at 24 h), whereas 64Cu-[2] showed minimal nuclear accumulation out to 24 h (1.3% +/- 0.1%). A427-7 cells were exposed to 0.40 Gy from 64Cu-[1] and exposed to 1.06 Gy from 64Cu-[2]. External beam irradiation of A427-7 cells showed <20% cell killing at 1 Gy. These results are consistent with our hypothesis that dissociation of 64Cu from TETA leads to nuclear localization. Dosimetry calculations indicated that the nuclear localization of 64Cu-[1] was not significant enough to increase the absorbed dose to the nuclei of A427-7 cells. These studies show that 64Cu localization to cell nuclei from internalizing, receptor-targeted radiopharmaceuticals is related to chelate stability.

Nuclear Heat Shock Response and Novel Nuclear Domain 10 Reorganization in Respiratory Syncytial Virus-Infected A549 Cells Identified by High-Resolution Two-Dimensional Gel Electrophoresis

PubMed Central

Brasier, Allan R.; Spratt, Heidi; Wu, Zheng; Boldogh, Istvan; Zhang, Yuhong; Garofalo, Roberto P.; Casola, Antonella; Pashmi, Jawad; Haag, Anthony; Luxon, Bruce; Kurosky, Alexander

2004-01-01

The pneumovirus respiratory syncytial virus (RSV) is a leading cause of epidemic respiratory tract infection. Upon entry, RSV replicates in the epithelial cytoplasm, initiating compensatory changes in cellular gene expression. In this study, we have investigated RSV-induced changes in the nuclear proteome of A549 alveolar type II-like epithelial cells by high-resolution two-dimensional gel electrophoresis (2DE). Replicate 2D gels from uninfected and RSV-infected nuclei were compared for changes in protein expression. We identified 24 different proteins by peptide mass fingerprinting after matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS), whose average normalized spot intensity was statistically significant and differed by ±2-fold. Notable among the proteins identified were the cytoskeletal cytokeratins, RNA helicases, oxidant-antioxidant enzymes, the TAR DNA binding protein (a protein that associates with nuclear domain 10 [ND10] structures), and heat shock protein 70- and 60-kDa isoforms (Hsp70 and Hsp60, respectively). The identification of Hsp70 was also validated by liquid chromatography quadropole-TOF tandem MS (LC-MS/MS). Separate experiments using immunofluorescence microscopy revealed that RSV induced cytoplasmic Hsp70 aggregation and nuclear accumulation. Data mining of a genomic database showed that RSV replication induced coordinate changes in Hsp family proteins, including the 70, 70-2, 90, 40, and 40-3 isoforms. Because the TAR DNA binding protein associates with ND10s, we examined the effect of RSV infection on ND10 organization. RSV induced a striking dissolution of ND10 structures with redistribution of the component promyelocytic leukemia (PML) and speckled 100-kDa (Sp100) proteins into the cytoplasm, as well as inducing their synthesis. Our findings suggest that cytoplasmic RSV replication induces a nuclear heat shock response, causes ND10 disruption, and redistributes PML and Sp100 to the cytoplasm. Thus, a high-resolution proteomics approach, combined with immunofluorescence localization and coupled with genomic response data, yielded unexpected novel insights into compensatory nuclear responses to RSV infection. PMID:15479789

Recent advances in understanding nuclear size and shape

PubMed Central

Mukherjee, Richik N.; Chen, Pan; Levy, Daniel L.

2016-01-01

ABSTRACT Size and shape are important aspects of nuclear structure. While normal cells maintain nuclear size within a defined range, altered nuclear size and shape are associated with a variety of diseases. It is unknown if altered nuclear morphology contributes to pathology, and answering this question requires a better understanding of the mechanisms that control nuclear size and shape. In this review, we discuss recent advances in our understanding of the mechanisms that regulate nuclear morphology, focusing on nucleocytoplasmic transport, nuclear lamins, the endoplasmic reticulum, the cell cycle, and potential links between nuclear size and size regulation of other organelles. We then discuss the functional significance of nuclear morphology in the context of early embryonic development. Looking toward the future, we review new experimental approaches that promise to provide new insights into mechanisms of nuclear size control, in particular microfluidic-based technologies, and discuss how altered nuclear morphology might impact chromatin organization and physiology of diseased cells. PMID:26963026

Three-dimensional telomere architecture of esophageal squamous cell carcinoma: comparison of tumor and normal epithelial cells.

PubMed

Sunpaweravong, S; Sunpaweravong, P; Sathitruangsak, C; Mai, S

2016-05-01

Telomeres are repetitive nucleotide sequences (TTAGGG)n located at the ends of chromosomes that function to preserve chromosomal integrity and prevent terminal end-to-end fusions. Telomere loss or dysfunction results in breakage-bridge-fusion cycles, aneuploidy, gene amplification and chromosomal rearrangements, which can lead to genomic instability and promote carcinogenesis. Evaluating the hypothesis that changes in telomeres contribute to the development of esophageal squamous cell carcinoma (ESCC) and to determine whether there are differences between young and old patients, we compared the three-dimensional (3D) nuclear telomere architecture in ESCC tumor cells with that of normal epithelial cells obtained from the same patient. Patients were equally divided by age into two groups, one comprising those less than 45 years of age and the other consisting of those over 80 years of age. Tumor and normal epithelial cells located at least 10 cm from the border of the tumor were biopsied in ESCC patients. Hematoxylin and eosin staining was performed for each sample to confirm and identify the cancer and normal epithelial cells. This study was based on quantitative 3D fluorescence in situ hybridization (Q-FISH), 3D imaging and 3D analysis of paraffin-embedded slides. The 3D telomere architecture data were computer analyzed using 100 nuclei per slide. The following were the main parameters compared: the number of signals (number of telomeres), signal intensity (telomere length), number of telomere aggregates, and nuclear volume. Tumor and normal epithelial samples from 16 patients were compared. The normal epithelial cells had more telomere signals and higher intensities than the tumor cells, with P-values of P < 0.0001 and P = 0.0078, respectively. There were no statistically significant differences in the numbers of telomere aggregates or the nuclear volumes between the tumor and normal epithelial cells. Secondary analyses examined the effects of age on 3D telomere architecture and found no statistically significant differences in any parameter tested between the young and old patients in either the tumor or epithelial cells. The 3D nuclear telomeric signature was able to detect differences in telomere architecture between the ESCC and normal epithelial tissues. However, there were no differences observed between the young and old patients. © 2015 International Society for Diseases of the Esophagus.

Scanning nuclear resonance imaging of a hyperfine-coupled quantum Hall system.

PubMed

Hashimoto, Katsushi; Tomimatsu, Toru; Sato, Ken; Hirayama, Yoshiro

2018-06-07

Nuclear resonance (NR) is widely used to detect and characterise nuclear spin polarisation and conduction electron spin polarisation coupled by a hyperfine interaction. While the macroscopic aspects of such hyperfine-coupled systems have been addressed in most relevant studies, the essential role of local variation in both types of spin polarisation has been indicated in 2D semiconductor systems. In this study, we apply a recently developed local and highly sensitive NR based on a scanning probe to a hyperfine-coupled quantum Hall (QH) system in a 2D electron gas subject to a strong magnetic field. We succeed in imaging the NR intensity and Knight shift, uncovering the spatial distribution of both the nuclear and electron spin polarisation. The results reveal the microscopic origin of the nonequilibrium QH phenomena, and highlight the potential use of our technique in microscopic studies on various electron spin systems as well as their correlations with nuclear spins.

Clustering and pasta phases in nuclear density functional theory

DOE PAGES

Schuetrumpf, Bastian; Zhang, Chunli; Nazarewicz, Witold

2017-05-23

Nuclear density functional theory is the tool of choice in describing properties of complex nuclei and intricate phases of bulk nucleonic matter. It is a microscopic approach based on an energy density functional representing the nuclear interaction. An attractive feature of nuclear DFT is that it can be applied to both finite nuclei and pasta phases appearing in the inner crust of neutron stars. While nuclear pasta clusters in a neutron star can be easily characterized through their density distributions, the level of clustering of nucleons in a nucleus can often be difficult to assess. To this end, we usemore » the concept of nucleon localization. We demonstrate that the localization measure provides us with fingerprints of clusters in light and heavy nuclei, including fissioning systems. Furthermore we investigate the rod-like pasta phase using twist-averaged boundary conditions, which enable calculations in finite volumes accessible by state of the art DFT solvers.« less

Evidence for GeV cosmic rays from white dwarfs in the local cosmic ray spectra and in the gamma-ray emissivity of the inner Galaxy

NASA Astrophysics Data System (ADS)

Kamae, Tuneyoshi; Lee, Shiu-Hang; Makishima, Kazuo; Shibata, Shinpei; Shigeyama, Toshikazu

2018-03-01

Recent observations found that electrons are accelerated to ˜10 GeV and emit synchrotron hard X-rays in two magnetic white dwarfs (WDs), also known as cataclysmic variables (CVs). In nova outbursts of WDs, multi-GeV gamma-rays were detected, implying that protons are accelerated to 100 GeV or higher. In recent optical surveys, the WD density is found to be higher near the Sun than in the Galactic disk by a factor ˜2.5. The cosmic rays (CRs) produced by local CVs and novae will accumulate in the local bubble for 106-107 yr. On these findings, we search for CRs from historic CVs and novae in the observed CR spectra. We model the CR spectra at the heliopause as sums of Galactic and local components based on observational data as much as possible. The initial Galactic CR electron and proton spectra are deduced from the gamma-ray emissivity, the local electron spectrum from the hard X-ray spectra at the CVs, and the local proton spectrum from gamma-ray spectra at novae. These spectral shapes are then expressed in a simple set of polynomial functions of CR energy and regressively fitted until the high-energy (>100 GeV) CR spectra near Earth and the Voyager-1 spectra at the heliopause are reproduced. We then extend the modeling to nuclear CR spectra and find that one spectral shape fits all local nuclear CRs, and that the apparent hardening of the nuclear CR spectra is caused by the roll-down of local nuclear spectra around 100-200 GeV. All local CR spectra populate a limited energy band below 100-200 GeV and enhance gamma-ray emissivity below ˜10 GeV. Such an enhancement is observed in the inner Galaxy, suggesting the CR fluxes from CVs and novae are substantially higher there.

Subjective and Quantitative Measurement of Wavefront Aberrations in Nuclear Cataracts – A Retrospective Case Controlled Study

PubMed Central

Wali, Upender K.; Bialasiewicz, Alexander A.; Al-Kharousi, Nadia; Rizvi, Syed G.; Baloushi, Habiba

2009-01-01

Purpose: To measure, quantify and compare Ocular Aberrations due to nuclear cataracts. Setting: Department of ophthalmology and school for ophthalmic technicians, college of medicine and health sciences, Sultan Qaboos University, Muscat, Oman. Design: Retrospective case controlled study. Methods: 113 eyes of 77 patients with nuclear cataract (NC) were recruited from outpatient clinic of a major tertiary referral center for Ophthalmology. Patients having NC with no co-existing ocular pathologies were selected. All patients were subjected to wavefront aberrometry (make) using Hartmann-Shack (HS) aberrometer. Consents were taken from all patients. Higher order Aberrations (HOA) were calculated with Zernike polynomials up to the fourth order. For comparison 28 eyes of 15 subjects with no lenticular opacities (control group) were recruited and evaluated in an identical manner. No pupillary mydriasis was done in both groups. Results: Total aberrations were almost six times higher in NC group compared to control (normal) subjects. The HOA were 21 times higher in NC group, and coma was significantly higher in NC eyes compared to normal (control) group. The pupillary diameter was significantly larger in control group (5.48mm ± 1.0024, p<.001) compared to NC (3.05mm ± 1.9145) subjects (probably due to younger control age group). Amongst Zernike coefficients up to fourth order, two polynomials, defocus (Z20) and spherical aberration (Z42) were found to be significantly greater amongst NC group, compared to normal control group. Conclusion: Nuclear cataracts predominantly produce increased defocus and spherical aberrations. This could explain visual symptoms like image deterioration in spite of normal Visual acuity. PMID:20142953

The nuclear lamina and heterochromatin: a complex relationship.

PubMed

Bank, Erin M; Gruenbaum, Yosef

2011-12-01

In metazoan cells, the heterochromatin is generally localized at the nuclear periphery, whereas active genes are preferentially found in the nuclear interior. In the present paper, we review current evidence showing that components of the nuclear lamina interact directly with heterochromatin, which implicates the nuclear lamina in a mechanism of specific gene retention at the nuclear periphery and release to the nuclear interior upon gene activation. We also discuss recent data showing that mutations in lamin proteins affect gene positioning and expression, providing a potential mechanism for how these mutations lead to tissue-specific diseases.

Encoded physics knowledge in checking codes for nuclear cross section libraries at Los Alamos

NASA Astrophysics Data System (ADS)

Parsons, D. Kent

2017-09-01

Checking procedures for processed nuclear data at Los Alamos are described. Both continuous energy and multi-group nuclear data are verified by locally developed checking codes which use basic physics knowledge and common-sense rules. A list of nuclear data problems which have been identified with help of these checking codes is also given.

Localization of proteasomes and proteasomal proteolysis in the mammalian interphase cell nucleus by systematic application of immunocytochemistry.

PubMed

Scharf, Andrea; Rockel, Thomas Dino; von Mikecz, Anna

2007-06-01

Proteasomes are ATP-driven, multisubunit proteolytic machines that degrade endogenous proteins into peptides and play a crucial role in cellular events such as the cell cycle, signal transduction, maintenance of proper protein folding and gene expression. Recent evidence indicates that the ubiquitin-proteasome system is an active component of the cell nucleus. A characteristic feature of the nucleus is its organization into distinct domains that have a unique composition of macromolecules and dynamically form as a response to the requirements of nuclear function. Here, we show by systematic application of different immunocytochemical procedures and comparison with signature proteins of nuclear domains that during interphase endogenous proteasomes are localized diffusely throughout the nucleoplasm, in speckles, in nuclear bodies, and in nucleoplasmic foci. Proteasomes do not occur in the nuclear envelope region or the nucleolus, unless nucleoplasmic invaginations expand into this nuclear body. Confirmedly, proteasomal proteolysis is detected in nucleoplasmic foci, but is absent from the nuclear envelope or nucleolus. The results underpin the idea that the ubiquitin-proteasome system is not only located, but also proteolytically active in distinct nuclear domains and thus may be directly involved in gene expression, and nuclear quality control.

Calculations of atomic magnetic nuclear shielding constants based on the two-component normalized elimination of the small component method

NASA Astrophysics Data System (ADS)

Yoshizawa, Terutaka; Zou, Wenli; Cremer, Dieter

2017-04-01

A new method for calculating nuclear magnetic resonance shielding constants of relativistic atoms based on the two-component (2c), spin-orbit coupling including Dirac-exact NESC (Normalized Elimination of the Small Component) approach is developed where each term of the diamagnetic and paramagnetic contribution to the isotropic shielding constant σi s o is expressed in terms of analytical energy derivatives with regard to the magnetic field B and the nuclear magnetic moment 𝝁 . The picture change caused by renormalization of the wave function is correctly described. 2c-NESC/HF (Hartree-Fock) results for the σiso values of 13 atoms with a closed shell ground state reveal a deviation from 4c-DHF (Dirac-HF) values by 0.01%-0.76%. Since the 2-electron part is effectively calculated using a modified screened nuclear shielding approach, the calculation is efficient and based on a series of matrix manipulations scaling with (2M)3 (M: number of basis functions).

Isolation and characterization of a carrot nucleolar protein with structural and sequence similarity to the vertebrate PESCADILLO protein.

PubMed

Ueda, Kenji; Xu, Zheng-Jun; Miyagi, Nobuaki; Ono, Michiyuki; Wabiko, Hiroetsu; Masuda, Kiyoshi; Inoue, Masayasu

2013-07-01

The nuclear matrix is involved in many nuclear events, but its protein architecture in plants is still not fully understood. A cDNA clone was isolated by immunoscreening with a monoclonal antibody raised against nuclear matrix proteins of Daucus carota L. Its deduced amino acid sequence showed about 40% identity with the PESCADILLO protein of zebrafish and humans. Primary structure analysis of the protein revealed a Pescadillo N-terminus domain, a single breast cancer C-terminal domain, two nuclear localization signals, and a potential coiled-coil region as also found in animal PESCADILLO proteins. Therefore, we designated this gene DcPES1. Although DcPES1 mRNA was detected in all tissues examined, its levels were highest in tissues with proliferating cells. Immunofluorescence using specific antiserum against the recombinant protein revealed that DcPES1 localized exclusively in the nucleolus. Examination of fusion proteins with green fluorescent protein revealed that the N-terminal portion was important for localization to the nucleoli of tobacco and onion cells. Moreover, when the nuclear matrix of carrot cells was immunostained with an anti-DcPES1 serum, the signal was detected in the nucleolus. Therefore, the DcPES1 protein appears to be a component of or tightly bound to components of the nuclear matrix. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Localization of nuclear subunits of cyclic AMP-dependent protein kinase by the immunocolloidal gold method

PubMed Central

1985-01-01

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP- dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density. PMID:2993318

Interactions and Nuclear Import of the N and P Proteins of Sonchus Yellow Net Virus, a Plant Nucleorhabdovirus

PubMed Central

Goodin, Michael M.; Austin, Jennifer; Tobias, Renée; Fujita, Miki; Morales, Christina; Jackson, Andrew O.

2001-01-01

We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione S-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization. PMID:11533202

Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity

PubMed Central

Görner, Wolfram; Durchschlag, Erich; Martinez-Pastor, Maria Teresa; Estruch, Francisco; Ammerer, Gustav; Hamilton, Barbara; Ruis, Helmut; Schüller, Christoph

1998-01-01

Msn2p and the partially redundant factor Msn4p are key regulators of stress-responsive gene expression in Saccharomyces cerevisiae. They are required for the transcription of a number of genes coding for proteins with stress-protective functions. Both Msn2p and Msn4p are Cys2His2 zinc finger proteins and bind to the stress response element (STRE). In vivo footprinting studies show that the occupation of STREs is enhanced in stressed cells and dependent on the presence of Msn2p and Msn4p. Both factors accumulate in the nucleus under stress conditions, such as heat shock, osmotic stress, carbon-source starvation, and in the presence of ethanol or sorbate. Stress-induced nuclear localization was found to be rapid, reversible, and independent of protein synthesis. Nuclear localization of Msn2p and Msn4p was shown to be correlated inversely to cAMP levels and protein kinase A (PKA) activity. A region with significant homologies shared between Msn2p and Msn4p is sufficient to confer stress-regulated localization to a SV40–NLS–GFP fusion protein. Serine to alanine or aspartate substitutions in a conserved PKA consensus site abolished cAMP-driven nuclear export and cytoplasmic localization in unstressed cells. We propose stress and cAMP-regulated intracellular localization of Msn2p to be a key step in STRE-dependent transcription and in the general stress response. PMID:9472026

Binding of Y-P30 to Syndecan 2/3 Regulates the Nuclear Localization of CASK

PubMed Central

Landgraf, Peter; Mikhaylova, Marina; Macharadze, Tamar; Borutzki, Corinna; Zenclussen, Ana-Claudia; Wahle, Petra; Kreutz, Michael R.

2014-01-01

The survival promoting peptide Y-P30 has documented neuroprotective effects as well as cell survival and neurite outgrowth promoting activity in vitro and in vivo. Previous work has shown that multimerization of the peptide with pleiotrophin (PTN) and subsequent binding to syndecan (SDC) -2 and -3 is involved in its neuritogenic effects. In this study we show that Y-P30 application regulates the nuclear localization of the SDC binding partner Calcium/calmodulin-dependent serine kinase (CASK) in neuronal primary cultures during development. In early development at day in vitro (DIV) 8 when mainly SDC-3 is expressed supplementation of the culture medium with Y-P30 reduces nuclear CASK levels whereas it has the opposite effect at DIV 18 when SDC-2 is the dominant isoform. In the nucleus CASK regulates gene expression via its association with the T-box transcription factor T-brain-1 (Tbr-1) and we indeed found that gene expression of downstream targets of this complex, like the GluN2B NMDA-receptor, exhibits a corresponding down- or up-regulation at the mRNA level. The differential effect of Y-P30 on the nuclear localization of CASK correlates with its ability to induce shedding of the ectodomain of SDC-2 but not -3. shRNA knockdown of SDC-2 at DIV 18 and SDC-3 at DIV 8 completely abolished the effect of Y-P30 supplementation on nuclear CASK levels. During early development a protein knockdown of SDC-3 also attenuated the effect of Y-P30 on axon outgrowth. Taken together these data suggest that Y-P30 can control the nuclear localization of CASK in a SDC-dependent manner. PMID:24498267

FBI-1 enhances transcription of the nuclear factor-kappaB (NF-kappaB)-responsive E-selectin gene by nuclear localization of the p65 subunit of NF-kappaB.

PubMed

Lee, Dong-Kee; Kang, Jae-Eun; Park, Hye-Jin; Kim, Myung-Hwa; Yim, Tae-Hee; Kim, Jung-Min; Heo, Min-Kyu; Kim, Kyu-Yeun; Kwon, Ho Jeong; Hur, Man-Wook

2005-07-29

The POZ domain is a highly conserved protein-protein interaction motif found in many regulatory proteins. Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of a variety of genes in response to infection, inflammation, and stressful conditions. We found that the POZ domain of FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) interacted with the Rel homology domain of the p65 subunit of NF-kappaB in both in vivo and in vitro protein-protein interaction assays. FBI-1 enhanced NF-kappaB-mediated transcription of E-selectin genes in HeLa cells upon phorbol 12-myristate 13-acetate stimulation and overcame gene repression by IkappaB alpha or IkappaB beta. In contrast, the POZ domain of FBI-1, which is a dominant-negative form of FBI-1, repressed NF-kappaB-mediated transcription, and the repression was cooperative with IkappaB alpha or IkappaB beta. In contrast, the POZ domain tagged with a nuclear localization sequence polypeptide of FBI-1 enhanced NF-kappaB-responsive gene transcription, suggesting that the molecular interaction between the POZ domain and the Rel homology domain of p65 and the nuclear localization by the nuclear localization sequence are important in the transcription enhancement mediated by FBI-1. Confocal microscopy showed that FBI-1 increased NF-kappaB movement into the nucleus and increased the stability of NF-kappaB in the nucleus, which enhanced NF-kappaB-mediated transcription of the E-selectin gene. FBI-1 also interacted with IkappaB alpha and IkappaB beta.

Identification of essential sequences for cellular localization in the muscle-specific ubiquitin E3 ligase MAFbx/Atrogin 1.

PubMed

Julie, Lagirand-Cantaloube; Sabrina, Batonnet-Pichon; Marie-Pierre, Leibovitch; Leibovitch, Serge A

2012-02-17

In skeletal muscle atrophy, upregulation and nuclear accumulation of the Ubiquitin E3 ligase MAFbx is essential for accelerated muscle protein loss, but the nuclear/cytoplasmic shuttling of MAFbx is undefined. Here we found that MAFbx contains two functional nuclear localization signals (NLS). Mutation or deletion of only one NLS induced cytoplasmic localization of MAFbx. We identified a non-classical NES located in the leucine charged domain (LCD) of MAFbx, which is leptomycin B insensitive. We demonstrated that mutation (L169Q) in LLXXL motif of LCD suppressed cytoplasmic retention of MAFbx. Nucleocytoplasmic shuttling of MAFbx represents a novel mechanism for targeting its substrates and its cytosolic partners in muscle atrophy. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Seismic risk management solution for nuclear power plants

DOE PAGES

Coleman, Justin; Sabharwall, Piyush

2014-12-01

Nuclear power plants should safely operate during normal operations and maintain core-cooling capabilities during off-normal events, including external hazards (such as flooding and earthquakes). Management of external hazards to expectable levels of risk is critical to maintaining nuclear facility and nuclear power plant safety. Seismic risk is determined by convolving the seismic hazard with seismic fragilities (capacity of systems, structures, and components). Seismic isolation (SI) is one protective measure showing promise to minimize seismic risk. Current SI designs (used in commercial industry) reduce horizontal earthquake loads and protect critical infrastructure from the potentially destructive effects of large earthquakes. The benefitmore » of SI application in the nuclear industry is being recognized and SI systems have been proposed in American Society of Civil Engineer Standard 4, ASCE-4, to be released in the winter of 2014, for light water reactors facilities using commercially available technology. The intent of ASCE-4 is to provide criteria for seismic analysis of safety related nuclear structures such that the responses to design basis seismic events, computed in accordance with this standard, will have a small likelihood of being exceeded. The U.S. nuclear industry has not implemented SI to date; a seismic isolation gap analysis meeting was convened on August 19, 2014, to determine progress on implementing SI in the U.S. nuclear industry. The meeting focused on the systems and components that could benefit from SI. As a result, this article highlights the gaps identified at this meeting.« less

Regulatory Phosphorylation of Ikaros by Bruton's Tyrosine Kinase

PubMed Central

Zhang, Jian; Ishkhanian, Rita; Uckun, Fatih M.

2013-01-01

Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK) as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4) within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros. PMID:23977012

Table of superdeformed nuclear bands and fission isomers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Firestone, R.B.; Singh, B.

A minimum in the second potential well of deformed nuclei was predicted and the associated shell gaps are illustrated in the harmonic oscillator potential shell energy surface calculations shown in this report. A strong superdeformed minimum in {sup 152}Dy was predicted for {beta}{sub 2}-0.65. Subsequently, a discrete set of {gamma}-ray transitions in {sup 152}DY was observed and, assigned to the predicted superdeformed band. Extensive research at several laboratories has since focused on searching for other mass regions of large deformation. A new generation of {gamma}-ray detector arrays is already producing a wealth of information about the mechanisms for feeding andmore » deexciting superdeformed bands. These bands have been found in three distinct regions near A=l30, 150, and 190. This research extends upon previous work in the actinide region near A=240 where fission isomers were identified and also associated with the second potential well. Quadrupole moment measurements for selected cases in each mass region are consistent with assigning the bands to excitations in the second local minimum. As part of our committment to maintain nuclear structure data as current as possible in the Evaluated Nuclear Structure Reference File (ENSDF) and the Table of Isotopes, we have updated the information on superdeformed nuclear bands. As of April 1994, we have complied data from 86 superdeformed bands and 46 fission isomers identified in 73 nuclides for this report. For each nuclide there is a complete level table listing both normal and superdeformed band assignments; level energy, spin, parity, half-life, magneto moments, decay branchings; and the energies, final levels, relative intensities, multipolarities, and mixing ratios for transitions deexciting each level. Mass excess, decay energies, and proton and neutron separation energies are also provided from the evaluation of Audi and Wapstra.« less

Re-suspension of Cesium-134/137 into the Canadian Environment and the Contribution Stemming from the Fukushima-Daiichi Nuclear Incident

NASA Astrophysics Data System (ADS)

Mercier, Jean-Francois; Zhang, Weihua; Loignon-Houle, Francis; Cooke, Michael W.; Ungar, Kurt R.; Pellerin, Eric R.

2013-04-01

Cesium-137 (t1/2 = 30 yr) and cesium-134 (t1/2 = 2yr) constitute major fission by-products observed as the result of a nuclear incident. Such radioisotopes become integrated into the soil and biomass, and can therefore undergo re-suspension into the environment via activities such as forest fires. The Canadian Radiological Monitoring Network (CRMN), which consists of 26 environmental monitoring stations spread across the country, commonly observes cesium-137 in air filters due to re-suspension of material originating from long-past weapons testing. Cesium-134 is not observed owing to its relatively short half-life. The Fukushima-Daiichi nuclear power plant incident of March 2011 caused a major release of radioactive materials into the environment. In Canada, small quantities of both cesium-137 and cesium-134 fallout were detected with great frequency in the weeks which followed, falling off rapidly beginning in July 2011. Since September 2011, the CRMN has detected both cesium-137 and cesium-134 from air filters collected at Yellowknife, Resolute, and Quebec City locations. Using the known initial cesium-134/cesium-137 ratio stemming from this incident, along with a statistical assessment of the normality of the data distribution, we herein present evidence that strongly suggests that these activity spikes are due to re-suspended hot particles originating from the Fukushima-Daiichi nuclear power plant incident. Moreover, we have evidence to suggest that this re-suspension is localized in nature. This study provided empirical insight into the transport and uptake of radionuclides over vast distances, and it demonstrates that the CRMN was able to detect evidence of a re-suspension of Fukushima-Daiichi related isotopes.

The Oncogene PDRG1 Is an Interaction Target of Methionine Adenosyltransferases

PubMed Central

Garrido, Francisco; Reytor, Edel; Portillo, Francisco; Pajares, María A.

2016-01-01

Methionine adenosyltransferases MAT I and MAT III (encoded by Mat1a) catalyze S-adenosylmethionine synthesis in normal liver. Major hepatic diseases concur with reduced levels of this essential methyl donor, which are primarily due to an expression switch from Mat1a towards Mat2a. Additional changes in the association state and even in subcellular localization of these isoenzymes are also detected. All these alterations result in a reduced content of the moderate (MAT I) and high Vmax (MAT III) isoenzymes, whereas the low Vmax (MAT II) isoenzyme increases and nuclear accumulation of MAT I is observed. These changes derive in a reduced availability of cytoplasmic S-adenosylmethionine, together with an effort to meet its needs in the nucleus of damaged cells, rendering enhanced levels of certain epigenetic modifications. In this context, the putative role of protein-protein interactions in the control of S-adenosylmethionine synthesis has been scarcely studied. Using yeast two hybrid and a rat liver library we identified PDRG1 as an interaction target for MATα1 (catalytic subunit of MAT I and MAT III), further confirmation being obtained by immunoprecipitation and pull-down assays. Nuclear MATα interacts physically and functionally with the PDRG1 oncogene, resulting in reduced DNA methylation levels. Increased Pdrg1 expression is detected in acute liver injury and hepatoma cells, together with decreased Mat1a expression and nuclear accumulation of MATα1. Silencing of Pdrg1 expression in hepatoma cells alters their steady-state expression profile on microarrays, downregulating genes associated with tumor progression according to GO pathway analysis. Altogether, the results unveil the role of PDRG1 in the control of the nuclear methylation status through methionine adenosyltransferase binding and its putative collaboration in the progression of hepatic diseases. PMID:27548429

40 CFR 190.10 - Standards for normal operations.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 25 2014-07-01 2014-07-01 false Standards for normal operations. 190.10 Section 190.10 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) RADIATION PROTECTION PROGRAMS ENVIRONMENTAL RADIATION PROTECTION STANDARDS FOR NUCLEAR POWER OPERATIONS Environmental...

40 CFR 190.10 - Standards for normal operations.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Standards for normal operations. 190.10 Section 190.10 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) RADIATION PROTECTION PROGRAMS ENVIRONMENTAL RADIATION PROTECTION STANDARDS FOR NUCLEAR POWER OPERATIONS Environmental Standards...

Antibody-based analysis reveals “filamentous vs. non-filamentous” and “cytoplasmic vs. nuclear” crosstalk of cytoskeletal proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumeta, Masahiro, E-mail: kumeta@lif.kyoto-u.ac.jp; Hirai, Yuya; Yoshimura, Shige H.

2013-12-10

To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do notmore » take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus. - Highlights: • A set of monoclonal antibodies were raised against nuclear scaffold proteins. • Helix-based cytoskeletal proteins were involved in nuclear scaffold. • Many cytoskeletal components shuttle into the nucleus in a CRM1-dependent manner. • Sets of antibodies distinguished distinct subcellular localization of a single isoform. • Nuclear keratin is soluble and does not form an obvious filamentous structure.« less

Amuse-Virgo: Downsizing In Black Hole Accretion

NASA Astrophysics Data System (ADS)

Gallo, Elena

2010-03-01

An issue of fundamental importance in understanding the galaxy-black hole connection is the duty cycle of accretion. If black holes are indeed ubiquitous in galactic nuclei, little is known about the frequency and intensity of their activity, the more so at the low-mass/low-luminosity end. I will present new results from AMUSE-Virgo, a Chandra survey of (formally) inactive early type galaxies in the Virgo cluster. Out of 100 objects, 32 show a nuclear X-ray source, including 6 hybrid nuclei which also host a massive nuclear cluster as visible from archival HST images. After carefully accounting for contamination from nuclear low mass X-ray binaries based on the shape and normalization of their X-ray luminosity function, we conclude that between 24-34% of the galaxies in our sample host a X-ray active super-massive black hole. This sets a firm lower limit to the black hole occupation fraction in nearby bulges within a cluster environment. At face value, the active fraction is found to increase with host stellar mass. However, taking into account selection effects, we find that the average Eddington-scaled X-ray luminosity scales with black hole mass to the power -0.62, with an intrinsic scatter of 0.46 dex. This represents the first observational evidence for down-sizing of black hole accretion in local early types, that is, the fraction of active galaxies, defined as those above a fixed X-ray Eddington ratio, decreases with increasing host galaxy mass.

C. elegans EAK-3 inhibits dauer arrest via nonautonomous regulation of nuclear DAF-16/FoxO activity

PubMed Central

Zhang, Yanmei; Xu, Jinling; Puscau, Cristina; Kim, Yongsoon; Wang, Xi; Alam, Hena; Hu, Patrick J.

2008-01-01

SUMMARY Insulin regulates development, metabolism, and lifespan via a conserved PI3K/Akt pathway that promotes cytoplasmic sequestration of FoxO transcription factors. The regulation of nuclear FoxO is poorly understood. In the nematode Caenorhabditis elegans, insulin-like signaling functions in larvae to inhibit dauer arrest and acts during adulthood to regulate lifespan. In a screen for genes that modulate C. elegans insulin-like signaling, we identified eak-3, which encodes a novel protein that is specifically expressed in the two endocrine XXX cells. The dauer arrest phenotype of eak-3 mutants is fully suppressed by mutations in daf-16/FoxO, which encodes the major target of C. elegans insulin-like signaling, and daf-12, which encodes a nuclear receptor regulated by steroid hormones known as dafachronic acids. eak-3 mutation does not affect DAF-16/FoxO subcellular localization but enhances expression of the direct DAF-16/FoxO target sod-3 in a daf-16/FoxO- and daf-12-dependent manner. eak-3 mutants have normal lifespans, suggesting that EAK-3 decouples insulin-like regulation of development and longevity. We propose that EAK-3 activity in the XXX cells promotes the synthesis and/or secretion of a hormone that acts in parallel to AKT-1 to inhibit the expression of DAF-16/FoxO target genes. Similar hormonal pathways may regulate FoxO target gene expression in mammals. PMID:18241854

Matrix Stiffness Corresponding to Strictured Bowel Induces a Fibrogenic Response in Human Colonic Fibroblasts

PubMed Central

Johnson, Laura A.; Rodansky, Eva S.; Sauder, Kay L.; Horowitz, Jeffrey C.; Mih, Justin D.; Tschumperlin, Daniel J.; Higgins, Peter D.

2013-01-01

Background Crohn’s disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progress to intestinal fibrosis. This inexorable progression towards fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that matrix stiffness regulates this auto-propagation of intestinal fibrosis. Methods The stiffness of fresh ex vivo samples from normal human small intestine, Crohn’s disease strictures, and the unaffected margin were measured with a microelastometer. Normal human colonic fibroblasts were cultured on physiologically normal or pathologically stiff matrices corresponding to the physiological stiffness of normal or fibrotic bowel. Cellular response was assayed for changes in cell morphology, α-smooth muscle actin (αSMA) staining, and gene expression. Results Microelastometer measurements revealed a significant increase in colonic tissue stiffness between normal human colon and Crohn’s strictures as well as between the stricture and adjacent tissue margin. In Ccd-18co cells grown on stiff matrices corresponding to Crohn’s strictures, cellular proliferation increased. Pathologic stiffness induced a marked change in cell morphology and increased αSMA protein expression. Growth on a stiff matrix induced fibrogenic gene expression, decreased matrix metalloproteinase and pro-inflammatory gene expression, and was associated with nuclear localization of the transcriptional cofactor MRTF-A. Conclusions Matrix stiffness, representative of the pathological stiffness of Crohn’s strictures, activates human colonic fibroblasts to a fibrogenic phenotype. Matrix stiffness affects multiple pathways suggesting the mechanical properties of the cellular environment are critical to fibroblast function and may contribute to autopropagation of intestinal fibrosis in the absence of inflammation, thereby contributing to the intractable intestinal fibrosis characteristic of Crohn’s disease. PMID:23502354

Nebraska Prostate Cancer Research Program

DTIC Science & Technology

2012-05-01

Powell. (2012). Dioxin exposure enhances nuclear localization of androgen receptor. The 8th Annual National Symposium on Prostate Cancer by CCRTD...cholesterol. Mol . Cellu. Endo. 295:115-120. 2. Siegel, R., Naishadham, D., and Jemal, A. (2012). Cancer Statistics, 2012. CA Cancer J Clin 62: 10-29...Ul DIOXIN J!1XPOSURE EN CES NUCLEAR LOCALIZATION OF ANDROGEN RECEPTOR\\~f..aTayia Aaron, nd Joann Powell, Center for Cancer Research and Therapeutic

Two short protein domains are responsible for the nuclear localization of the mouse spermine oxidase mu isoform.

PubMed

Bianchi, Marzia; Amendola, Roberto; Federico, Rodolfo; Polticelli, Fabio; Mariottini, Paolo

2005-06-01

In mouse, at least two catalytically active splice variants (mSMOalpha and mSMOmicro) of the flavin-containing spermine oxidase enzyme are present. We have demonstrated previously that the cytosolic mSMOalpha is the major isoform, while the mSMOmicro enzyme is present in both nuclear and cytoplasmic compartments and has an extra protein domain corresponding to the additional exon VIa. By amino acid sequence comparison and molecular modeling of mSMO proteins, we identified a second domain that is necessary for nuclear localization of the mSMOmicro splice variant. A deletion mutant enzyme of this region was constructed to demonstrate its role in protein nuclear targeting by means of transient expression in the murine neuroblastoma cell line, N18TG2.

A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

PubMed

Böhm, Jennifer; Thavaraja, Ramya; Giehler, Susanne; Nalaskowski, Marcus M

2017-09-15

Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

PubMed

Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R Max; Tu, Benjamin P; MacMillan, John B; De Brabander, Jef K; Veech, Richard L; Uyeda, Kosaku

2016-05-13

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Job Aids for Using Preventive Radiological/Nuclear Detection Equipment for Consequence Management

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buddemeier, Brooke R.; Haynes, Daniel; Wood-Zika, Annmarie R.

The overall objective of this project is to research, evaluate, and test first responder preventive radiological/nuclear detection equipment (PRND) to provide state and local agencies with guidance on how to best use this equipment for response after a radiological/nuclear release or detonation.

10 CFR 2.106 - Notice of issuance.

Code of Federal Regulations, 2013 CFR

2013-01-01

... issuance. (a) The Director, Office of New Reactors, Director, Office of Nuclear Reactor Regulation... 10 Energy 1 2013-01-01 2013-01-01 false Notice of issuance. 2.106 Section 2.106 Energy NUCLEAR... of Nuclear Material Safety and Safeguards, as appropriate, will inform the State and local officials...

Featured Article: Nuclear export of opioid growth factor receptor is CRM1 dependent.

PubMed

Kren, Nancy P; Zagon, Ian S; McLaughlin, Patricia J

2016-02-01

Opioid growth factor receptor (OGFr) facilitates growth inhibition in the presence of its specific ligand opioid growth factor (OGF), chemically termed [Met(5)]-enkephalin. The function of the OGF-OGFr axis requires the receptor to translocate to the nucleus. However, the mechanism of nuclear export of OGFr is unknown. In this study, endogenous OGFr, as well as exogenously expressed OGFr-EGFP, demonstrated significant nuclear accumulation in response to leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export, suggesting that OGFr is exported in a CRM1-dependent manner. One consensus sequence for a nuclear export signal (NES) was identified. Mutation of the associated leucines, L217 L220 L223 and L225, to alanine resulted in decreased nuclear accumulation. NES-EGFP responded to LMB, indicating that this sequence is capable of functioning as an export signal in isolation. To determine why the sequence functions differently in isolation than as a full length protein, the localization of subNES was evaluated in the presence and absence of MG132, a potent inhibitor of proteosomal degradation. MG132 had no effect of subNES localization. The role of tandem repeats located at the C-terminus of OGFr was examined for their role in nuclear trafficking. Six of seven tandem repeats were removed to form deltaTR. DeltaTR localized exclusively to the nucleus indicating that the tandem repeats may contribute to the localization of the receptor. Similar to the loss of cellular proliferation activity (i.e. inhibition) recorded with subNES, deltaTR also demonstrated a significant loss of inhibitory activity indicating that the repeats may be integral to receptor function. These experiments reveal that OGFr contains one functional NES, L217 L220 L223 and L225 and can be exported from the nucleus in a CRM1-dependent manner. © 2015 by the Society for Experimental Biology and Medicine.

Fernandina caldera collapse morphology in geometric and dynamic comparison to sandbox models, subsidence sinks over nuclear-explosion cavities, and some other calderas

NASA Astrophysics Data System (ADS)

Howard, K. A.

2009-12-01

The 1968 collapse structure of Fernandina caldera (1.5 km3 collapsed) and also the smaller Darwin Bay caldera in Galápagos each closely resembles morphologically the structural zoning of features found in depressions collapsed into nuclear-explosion cavities (“sinks” of Houser, 1969) and in coherent sandbox-collapse models. Coherent collapses characterized by faulting, folding, and organized structure contrast with spalled pit craters (and lab experiments with collapsed powder) where disorganized piles of floor rubble result from tensile failure of the roof. Subsidence in coherent mode, whether in weak sand in the lab, stronger desert alluvium for nuclear-test sinks, or in hard rock for calderas, exhibits consistent morphologic zones. Characteristically in the sandbox and the nuclear-test analogs these include a first-formed central plug that drops along annular reverse faults. This plug and a surrounding inward-tilted or monoclinal ring (hanging wall of the reverse fault) contract as the structure expands outward by normal faulting, wherein peripheral rings of distending material widen the upper part of the structure along inward-dipping normal faults and compress inner zones and help keep them intact. In Fernandina, a region between the monocline and the outer zone of normal faulting is interpreted, by comparison to the analogs, to overlie the deflation margin of an underlying magma chamber. The same zoning pattern is recognized in structures ranging from sandbox subsidence features centimeters across, to Alae lave lake and nuclear-test sinks tens to hundreds of meters across, to Fenandina’s 2x4 km-wide collapse, to Martian calderas tens of kilometers across. Simple dimensional analysis using the height of cliffs as a proxie for material strength implies that the geometric analogs are good dynamic analogs, and validates that the pattern of both reverse and normal faulting that has been reported consistently from sandbox modeling applies widely to calderas.

A PPARgamma mutant serves as a dominant negative inhibitor of PPAR signaling and is localized in the nucleus.

PubMed

Berger, J; Patel, H V; Woods, J; Hayes, N S; Parent, S A; Clemas, J; Leibowitz, M D; Elbrecht, A; Rachubinski, R A; Capone, J P; Moller, D E

2000-04-25

The peroxisomal proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that act as ligand-activated transcription factors. PPARgamma plays a critical role in regulating adipocyte differentiation and lipid metabolism. Recently, thiazolidinedione (TZD) and select non-TZD antidiabetic agents have been identified as PPARgamma agonists. To further characterize this receptor subclass, a mutant hPPARgamma lacking five carboxyl-terminal amino acids was produced (hPPARgamma2Delta500). In COS-1 cells transfected with PPAR-responsive reporter constructs, the mutant receptor could not be activated by a potent PPARgamma agonist. When cotransfected with hPPARgamma2 or hPPARalpha, hPPARgamma2Delta500 abrogated wild-type receptor activity in a dose-responsive manner. hPPARgamma2Delta500 was also impaired with respect to binding of a high-affinity radioligand. In addition, its conformation was unaffected by normally saturating concentrations of PPARgamma agonist as determined by protease protection experiments. Electrophoretic mobility shift assays demonstrated that hPPARgamma2Delta500 and hPPARgamma2 both formed heterodimeric complexes with human retinoidxreceptor alpha (hRXRalpha) and could bind a peroxisome proliferator-responsive element (PPRE) with similar affinity. Therefore, hPPARgamma2Delta500 appears to repress PPAR activity by competing with wild type receptor to dimerize with RXR and bind the PPRE. In addition, the mutant receptor may titrate out factors required for PPAR-regulated transcriptional activation. Both hPPARgamma2 and hPPARgamma2Delta500 localized to the nucleus of transiently transfected COS-1 cells as determined by immunofluorescence using a PPARgamma-specific antibody. Thus, nuclear localization of PPARgamma occurs independently of its activation state. The dominant negative mutant, hPPARgamma2Delta500, may prove useful in further studies to characterize PPAR functions both in vitro and in vivo

Axonal localization and mitochondrial association of precursor microRNA 338

PubMed Central

Vargas, Jose Norberto S.; Kar, Amar N.; Kowalak, Jeffrey A.; Gale, Jenna R.; Aschrafi, Armaz; Chen, Cai-Yun; Gioio, Anthony E.; Kaplan, Barry B.

2016-01-01

microRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can be locally processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatio-temporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs. PMID:27229124

Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts.

PubMed

Duan, Zhiqiang; Xu, Haixu; Ji, Xinqin; Zhao, Jiafu; Xu, Houqiang; Hu, Yan; Deng, Shanshan; Hu, Shunlin; Liu, Xiufan

2018-12-31

The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336-433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.

Nuclear reactor control apparatus

DOEpatents

Sridhar, Bettadapur N.

1983-11-01

Nuclear reactor core safety rod release apparatus comprises a control rod having a detent notch in the form of an annular peripheral recess at its upper end, a control rod support tube for raising and lowering the control rod under normal conditions, latches pivotally mounted on the control support tube with free ends thereof normally disposed in the recess in the control rod, and cam means for pivoting the latches out of the recess in the control rod when a scram condition occurs. One embodiment of the invention comprises an additional magnetically-operated latch for releasing the control rod under two different conditions, one involving seismic shock.

The effects of nuclear magnetic resonance on patients with cardiac pacemakers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pavlicek, W.; Geisinger, M.; Castle, L.

1983-04-01

The effect of nuclear magnetic resonance (NMR) imaging on six representative cardiac pacemakers was studied. The results indicate that the threshold for initiating the asynchronous mode of a pacemaker is 17 gauss. Radiofrequency levels are present in an NMR unit and may confuse or possibly inhibit demand pacemakers, although sensing circuitry is normally provided with electromagnetic interference discrimination. Time-varying magnetic fields can generate pulse amplitudes and frequencies to mimic cardiac activity. A serious limitation in the possibility of imaging a patient with a pacemaker would be the alteration of normal pulsing parameters due to time-varying magnetic fields.

The Homothorax homeoprotein activates the nuclear localization of another homeoprotein, Extradenticle, and suppresses eye development in Drosophila

PubMed Central

Pai, Chi-Yun; Kuo, Tung-Sheng; Jaw, Thomas J.; Kurant, Estee; Chen, Cheng-Tse; Bessarab, Dmitri A.; Salzberg, Adi; Sun, Y. Henry

1998-01-01

The Extradenticle (Exd) protein in Drosophila acts as a cofactor to homeotic proteins. Its nuclear localization is regulated. We report the cloning of the Drosophila homothorax (hth) gene, a homolog of the mouse Meis1 proto-oncogene that has a homeobox related to that of exd. Comparison with Meis1 finds two regions of high homology: a novel MH domain and the homeodomain. In imaginal discs, hth expression coincides with nuclear Exd. hth and exd also have virtually identical, mutant clonal phenotypes in adults. These results suggest that hth and exd function in the same pathway. We show that hth acts upstream of exd and is required and sufficient for Exd protein nuclear localization. We also show that hth and exd are both negative regulators of eye development; their mutant clones caused ectopic eye formation. Targeted expression of hth, but not of exd, in the eye disc abolished eye development completely. We suggest that hth acts with exd to delimit the eye field and prevent inappropriate eye development. PMID:9450936

Nucleocytoplasmic transfer of cyclin dependent kinase 5 and its binding to puromycin-sensitive aminopeptidase in Dictyostelium discoideum.

PubMed

Huber, Robert J; O'Day, Danton H

2011-08-01

The Dictyostelium discoideum homolog of mammalian cyclin dependent kinase 5 (Cdk5) has previously been shown to be required for optimal growth and differentiation in this model organism, however, the subcellular localization of the protein has not previously been studied. In this study, immunolocalizations and a GFP fusion construct localized Cdk5 predominantly to the nucleus of vegetative cells. Western blots showed that Cdk5 was present in both nuclear and non-nuclear fractions, suggesting a functional role in both cellular locales. During the early stages of mitosis, Cdk5 gradually moved from a punctate nucleoplasmic distribution to localize adjacent to the inner nuclear envelope. During anaphase and telophase, Cdk5 localized to the cytoplasm and was not detected in the nucleoplasm. Cdk5 returned to the nucleus during cytokinesis. Proteolytic activity has been shown to be a critical regulator of the cell cycle. Immunoprecipitations coupled with immunolocalizations identified puromycin-sensitive aminopeptidase A (PsaA) as a potential Cdk5 binding partner in Dictyostelium. Immunoprecipitations also identified two phosphotyrosine proteins (35 and 18 kDa) that may interact with Cdk5 in vivo. Together, this work provides new insight into the localization of Cdk5, its function during cell division, and its binding to a proteolytic enzyme in Dictyostelium.