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Sample records for cell nuclear proteins

  1. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

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    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M. [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany); Wehnert, Manfred [Institute of Human Genetics, University of Greifswald, Greifswald (Germany); Huebner, Stefan, E-mail: stefan.huebner@mail.uni-wuerzburg.de [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)

    2009-08-15

    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-associated 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.

  2. Multidimensional profiling of cell surface proteins and nuclear markers

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    Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Helen Barcellos-Hoff, Mary; Parvin, Bahram

    2009-01-30

    Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables.

  3. Aberrant expression of nuclear matrix proteins during HMBA-induced differentiation of gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate the aberrant expression of nuclear matrix proteins in human gastric cancer cells before and after hexamethylene bisacetamide (HMBA) treatment.METHODS: Proteomics analysis of differential nuclear matrix proteins was performed by two dimensional electrophoresis polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The expression levels of three nuclear matrix proteins were further confirmed by Western blotting and their location...

  4. Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Rebekka Müller

    Full Text Available Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM. Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.

  5. Alteration of nuclear matrix-intermediate filament system and differential expression of nuclear matrix proteins during human hepatocarcinoma cell differentiation

    Institute of Scientific and Technical Information of China (English)

    Jian Tang; Jing-Wen Niu; Dong-Hui Xu; Zhi-Xing Li; Qi-Fu Li; Jin-An Chen

    2007-01-01

    AIM:To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells.METHODS: Cells cultured with or without 5 × 10-3mmol/L of hexamethylene bisacetamide (HMBA) on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin.The peptides were analyzed by matrix-assisted laserdesorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Data were submitted for database searching using Mascot tool (www. Matrixscience.com).RESULTS: The nuclear matrix (NM) and intermediate filament (IF) in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly.The nuclear lamina was non-uniform, and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA, the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments, including highly branched utrathin filaments could also be seen in the regular meshwork. The connection between the two kinds of filaments and the relatively thin, condensed and sharply demarcated lamina composed of intermediatesized filaments was relatively fastened. Meanwhile, 21NM proteins changed remarkably during SMMC-7721cell differentiation. Four proteins, I.e. Mutant Pyst1,hypothetical protein, nucleophosmin1, and LBP were downregulated, whereas four other proteins, eIF6, p44subunit, β-tubulin, and SIN3B were upregulated with the last one, SR2/ASF found only in the differentiated SMMC-7721 cells.CONCLUSION: The induced differentiation of SMMC-7721cells by HMBA is

  6. Proteomic analysis of nuclear matrix proteins during arsenic trioxide induced apoptosis in leukemia K562 cells

    Institute of Scientific and Technical Information of China (English)

    WANG Zi-hui; YU Ding; CHEN Yan; HAO Jian-zhong

    2005-01-01

    Background Arsenic trioxide (As2O3) has been identified as a very potent anti-acute leukemic agent. However its role in apoptosis needs to be elucidated. As2O3 interferes with the proliferation and survival of tumor cells via a variety of mechanisms. Drug-target interactions at the level of nuclear matrix (NM) may be critical events in the induction of cell death by As2O3. This study dealt with As2O3-target interactions at the level of NM in chronic myelogenous leukemia cell line K562 by proteomics. Methods K562 cells were cultured in MEM and treated with different concentrations of As2O3. The nuclear matrix proteins were analyzed by high-resolution two-dimensional gel electrophoresis and computer-assisted image analysis. Results As2O3 significantly inhibited the growth of chronic myelogenous leukemia cell line K562 at low concentrations. While more than 200 protein spots were shared among the nuclear matrices, about 18 distinct spots in the nuclear matrices were found characteristic for As2O3 treated cells. Conclusions: As2O3 induces apoptosis in K562 cells in a dose and time-dependent manner. Our results demonstrated that for the detection of the onset of apoptosis, the alteration in the composition of nuclear matrix proteins was a more sensitive indicator than nucleosomal DNA fragmentation test. These results indicated that As2O3 might be clinically useful in the treatment of chronic myelogenous leukemia. The changes of nuclear matrix proteins in the treated cells can be used as a useful indicator for this treatment.

  7. Localization of influenza virus proteins to nuclear dot 10 structures in influenza virus-infected cells

    International Nuclear Information System (INIS)

    We studied influenza virus M1 protein by generating HeLa and MDCK cell lines that express M1 genetically fused to green fluorescent protein (GFP). GFP-M1 was incorporated into virions produced by influenza virus infected MDCK cells expressing the fusion protein indicating that the fusion protein is at least partially functional. Following infection of either HeLa or MDCK cells with influenza A virus (but not influenza B virus), GFP-M1 redistributes from its cytosolic/nuclear location and accumulates in nuclear dots. Immunofluorescence revealed that the nuclear dots represent nuclear dot 10 (ND10) structures. The colocalization of authentic M1, as well as NS1 and NS2 protein, with ND10 was confirmed by immunofluorescence following in situ isolation of ND10. These findings demonstrate a previously unappreciated involvement of influenza virus with ND10, a structure involved in cellular responses to immune cytokines as well as the replication of a rapidly increasing list of viruses

  8. Altered profiles of nuclear matrix proteins during the differentiation of human gastric mucous adenocarcinoma MGc80-3 cells

    Institute of Scientific and Technical Information of China (English)

    Chun-Hong Zhao; Qi-Fu Li

    2005-01-01

    AIM: To find and identify specific nuclear matrix proteins associated with proliferation and differentiation of carcinoma cells, which will be potential markers for cancer diagnosis and targets in cancer therapy.METHODS: Nuclear matrix proteins were selectively extracted from MGc80-3 cells treated with or without hexamethylamine bisacetamide (HMBA), and subjected to 2-D gel electrophoresis. The resulted protein patterns were analyzed by Melanie software. Spots of nuclear matrix proteins differentially expressed were excised and subjected to in situ digestion with trypsin. Peptide masses were obtained by matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis and submitted for database searching using Mascot tool.RESULTS: The MGc80-3 cells were induced into differentiation by HMBA. There were 22 protein spots which changed remarkably in the nuclear matrix, from differentiation of MGc80-3 cells compared to control.Eleven of which were identified. Seven proteins -actin, prohibitin, porin 31HL, heterogeneous nuclear ribonucleoprotein A2/B1, vimentin, ATP synthase, and heatshock protein 60 were downregulated, whereas three proteins - heat shock protein gp96, heat shock protein 90-beta, and valosin-containing protein were upregulated,and the oxygen-regulated protein was only found in the differentiated MGc80-3 cells.CONCLUSION: The induced differentiation of carcinoma cells is accompanied by the changes of nuclear matrix proteins. Further characterization of those proteins will show the mechanism of cellular proliferation and differentiation, as well as cancer differentiation.

  9. Nuclear matrix associated protein PML: an arsenic trioxide apoptosis therapeutic target protein in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    于鼎; 王子慧; 朱立元; 邱殷庆

    2003-01-01

    Objective To investigate arsenic trioxide (As2O3)-induced apoptosis and the effects on cell nuclear matrix related protein promyelocytic leukaemia (PML). Methods HepG2 cells were cultured in MEM medium and treated with 0.5, 2, 5 and 10 μmol/L As2O3 for either 24 h or 96 h at each concentration. In situ terminal deoxynucleotidyl transferase (TdT) labeling (TUNEL) and DNA ladders were used to detect apoptosis. Confocal microscopy and Western blotting were used to observe the expression of PML. Results The growth rates of HepG2 cells were slower in the As2O3 treated than the untreated control group. DNA ladder and TUNEL positive apoptotic cells could be detected in As2O3 treated groups. The expression of PML decreased in HepG2 cells with 2 μmol/L As2O3 treatment. Confocal images demonstrated that the expression of PML protein in HepG2 cell nuclei decreased after treatment with 2 μmol/L As2O3, and micropunctates characteristic of PML protein in HepG2 cell nuclei disappeared after treatment with 5 μmol/L As2O3.Conclusions Our results show that arsenic trioxide can significantly inhibit the growth of HepG2 cells in vitro. As2O3 induces apoptosis in HepG2 tumor cells in a time and concentration dependent manner. As2O3 may degrade the PML protein in HepG2 cell nuclei. The decreased expression of PML in As2O3 treated tumor cells is most likely to be caused by apoptosis. Nuclear matrix associated protein PML could be the target of As2O3 therapy.

  10. Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

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    C. Soldani

    2010-05-01

    Full Text Available Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP-containing components (PANA, hnRNP-core proteins, fibrillarin or RNP-associated nuclear proteins (SC-35 splicing factor. Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures.

  11. Abnormal expressions of proliferating cell nuclear antigen and P27 protein in brain glioma

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    BACKGROUND: Both proliferating cell nuclear antigen and P27 protein are important factors to regulate cell cycle. While, the combination of them can provide exactly objective markers to evaluate prognosis of patients with brain glioma needs to be further studied based on pathological level.OBJECTIVE: To observe the expressions of proliferating cell nuclear antigen and P27 protein in both injured and normal brain glioma tissues and analyze the effect of them on onset and development of brain glioma.DESIGN: Case contrast observation.SETTING: Department of Neurosurgery, the Second Affiliated Hospital of Xi'an Jiaotong University.PARTICIPANTS: A total of 63 patients with brain glioma were selected from Department of Neurosurgery,the Second Affiliated Hospital of Xi'an Jiaotong University from July 1996 to June 2000. There were 38 males and 25 females and their ages ranged from 23 to 71 years. Based on pathological classification and grading standards of brain glioma, patients were divided into grade Ⅰ - tⅡ (n =30) and grade Ⅲ - Ⅳ (n =33). All cases received one operation but no radiotherapy and chemiotherapy before operation. Sample tissues were collected from tumor parenchyma. Non-neoplastic brain tissues were collected from another 12 non-tumor subjects who received craniocerebral trauma infra-decompression and regarded as the control group. There were 10 males and 2 females and their ages ranged from 16 to 54 years. The experiment had got confirmed consent from local ethic committee and the collection was provided confirmed consent from patients and their relatives. All samples were restained with HE staining so as to diagnose as the brain glioma.While, all patients with brain glioma received radiotherapy after operation and their survival periods were followed up.METHODS: Primary lesion wax of brain glioma was cut into serial sections and stained with S-P immunohistochemical staining. Brown substance which was observed in tumor nucleus was regarded as the

  12. Changes of Nuclear Matrix Proteins Following the Differentiation of Human Osteosarcoma MG-63 Cells

    Institute of Scientific and Technical Information of China (English)

    Chun-Hong Zhao; Qi-Fu Li; Yan Zhao; Jing-Wen Niu; Zhi-Xing Li; Jin-An Chen

    2006-01-01

    Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide (HMBA). Their nuclear matrix proteins (NMPs) were selectively extracted and subjected to two-dimensional gel electrophoresis analysis.The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOFMS analysis, and were submitted for NCBI database searches by Mascot tool.There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.

  13. SUMOylation regulates the nuclear mobility of CREB binding protein and its association with nuclear bodies in live cells

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    Ryan, Colm M.; Kindle, Karin B.; Collins, Hilary M. [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom); Heery, David M., E-mail: david.heery@nottingham.ac.uk [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom)

    2010-01-01

    The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP-CBP{Delta}998-1087 had a retarded recovery time in the nucleus, as compared to YFP-CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.

  14. Reassembly and protection of small nuclear ribonucleoprotein particles by heat shock proteins in yeast cells.

    OpenAIRE

    Bracken, A P; Bond, U

    1999-01-01

    The process of mRNA splicing is sensitive to in vivo thermal inactivation, but can be protected by pretreatment of cells under conditions that induce heat-shock proteins (Hsps). This latter phenomenon is known as "splicing thermotolerance". In this article we demonstrate that the small nuclear ribonucleoprotein particles (snRNPs) are in vivo targets of thermal damage within the splicing apparatus in heat-shocked yeast cells. Following a heat shock, levels of the tri-snRNP (U4/U6.U5), free U6 ...

  15. The folate-coupled enzyme MTHFD2 is a nuclear protein and promotes cell proliferation.

    Science.gov (United States)

    Gustafsson Sheppard, Nina; Jarl, Lisa; Mahadessian, Diana; Strittmatter, Laura; Schmidt, Angelika; Madhusudan, Nikhil; Tegnér, Jesper; Lundberg, Emma K; Asplund, Anna; Jain, Mohit; Nilsson, Roland

    2015-01-01

    Folate metabolism is central to cell proliferation and a target of commonly used cancer chemotherapeutics. In particular, the mitochondrial folate-coupled metabolism is thought to be important for proliferating cancer cells. The enzyme MTHFD2 in this pathway is highly expressed in human tumors and broadly required for survival of cancer cells. Although the enzymatic activity of the MTHFD2 protein is well understood, little is known about its larger role in cancer cell biology. We here report that MTHFD2 is co-expressed with two distinct gene sets, representing amino acid metabolism and cell proliferation, respectively. Consistent with a role for MTHFD2 in cell proliferation, MTHFD2 expression was repressed in cells rendered quiescent by deprivation of growth signals (serum) and rapidly re-induced by serum stimulation. Overexpression of MTHFD2 alone was sufficient to promote cell proliferation independent of its dehydrogenase activity, even during growth restriction. In addition to its known mitochondrial localization, we found MTHFD2 to have a nuclear localization and co-localize with DNA replication sites. These findings suggest a previously unknown role for MTHFD2 in cancer cell proliferation, adding to its known function in mitochondrial folate metabolism.

  16. Novel nuclear protein ALC-INTERACTING PROTEIN1 is expressed in vascular and mesocarp cells in Arabidopsis.

    Science.gov (United States)

    Wang, Fang; Shi, Dong-Qiao; Liu, Jie; Yang, Wei-Cai

    2008-07-01

    Pod shattering is an agronomical trait that is a result of the coordinated action of cell differentiation and separation. In Arabidopsis, pod shattering is controlled by a complex genetic network in which ALCATRAZ (ALC), a member of the basic helix-loop-helix family, is critical for cell separation during fruit dehiscence. Herein, we report the identification of ALC-INTERACTING PROTEIN1 (ACI1) via the yeast two-hybrid screen. ACI1 encodes a nuclear protein with a lysine-rich domain and a C-terminal serine-rich domain. ACI1 is mainly expressed in the vascular system throughout the plant and mesocarp of the valve in siliques. Our data showed that ACI1 interacts strongly with the N-terminal portion of ALC in yeast cells and in plant cells in the nucleus as demonstrated by bimolecular fluorescence complementation assay. Both ACI1 and ALC share an overlapping expression pattern, suggesting that they likely function together in planta. However, no detectable phenotype was found in plants with reduced ACI1 expression by RNA interference technology, suggesting that ACI1 may be redundant. Taken together, these data indicate that ALC may interact with ACI1 and its homologs to control cell separation during fruit dehiscence in Arabidopsis. PMID:18713402

  17. Novel Nuclear Protein ALC-INTERACTING PROTEIN1 is Expressed in Vascular and Mesocarp Cells in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Fang Wang; Dong-Qiao Shi; Jie Liu; Wei-Cai Yang

    2008-01-01

    Pod shattering is an agronomical trait that is a result of the coordinated action of cell differentiation and separation. In Arabidopsis, pod shattering is controlled by a complex genetic network in which ALCATRAZ (ALC), a member of the basic helix-loop-helix family, is critical for cell separation during fruit dehiscence. Herein, we report the identification of ALC-INTERACTiNG PROTEIN1 (ACI1) via the yeast two-hybrid screen. ACI1 encodes a nuclear protein with a lysine-rich domain and a C-terminal serine-rich domain. ACI1 is mainly expressed in the vascular system throughout the plant and mesocarp of the valve in siliques. Our data showed that ACI1 interacts strongly with the N-terminal portion of ALC in yeast cells and in plant cells in the nucleus as demonstrated by bimolecular fluorescence complementation assay. Both ACl1 and ALC share an overlapping expression pattern, suggesting that they likely function together in planta. However, no detectable phenotype was found in plants with reduced ACI1 expression by RNA interference technology, suggesting that ACI1 may be redundant. Taken together, these data indicate that ALC may interact with ACll and its homologs to control cell separation during fruit dehiscence in Arabidopsis.

  18. EXPRESSION OF P53 PROTEIN AND PROLIFERATING CELL NUCLEAR ANTIGEN IN HUMAN GESTATION TROPHOBLASTIC DISEASE

    Institute of Scientific and Technical Information of China (English)

    黄铁军; 王志忠; 方光光; 刘志恒

    2004-01-01

    Objective: To study the relationship between p53 protein, proliferating cell nuclear antigen (PCNA) expression and benign or malignant gestational trophoblastic disease (MGTD). Methods: The histotomic sections of 48 patients with gestational trophoblastic disease and 24 patients of normal chorionic villi were stained using immunohistochemistry. The monoclonal antibodies were used to determine p53 protein and PCNA. Results: The frequency of p53 and PCNA positive expression were significantly different among the chorionic villi of normal pregnancy, hydratidiform mole (HM) and MGTD. But neither p53 nor PCNA has any relation with the clinical staging or metastasis of MGTD. Conclusion: Both P53 and PCNA are valuable in diagnosis of human gestational trophoblastic disease.

  19. Determining nuclear shape: The role of farnesylated nuclear membrane proteins

    OpenAIRE

    Polychronidou, Maria; Großhans, Jörg

    2011-01-01

    Changes in nuclear morphology are observed in diverse developmental processes as well as in pathological conditions. Modification of nuclear membrane and nuclear lamina protein levels results in altered nuclear shapes, as it has been demonstrated in experimental systems ranging from yeast to human cells. The important role of nuclear membrane components in regulating nuclear morphology is additionally highlighted by the abnormally shaped nuclei observed in diseases where nuclear lamina protei...

  20. Green fluorescent protein (GFP) transgenic pig produced by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    LIU ZhongHua; SUN Shuang; LI YuTian; WANG HongBin; R S PRATHER; SONG Jun; WANG ZhenKun; TIAN JiangTian; KONG QingRan; ZHENG Zhong; YIN Zhi; GAO Li; MA HaiKun

    2008-01-01

    Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm ani-mals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfection system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe-tal-derived fibroblast cells cultured with 4.0 plJmL liposome and 1.6 pg/mL plasmid DNA for 6 h re-sulted in the highest transfection rate (3.6%). The percentage of GFP reconstructed embryos that de-veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Re-constructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 de-livered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.

  1. A Peptide Mimicking a Region in Proliferating Cell Nuclear Antigen Specific to Key Protein Interactions Is Cytotoxic to Breast Cancer

    OpenAIRE

    Smith, Shanna J.; Gu, Long; Phipps, Elizabeth A.; Lacey E Dobrolecki; Mabrey, Karla S.; Gulley, Pattie; Dillehay, Kelsey L; Dong, Zhongyun; Fields, Gregg B.; Chen, Yun-Ru; Ann, David; Hickey, Robert J.; Malkas, Linda H.

    2015-01-01

    Proliferating cell nuclear antigen (PCNA) is a highly conserved protein necessary for proper component loading during the DNA replication and repair process. Proteins make a connection within the interdomain connector loop of PCNA, and much of the regulation is a result of the inherent competition for this docking site. If this target region of PCNA is modified, the DNA replication and repair process in cancer cells is potentially altered. Exploitation of this cancer-associated region has imp...

  2. Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Yoon, Hyun-Joo [TissueGene Inc. 9605 Medical Center Dr., Rockville, MD 20850 (United States); Yoo, Hae Yong [Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Samsung Medical Center, Sungkyunkwan University, Seoul 135-710 (Korea, Republic of); Choi, Cheol Yong, E-mail: choicy@skku.ac.kr [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2014-01-03

    Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

  3. Levels of synthesis of primate-specific nuclear proteins differ between growth-arrested and proliferating cells

    International Nuclear Information System (INIS)

    A monoclonal antibody that reacts specifically with the proliferation-sensitive nuclear proteins, isoelectric focusing (IEF) 8Z31 (molecular weight (MW), 76,000 charge variants, HeLa protein catalogue number) has been characterized. As determined by indirect immunofluorescence, the antibody stains the nucleolus and nucleoplasm of interphase-cultured cells of primate origin, but does not react with cells of other species. Proteins having similar MWs and isoelectric points as the human or monkey (primates) proteins were not observed in cultured cells of the following species: aves, bat, dog, dolphin, goat, hamster, mink, mouse, pisces, potoroo, rabbit and rat. Quantitative two-dimensional (2D) gel electrophoretic analysis of [35S]methionine-labelled proteins synthesized by normal (quiescent, proliferating) and SV40-transformed human MRC-5 fibroblasts revealed significant differences in the levels of synthesis of both IEF 8Z30 and 8Z31. In quiescent cells the main labelled product corresponded to IEF 8Z31 (ratio IEF 8Z31/8Z30, 2.3), while in the transformed cells the major product was IEF 8Z30 (ratio, 0.62). Normal proliferating fibroblasts exhibited similar levels of both proteins (ratio, 1.21). Combined levels of synthesis of both proteins were 1.50 and 1.20 times as high in the transformed cells as in the quiescent and proliferating cells, respectively. Modulation of the levels of synthesis of these proteins may play a role in cell proliferation

  4. Rhinovirus 3C protease facilitates specific nucleoporin cleavage and mislocalisation of nuclear proteins in infected host cells.

    Directory of Open Access Journals (Sweden)

    Erin J Walker

    Full Text Available Human Rhinovirus (HRV infection results in shut down of essential cellular processes, in part through disruption of nucleocytoplasmic transport by cleavage of the nucleoporin proteins (Nups that make up the host cell nuclear pore. Although the HRV genome encodes two proteases (2A and 3C able to cleave host proteins such as Nup62, little is known regarding the specific contribution of each. Here we use transfected as well as HRV-infected cells to establish for the first time that 3C protease is most likely the mediator of cleavage of Nup153 during HRV infection, while Nup62 and Nup98 are likely to be targets of HRV2A protease. HRV16 3C protease was also able to elicit changes in the appearance and distribution of the nuclear speckle protein SC35 in transfected cells, implicating it as a key mediator of the mislocalisation of SC35 in HRV16-infected cells. In addition, 3C protease activity led to the redistribution of the nucleolin protein out of the nucleolus, but did not affect nuclear localisation of hnRNP proteins, implying that complete disruption of nucleocytoplasmic transport leading to relocalisation of hnRNP proteins from the nucleus to the cytoplasm in HRV-infected cells almost certainly requires 2A in addition to 3C protease. Thus, a specific role for HRV 3C protease in cleavage and mislocalisation of host cell nuclear proteins, in concert with 2A, is implicated for the first time in HRV pathogenesis.

  5. The SMN protein is a key regulator of nuclear architecture in differentiating neuroblastoma cells

    OpenAIRE

    Clelland, Allyson K.; Kinnear, Nicholas P; Oram, Lisa; Burza, Julie; Sleeman, Judith Elizabeth

    2009-01-01

    The cell nucleus contains two closely related structures, Cajal bodies (CBs) and gems. CBs are the first site of accumulation of newly assembled splicing snRNPs (small nuclear ribonucleoproteins) following their import into the nucleus, before they form their steady-state localization in nuclear splicing speckles. Gems are the nuclear site of accumulation of survival motor neurons (SMNs), an insufficiency of which leads to the inherited neurodegenerative condition, spinal muscular atrophy (SM...

  6. Nuclear envelope proteins modulate proliferation of vascular smooth muscle cells during cyclic stretch application.

    Science.gov (United States)

    Qi, Ying-Xin; Yao, Qing-Ping; Huang, Kai; Shi, Qian; Zhang, Ping; Wang, Guo-Liang; Han, Yue; Bao, Han; Wang, Lu; Li, Hai-Peng; Shen, Bao-Rong; Wang, Yingxiao; Chien, Shu; Jiang, Zong-Lai

    2016-05-10

    Cyclic stretch is an important inducer of vascular smooth muscle cell (VSMC) proliferation, which is crucial in vascular remodeling during hypertension. However, the molecular mechanism remains unclear. We studied the effects of emerin and lamin A/C, two important nuclear envelope proteins, on VSMC proliferation in hypertension and the underlying mechano-mechanisms. In common carotid artery of hypertensive rats in vivo and in cultured cells subjected to high (15%) cyclic stretch in vitro, VSMC proliferation was increased significantly, and the expression of emerin and lamin A/C was repressed compared with normotensive or normal (5%) cyclic stretch controls. Using targeted siRNA to mimic the repressed expression of emerin or lamin A/C induced by 15% stretch, we found that VSMC proliferation was enhanced under static and 5%-stretch conditions. Overexpression of emerin or lamin A/C reversed VSMC proliferation induced by 15% stretch. Hence, emerin and lamin A/C play critical roles in suppressing VSMC hyperproliferation induced by hyperstretch. ChIP-on-chip and MOTIF analyses showed that the DNAs binding with emerin contain three transcription factor motifs: CCNGGA, CCMGCC, and ABTTCCG; DNAs binding with lamin A/C contain the motifs CVGGAA, GCCGCYGC, and DAAGAAA. Protein/DNA array proved that altered emerin or lamin A/C expression modulated the activation of various transcription factors. Furthermore, accelerating local expression of emerin or lamin A/C reversed cell proliferation in the carotid artery of hypertensive rats in vivo. Our findings establish the pathogenetic role of emerin and lamin A/C repression in stretch-induced VSMC proliferation and suggest mechanobiological mechanism underlying this process that involves the sequence-specific binding of emerin and lamin A/C to specific transcription factor motifs.

  7. 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

    Energy Technology Data Exchange (ETDEWEB)

    Eto, Masumi, E-mail: masumi.eto@jefferson.edu [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kim, Jee In [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu 700-422 (Korea, Republic of)

    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 concentrated 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.

  8. 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

    International Nuclear Information System (INIS)

    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 concentrated 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

  9. Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces.

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    Amalia Slomiany, Maria Grabska, Bronislaw L. Slomiany

    2006-01-01

    Full Text Available Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860. In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN, the outer nuclear membrane (ONM, the inner nuclear membrane (INM and the cell cytosol (CC. In contrast to Endoplasmic Reticulum (ER which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC, phosphatidylinositol (PI, phosphatidylinositol phosphates (PIPs and phosphatidic acid (PA. The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of

  10. High frequency of tumor cells with nuclear Egr-1 protein expression in human bladder cancer is associated with disease progression

    International Nuclear Information System (INIS)

    Egr-1 (early growth response-1 transcription factor) has been proposed to be involved in invasion and metastasis processes of human bladder cancer, but Egr-1 protein expression levels in human bladder cancer have not been investigated. In the present study we investigated the expression levels of Egr-1 protein in early stages of human bladder cancer and correlated it to later progression. Expression of Egr-1 protein in human bladder cancer was examined by immunohistochemistry, on a tissue microarray constructed from tumors from 289 patients with non-muscle invasive urothelial bladder cancer. The frequency of tumor cells with nuclear Egr-1 immunolabelling correlated to bladder cancer stage, grade and to later progression to muscle-invasive bladder cancer (T2-4). Stage T1 tumors exhibited significantly higher frequencies of tumor cells with nuclear Egr-1 immunolabelling than Ta tumors (P = 0.001). Furthermore, Kaplan-Meier survival analysis showed that a high frequency of tumor cells with nuclear Egr-1 immunolabelling was significantly associated with a higher risk of progression to stage T2-4 (log-rank test, P = 0.035). Tumor cells with nuclear Egr-1 immunolabelling were found to localize at the tumor front in some of the tumor biopsies. The results from this study support a potential involvement of Egr-1 in the progression from non-muscle invasive bladder cancers to muscle invasive bladder cancer

  11. Deguelin regulates nuclear pore complex proteins Nup98 and Nup88 in U937 cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Hong-li LIU; Yan CHEN; Guo-hui CUI; Qiu-ling WU; Jing HE; Wei-hua CHEN; Jian-feng ZHOU

    2005-01-01

    Aim: To investigate the anticancer effects and the molecular mechanisms of deguelin on human U937 leukemia cells, and to explore the underlying mechanism regulating nucleoporin 98 (Nup98) and nucleoporin 88 (Nup88) in vitro. Methods: The effects of deguelin on the growth of U937 cells were studied by MTT assay.The effect of deguelin on the cell cycle of U937 cells was studied by using a propidium iodide method. The localization of the nuclear pore complex proteins Nup98 and Nup88 was investigated by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 and Nup88 in U937 cells was investigated by using flow cytometry and Western blot. Results: The proliferation of U937 cells was inhibited in the deguelin-treated group, with a 24-h IC50 value of 21.61 nmol/L and a 36-h IC50 value of 17.07 nmol/L. U937 cells treated with deguelin had reduced percentages of cells in the G0/G1 phase, whereas cells accumulated in the S and G2/M phases. Nup88 and Nup98 were found on both the nuclear and cytoplasmic sides of the U937 cells by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 was upregulated and that of the Nup88 protein was downregulated in U937 cells treated with deguelin.Conclusion: Deguelin is able to inhibit the proliferation of U937 cells by regulating the cell cycle such that cells are arrested at the S and G2/M phases, so that the proportion of cells in the G0/G1 phase decreases. The antitumor effects of deguelin are related to upregulating the expression of Nup98 and downregulating the expression of Nup88 protein in U937 cells.

  12. Basic nuclear protein pattern and chromatin condensation in the male germ cells of a tropical abalone, Haliotis asinina.

    Science.gov (United States)

    Suphamungmee, Worawit; Apisawetakan, Somjai; Weerachatyanukul, Wattana; Wanichanon, Chaitip; Sretarugsa, Prapee; Poomtong, Tanes; Sobhon, Prasert

    2005-02-01

    The basic nuclear proteins (BNPs) in spermatozoa of a tropical abalone, Haliotis asinina, were composed of a majority of protamine-like (PL) protein and a small amount of histones H1 and H4. Abalone H1 and PL proteins exhibited strong immunological cross reactivities among themselves as well as with chick H5 and calf thymus H1. Thus, all these proteins may belong to the same family. Immunolocalization by indirect immunofluorescence and immunoelectron microscopy indicated that H1 and H4 were present in all steps of the male germ cells, however, with decreasing amount in late stage cells, particularly spermatids and spermatozoa. On the other hand, PL was present in middle step cells (secondary spermatocytes) with increasing amount in spermatids and spermatozoa when the chromatin became tightly packed. Thus, PL may be involved in the condensation of chromatin in the spermatozoa of this species.

  13. Nuclear Import Analysis of Two Different Fluorescent Marker Proteins into Hepatocyte Cell Lines (HuH-7 Cell

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    Aris Haryanto

    2015-10-01

    Full Text Available The application of fluorescent proteins as expression markers and protein fusion partners has provedimmensely valuable for resolving the organization of biological events in living cells. EGFP and DsRed2 arecommonly fluorescent marker protein which is used for biotechnology and cell biology research. The presentstudy was designed to identify the expression vector that suitable to ligate with DNA encoding HBV coreprotein for intracellular localization study in hepatocyte cell, which were expressed as fusion proteins. We alsocompared and quantified the expressed fluorescent protein which predominantly localized in the cellcompartment. The results indicated that DsRed2 shown as less than ideal for intracellular localization study ofthan EGFP, because of its tetrameric structure of the fluorescent protein and when fused to a protein of interest,the fusion protein often forms aggregates in the living cells. In contrast, EGFP fluorescent protein shown a muchhigher proportion of cytoplasmic localization, thus being more suitable for analysis of intracellular localizationthan DsRed2 fluorescent protein. EGFP fluorescent protein is also capable to produce a strong green fluorescencewhen excited by blue light, without any exogenously added substrate or cofactor, events inside living cell canthus be visualized in a non-invasive way. Based on our present quantitative data and some reasons above shownthat EGFP is more suitable than DsRed2 as a fluorescent marker protein for intracellular localization study intoHuH-7 cell.Keywords: EGFP, DsRed2 fluorescent protein , HuH-7 cell, HBV, intracellular localization

  14. The Nuclear Zinc Finger Protein Zfat Maintains FoxO1 Protein Levels in Peripheral T Cells by Regulating the Activities of Autophagy and the Akt Signaling Pathway.

    Science.gov (United States)

    Ishikura, Shuhei; Iwaihara, Yuri; Tanaka, Yoko; Luo, Hao; Nishi, Kensuke; Doi, Keiko; Koyanagi, Midori; Okamura, Tadashi; Tsunoda, Toshiyuki; Shirasawa, Senji

    2016-07-15

    Forkhead box O1 (FoxO1) is a key molecule for the development and functions of peripheral T cells. However, the precise mechanisms regulating FoxO1 expression in peripheral T cells remain elusive. We previously reported that Zfat(f/f)-CD4Cre mice showed a marked decline in FoxO1 protein levels in peripheral T cells, partially through proteasomal degradation. Here we have identified the precise mechanisms, apart from proteasome-mediated degradation, of the decreased FoxO1 levels in Zfat-deficient T cells. First, we confirmed that tamoxifen-inducible deletion of Zfat in Zfat(f/f)-CreERT2 mice coincidently decreases FoxO1 protein levels in peripheral T cells, indicating that Zfat is essential for maintaining FoxO1 levels in these cells. Although the proteasome-specific inhibitors lactacystin and epoxomicin only moderately increase FoxO1 protein levels, the inhibitors of lysosomal proteolysis bafilomycin A1 and chloroquine restore the decreased FoxO1 levels in Zfat-deficient T cells to levels comparable with those in control cells. Furthermore, Zfat-deficient T cells show increased numbers of autophagosomes and decreased levels of p62 protein, together indicating that Zfat deficiency promotes lysosomal FoxO1 degradation through autophagy. In addition, Zfat deficiency increases the phosphorylation levels of Thr-308 and Ser-473 of Akt and the relative amounts of cytoplasmic to nuclear FoxO1 protein levels, indicating that Zfat deficiency causes Akt activation, leading to nuclear exclusion of FoxO1. Our findings have demonstrated a novel role of Zfat in maintaining FoxO1 protein levels in peripheral T cells by regulating the activities of autophagy and the Akt signaling pathway. PMID:27226588

  15. Identification of interacting proteins of retinoid-related orphan nuclear receptor gamma in HepG2 cells

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    Ze-Min Huang1,#, Jun Wu2,#, Zheng-Cai Jia1, Yi Tian1, Jun Tang3, Yan Tang1, Ying Wang2, Yu-Zhang Wu1,* & Bing Ni1,*

    2012-06-01

    Full Text Available The retinoid-related orphan nuclear receptor gamma (RORγplays critical roles in regulation of development, immunity andmetabolism. As transcription factor usually forms a proteincomplex to function, thus capturing and dissecting of theRORγ protein complex will be helpful for exploring themechanisms underlying those functions. After construction ofthe recombinant tandem affinity purification (TAP plasmid,pMSCVpuro RORγ-CTAP(SG, the nuclear localization ofRORγ-CTAP(SG fusion protein was verified. Followingisolation of RORγ protein complex by TAP strategy, sevencandidate interacting proteins were identified. Finally, the heatshock protein 90 (HSP90 and receptor-interacting protein 140(RIP140 were confirmed to interplay with RORγ byco-immunoprecipitation. Interference of HSP90 or/and RIP140genes resulted in dramatically decreased expression ofCYP2C8 gene, the RORγ target gene. Data from this studydemonstrate that HSP90 and RIP140 proteins interact withRORγ protein in a complex format and function asco-activators in the RORγ-mediated regulatory processes ofHepG2 cells.

  16. Protein nanocages for self-triggered nuclear delivery of DNA-targeted chemotherapeutics in Cancer Cells.

    Science.gov (United States)

    Bellini, Michela; Mazzucchelli, Serena; Galbiati, Elisabetta; Sommaruga, Silvia; Fiandra, Luisa; Truffi, Marta; Rizzuto, Maria A; Colombo, Miriam; Tortora, Paolo; Corsi, Fabio; Prosperi, Davide

    2014-12-28

    A genetically engineered apoferritin variant consisting of 24 heavy-chain subunits (HFn) was produced to achieve a cumulative delivery of an antitumor drug, which exerts its cytotoxic action by targeting the DNA at the nucleus of human cancer cells with subcellular precision. The rationale of our approach is based on exploiting the natural arsenal of defense of cancer cells to stimulate them to recruit large amounts of HFn nanoparticles loaded with doxorubicin inside their nucleus in response to a DNA damage, which leads to a programmed cell death. After demonstrating the selectivity of HFn for representative cancer cells compared to healthy fibroblasts, doxorubicin-loaded HFn was used to treat the cancer cells. The results from confocal microscopy and DNA damage assays proved that loading of doxorubicin in HFn nanoparticles increased the nuclear delivery of the drug, thus enhancing doxorubicin efficacy. Doxorubicin-loaded HFn acts as a "Trojan Horse": HFn was internalized in cancer cells faster and more efficiently compared to free doxorubicin, then promptly translocated into the nucleus following the DNA damage caused by the partial release in the cytoplasm of encapsulated doxorubicin. This self-triggered translocation mechanism allowed the drug to be directly released in the nuclear compartment, where it exerted its toxic action. This approach was reliable and straightforward providing an antiproliferative effect with high reproducibility. The particular self-assembling nature of HFn nanocage makes it a versatile and tunable nanovector for a broad range of molecules suitable both for detection and treatment of cancer cells. PMID:25312541

  17. Pathogenic and Diagnostic Potential of BLCA-1 and BLCA-4 Nuclear Proteins in Urothelial Cell Carcinoma of Human Bladder

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    Matteo Santoni

    2012-01-01

    Full Text Available Transitional cell carcinoma (TCC of the bladder is one of the most common malignancies of genitourinary tract. Patients with bladder cancer need a life-long surveillance, directly due to the relatively high recurrence rate of this tumor. The use of cystoscopy represents the gold standard for the followup of previously treated patients. Nevertheless, several factors, including cost and invasiveness, render cystoscopy not ideal for routine controls. Advances in the identification of specific alterations in the nuclear structure of bladder cancer cells have opened novel diagnostic landscapes. The members of nuclear matrix protein family BLCA-1 and BLCA-4, are currently under evaluation as bladder cancer urinary markers. They are involved in tumour cell proliferation, survival, and angiogenesis. In this paper, we illustrate the role of BLCA-1 and BLCA-4 in bladder carcinogenesis and their potential exploitation as biomarkers in this cancer.

  18. Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: Members of the nuclear DAPC associate with the nuclear matrix

    International Nuclear Information System (INIS)

    Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. In this work, we report the identification and characterization of a novel DAPC present in the nuclei of HeLa cells, which contains dystrophin Dp71 as a key component. Using confocal microscopy and cell fractionation analyses, we found the presence of Dp71, β-sarcoglycan, β-dystroglycan, α- and β-syntrophin, α1- and β-dystrobrevin and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Next, we analyze the possible association of the nuclear DAPC with the nuclear matrix. We found the presence of Dp71, β-dystroglycan, nNOS, β-sarcoglycan, α/β syntrophin, α1-dystrobrevin and β-dystrobrevin in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells. Moreover, we found that Dp71, β-dystroglycan and β-dystrobrevin co-immunoprecipitated with the nuclear matrix proteins lamin B1 and actin. The association of members of the nuclear DAPC with the nuclear matrix indicates that they may work as scaffolding proteins involved in nuclear architecture

  19. Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

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    Rafael Nisa-Martínez

    Full Text Available Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

  20. Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

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    Raabe Timothy D

    2003-04-01

    Full Text Available Abstract Background Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97 have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. Results Using confocal microscopy and western analysis techniques, we determined 1 the immunolabeled structures are truly within the nucleus; 2 the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs and it can be identified on other intermediate filament proteins (IFs in other cell types; and 3 there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. Conclusion We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

  1. Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

    Science.gov (United States)

    Godoi, Paulo H C; Wilkie-Grantham, Rachel P; Hishiki, Asami; Sano, Renata; Matsuzawa, Yasuko; Yanagi, Hiroko; Munte, Claudia E; Chen, Ya; Yao, Yong; Marassi, Francesca M; Kalbitzer, Hans R; Matsuzawa, Shu-Ichi; Reed, John C

    2016-07-01

    B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins. PMID:27129202

  2. Knock-down of methyl CpG-binding protein 2 (MeCP2 causes alterations in cell proliferation and nuclear lamins expression in mammalian cells

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    Babbio Federica

    2012-07-01

    Full Text Available Abstract Background MeCP2 (CpG-binding protein 2 is a nuclear multifunctional protein involved in several cellular processes, like large-scale chromatin reorganization and architecture, and transcriptional regulation. In recent years, a non-neuronal role for MeCP2 has emerged in cell growth and proliferation. Mutations in the MeCP2 gene have been reported to determine growth disadvantages in cultured lymphocyte cells, and its functional ablation suppresses cell growth in glial cells and proliferation in mesenchymal stem cells and prostate cancer cells. MeCP2 interacts with lamin B receptor (LBR and with Heterochromatin Protein 1 (HP1 at the nuclear envelope (NE, suggesting that it could be part of complexes involved in attracting heterochromatin at the nuclear periphery and in mediating gene silencing. The nuclear lamins, major components of the lamina, have a role in maintaining NE integrity, in orchestrating mitosis, in DNA replication and transcription, in regulation of mitosis and apoptosis and in providing anchoring sites for chromatin domains. In this work, we inferred that MeCP2 might have a role in nuclear envelope stability, thereby affecting the proliferation pattern of highly proliferating systems. Results By performing knock-down (KD of MeCP2 in normal murine (NIH-3 T3 and in human prostate transformed cells (PC-3 and LNCaP, we observed a strong proliferation decrease and a defect in the cell cycle progression, with accumulation of cells in S/G2M, without triggering a strong apoptotic and senescent phenotype. In these cells, KD of MeCP2 evidenced a considerable decrease of the levels of lamin A, lamin C, lamin B1 and LBR proteins. Moreover, by confocal analysis we confirmed the reduction of lamin A levels, but we also observed an alteration in the shape of the nuclear lamina and an irregular nuclear rim. Conclusions Our results that indicate reduced levels of NE components, are consistent with a hypothesis that the deficiency of Me

  3. Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

    International Nuclear Information System (INIS)

    spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis

  4. Involvement of elevated expression of multiple cell-cycle regulator, DTL/RAMP (denticleless/RA-regulated nuclear matrix associated protein), in the growth of breast cancer cells.

    Science.gov (United States)

    Ueki, T; Nishidate, T; Park, J H; Lin, M L; Shimo, A; Hirata, K; Nakamura, Y; Katagiri, T

    2008-09-25

    To investigate the detailed molecular mechanism of mammary carcinogenesis and discover novel therapeutic targets, we previously analysed gene expression profiles of breast cancers. We here report characterization of a significant role of DTL/RAMP (denticleless/RA-regulated nuclear matrix associated protein) in mammary carcinogenesis. Semiquantitative RT-PCR and northern blot analyses confirmed upregulation of DTL/RAMP in the majority of breast cancer cases and all of breast cancer cell lines examined. Immunocytochemical and western blot analyses using anti-DTL/RAMP polyclonal antibody revealed cell-cycle-dependent localization of endogenous DTL/RAMP protein in breast cancer cells; nuclear localization was observed in cells at interphase and the protein was concentrated at the contractile ring in cytokinesis process. The expression level of DTL/RAMP protein became highest at G(1)/S phases, whereas its phosphorylation level was enhanced during mitotic phase. Treatment of breast cancer cells, T47D and HBC4, with small-interfering RNAs against DTL/RAMP effectively suppressed its expression and caused accumulation of G(2)/M cells, resulting in growth inhibition of cancer cells. We further demonstrate the in vitro phosphorylation of DTL/RAMP through an interaction with the mitotic kinase, Aurora kinase-B (AURKB). Interestingly, depletion of AURKB expression with siRNA in breast cancer cells reduced the phosphorylation of DTL/RAMP and decreased the stability of DTL/RAMP protein. These findings imply important roles of DTL/RAMP in growth of breast cancer cells and suggest that DTL/RAMP might be a promising molecular target for treatment of breast cancer.

  5. Expression of TGF-β2 mRNA and PCNA, FN Protein in Lens Epithelial Cells in Age-related Nuclear and Cortex Cataract

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    By using RT-PCR and immunohistochemistry, the expressions of transforming growth factor β2 (TGF-β2) mRNA, proliferating cell nuclear antigen (PCNA) and fibronection (FN) protein in lens epithelial cells (LECs) of age-related nuclear and cortex cataract were detected and compared. The results of RT-PCR revealed that the expression of TGF-β2 mRNA was higher in cortex cataract than in nuclear cataract. Immunohistochemistry demonstrated that the expression of PCNA protein was lower and the expression of FN protein was higher in cortex cataract than in nuclear cataract. It was suggested that TGF-β2, PCNA and FN might take important parts in the process of age-related cataract. Cortex cataract was related to the transdifferentiation of LECs, and nuclear cataract to the proliferation of LECs.

  6. Nuclear matrix binding protein SMAR1 regulates T-cell differentiation and allergic airway disease.

    Science.gov (United States)

    Chemmannur, S V; Badhwar, A J; Mirlekar, B; Malonia, S K; Gupta, M; Wadhwa, N; Bopanna, R; Mabalirajan, U; Majumdar, S; Ghosh, B; Chattopadhyay, S

    2015-11-01

    Asthma is a complex airway allergic disease involving the interplay of various cell types, cytokines, and transcriptional factors. Though many factors contribute to disease etiology, the molecular control of disease phenotype and responsiveness is not well understood. Here we report an essential role of the matrix attachment region (MAR)-binding protein SMAR1 in regulating immune response during allergic airway disease. Conditional knockout of SMAR1 in T cells rendered the mice resistant to eosinophilic airway inflammation against ovalbumin (OVA) allergen with low immunoglobulin E (IgE) and interleukin-5 (IL-5) levels. Moreover, a lower IgE/IgG2a ratio and higher interferon-γ (IFN-γ) response suggested aberrant skewing of T-cell differentiation toward type 1 helper T cell (Th1) response. We show that SMAR1 functions as a negative regulator of Th1 and Th17 differentiation by interacting with two potential and similar MAR regions present on the promoters of T-bet and IL-17. Thus, we present SMAR1 as a regulator of T-cell differentiation that favors the establishment of Th2 cells by modulating Th1 and Th17 responses. PMID:25736456

  7. Protein kinase C delta (PKCδ affects proliferation of insulin-secreting cells by promoting nuclear extrusion of the cell cycle inhibitor p21Cip1/WAF1.

    Directory of Open Access Journals (Sweden)

    Felicia Ranta

    Full Text Available BACKGROUND: High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ in β-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. METHODOLOGY AND PRINCIPAL FINDINGS: Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21(Cip1/WAF1. This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21(Cip1/WAF1 at Ser146. In kinase dead PKCδ (PKCδKN overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21(Cip1/WAF1 was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21(Cip1/WAF1 with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic. CONCLUSIONS AND SIGNIFICANCE: These observations disclose PKCδ as negative regulator of p21(Cip1/WAF1, which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role.

  8. Separation and identification of differentially expressed nuclear matrix proteins between human esophageal immortalized and carcinomatous cell lines

    Institute of Scientific and Technical Information of China (English)

    Xing-Dong Xiong; En-Min Li; Li-Yan Xu; Hai-Bin Chen; Ling Chen; Wei-Jia Cai; Ya-Li Han; Zhong-Ying Shen; Yi Zeng

    2003-01-01

    AIM: To separate and identify differentially expressed nuclear matrix proteins (NMPs) between the immortalized human esophageal epithelial cell line (SHEE) and the malignantly transformed esophageal carcinoma cell line (SHEEC), and to provide new ways for finding specific markers and the pathogenesis of esophageal carcinoma.METHODS: SHEE and SHEEC cell lines were used to extract NMPs. The quality of NMPs was monitored by Western blot analysis including DNA topoisomerase Ⅱα, proliferation cell nuclear antigen (PCNA) and histone. NMPs of SHEE and SHEEC were analyzed by two-dimensional electrophoresis (2-DE), silver staining and PDQuest6.2 image analysis software. Three spots in which the differentially expressed NMlPs were more obvious, were selected and analyzed with matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI- TOF-MS) and database search.RESULTS: Western blot analysis revealed that DNA topoisomerase Ⅱα and PCNA were detected, and the majority of histones were deleted in NMPs of SHEE and SHEEC. After 2-DE image analysis by PDQuest6.2 software, the 2-DE maps were detected with an average of 106±7.1 spots in SHEE and 132±5.0 spots in SHEEC. Most of them were matched one another (r=0.72), only 16 protein spots were found differing in intensity. Three NMPs including cytoskeletal tropomyosin,FK506bindingprotein6,similartoretinoblastoma binding protein 8 were preliminarily identified by MALDI- TOF-MS.CONCLUSION: These differentially expressed NMPs may play an important role during malignant transformation from SHEE to SHEEC. Their separation and identification will contribute to searching for specific markers and probing into the pathogenesis of esophageal carcinoma.

  9. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  10. Epstein-Barr virus nuclear protein EBNA3C directly induces expression of AID and somatic mutations in B cells.

    Science.gov (United States)

    Kalchschmidt, Jens S; Bashford-Rogers, Rachael; Paschos, Kostas; Gillman, Adam C T; Styles, Christine T; Kellam, Paul; Allday, Martin J

    2016-05-30

    Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma. PMID:27217538

  11. Nuclear variants of bone morphogenetic proteins

    Directory of Open Access Journals (Sweden)

    Meinhart Christopher A

    2010-03-01

    Full Text Available Abstract Background Bone morphogenetic proteins (BMPs contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts. Results In all three proteins, a bipartite nuclear localization signal (NLS was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins (designated nBmp2, nBmp4, and nGdf5 containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle. Conclusions The observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell.

  12. Expression of cell cycle regulator p57kip2, cyclinE protein and proliferating cell nuclear antigen in human pancreatic cancer: An immunohistochemical study

    Institute of Scientific and Technical Information of China (English)

    Hui Yue; Hui-Yong Jiang

    2005-01-01

    AIM: To investigate the effects of p57kip2, cyclinE protein and proliferating cell nuclear antigen (PCNA) on occurrence and progression of human pancreatic cancer.METHODS: The expression of p57kip2, cyclinE protein and PCNA in tumor tissues and adjacent tissues from 32patients with pancreatic cancer was detected by SP immunohistochemical technique.RESULTS: The positive expression rate of p57kip2 protein in tumor tissues was 46.9%, which was lower than that in adjacent pancreatic tissues (x2 = 5.317, P<0.05). P57kip2protein positive expression remarkably correlated with tumor cell differentiation (P<0.05), but not with lymph node metastasis (P>0.05). The positive expression rate of cyclinE protein in tumor tissues was 68.8%, which was higher than that in adjacent pancreatic tissues (x2 = 4.063,P<0.05). CyclinE protein positive expression significantly correlated with tumor cell differentiation and lymph node metastasis (P<0.05). The positive expression rate of PCNA in the tumor tissues was 71.9%, which was higher than that in adjacent pancreatic tissues (x2 = 5.189, P<0.05).PCNA positive expression remarkably correlated with tumor cell differentiation and lymph node metastasis (P<0.05).CONCLUSION: The decreased expression of p57kip2 and/or overexpression of cyclinE protein and PCNA may contribute to the occurrence and progression of pancreatic cancer.p57kip2, cyclinE protein, and PCNA play an important role in occurrence and progression of pancreatic cancer.

  13. Induction of DNA breakage in X-irradiated nucleoids selectively stripped of nuclear proteins in two mouse lymphoma cell lines differing in radiosensitivity

    International Nuclear Information System (INIS)

    The role of nuclear proteins in protection of DNA against ionizing radiation and their contribution to the radiation sensitivity was examined by an alkaline version of comet assay in two L5178Y (LY) mouse lymphoma cell lines differing in sensitivity t o ionizing radiation. LY-S cells are twice more sensitive to ionizing radiation than LY-R cells (D0 values of survival curves are 0.5 Gy and 1 Gy, respectively). Sequential removal of nuclear proteins by extraction with NaCl of different concentrations increased the X-ray induced DNA damage in LY-R nucleoids. In contrast, in the radiation sensitive LY-S cell line, depletion of nuclear proteins practically did not affect DNA damage. Although there is no doubt that the main cause of LY-S cells' sensitivity to ionizing radiation is a defect in the repair of double-strand breaks, our data support the concept that nuclear matrix organization may contribute to the cellular susceptibility to DNA damaging agents. (author)

  14. Brd4-Mediated Nuclear Retention of the Papillomavirus E2 Protein Contributes to Its Stabilization in Host Cells

    Directory of Open Access Journals (Sweden)

    Jing Li

    2014-01-01

    Full Text Available Papillomavirus E2 is a multifunctional viral protein that regulates many aspects of the viral life cycle including viral episome maintenance, transcriptional activation, and repression. E2 is degraded by the ubiquitin-proteasome pathway. Cellular bromodomain protein Brd4 has been implicated in the stabilization of the E2 protein. E2 normally shuttles between the cytoplasm and the nucleus. In this study, we demonstrate that E2 ubiquitylation mostly occurs in the cytoplasm. We also find that the interaction with Brd4 promotes nuclear retention of papillomavirus E2 proteins and contributes to their stabilization in the nucleus. Compared to wild type E2 proteins, nuclear-localization-defective mutants are rapidly degraded by the ubiquitin-proteasome pathway; however, co-expression of Brd4 redirects these mutants into the nucleus and significantly increases their stability. We further demonstrate that tethering E2 proteins to chromatin as either double-bromodomain fusion proteins or histone 2B (H2B fusion proteins significantly stabilizes the E2 proteins. Our studies suggest that chromatin recruitment of the E2 protein via interaction with Brd4 prevents E2 ubiquitylation and proteasomal degradation in the cytoplasm, leading to its stabilization in the nucleus. These studies bring new insights for understanding Brd4-mediated E2 stabilization, and provide an additional mechanism by which the chromatin-associated Brd4 regulates E2 functions.

  15. Brd4-mediated nuclear retention of the papillomavirus E2 protein contributes to its stabilization in host cells.

    Science.gov (United States)

    Li, Jing; Li, Qing; Diaz, Jason; You, Jianxin

    2014-01-01

    Papillomavirus E2 is a multifunctional viral protein that regulates many aspects of the viral life cycle including viral episome maintenance, transcriptional activation, and repression. E2 is degraded by the ubiquitin-proteasome pathway. Cellular bromodomain protein Brd4 has been implicated in the stabilization of the E2 protein. E2 normally shuttles between the cytoplasm and the nucleus. In this study, we demonstrate that E2 ubiquitylation mostly occurs in the cytoplasm. We also find that the interaction with Brd4 promotes nuclear retention of papillomavirus E2 proteins and contributes to their stabilization in the nucleus. Compared to wild type E2 proteins, nuclear-localization-defective mutants are rapidly degraded by the ubiquitin-proteasome pathway; however, co-expression of Brd4 redirects these mutants into the nucleus and significantly increases their stability. We further demonstrate that tethering E2 proteins to chromatin as either double-bromodomain fusion proteins or histone 2B (H2B) fusion proteins significantly stabilizes the E2 proteins. Our studies suggest that chromatin recruitment of the E2 protein via interaction with Brd4 prevents E2 ubiquitylation and proteasomal degradation in the cytoplasm, leading to its stabilization in the nucleus. These studies bring new insights for understanding Brd4-mediated E2 stabilization, and provide an additional mechanism by which the chromatin-associated Brd4 regulates E2 functions. PMID:24448221

  16. Nuclear Mechanics and Stem Cell Differentiation.

    Science.gov (United States)

    Mao, Xinjian; Gavara, Nuria; Song, Guanbin

    2015-12-01

    Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.

  17. BIG3 Inhibits the Estrogen-Dependent Nuclear Translocation of PHB2 via Multiple Karyopherin-Alpha Proteins in Breast Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Nam-Hee Kim

    Full Text Available We recently reported that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3 binds Prohibitin 2 (PHB2 in cytoplasm, thereby causing a loss of function of the PHB2 tumor suppressor in the nuclei of breast cancer cells. However, little is known regarding the mechanism by which BIG3 inhibits the nuclear translocation of PHB2 into breast cancer cells. Here, we report that BIG3 blocks the estrogen (E2-dependent nuclear import of PHB2 via the karyopherin alpha (KPNA family in breast cancer cells. We found that overexpressed PHB2 interacted with KPNA1, KPNA5, and KPNA6, thereby leading to the E2-dependent translocation of PHB2 into the nuclei of breast cancer cells. More importantly, knockdown of each endogenous KPNA by siRNA caused a significant inhibition of E2-dependent translocation of PHB2 in BIG3-depleted breast cancer cells, thereby enhancing activation of estrogen receptor alpha (ERα. These data indicated that BIG3 may block the KPNAs (KPNA1, KPNA5, and KPNA6 binding region(s of PHB2, thereby leading to inhibition of KPNAs-mediated PHB2 nuclear translocation in the presence of E2 in breast cancer cells. Understanding this regulation of PHB2 nuclear import may provide therapeutic strategies for controlling E2/ERα signals in breast cancer cells.

  18. On the mechanism of transport of Inner Nuclear Membrane Proteins

    NARCIS (Netherlands)

    Laba, Justyna Katarzyna

    2016-01-01

    The nucleus is usually the biggest, round-shaped organelle in the cell, which contains numerous proteins and nucleic acids and protects the DNA. Nuclear components are contained within the boarders of Nuclear Envelope (NE), a double membrane system, formed by the fusion of Outer Nuclear Membrane (OM

  19. MicroRNA-96 promotes the proliferation of colorectal cancer cells and targets tumor protein p53 inducible nuclear protein 1, forkhead box protein O1 (FOXO1) and FOXO3a.

    Science.gov (United States)

    Gao, Feng; Wang, Wenhui

    2015-02-01

    MicroRNAs (miRNAs) are a conserved class of small, endogenous, non protein-coding RNA molecules that are capable of regulating gene expression at post-transcriptional levels and are involved in diverse cellular processes, including cancer pathogenesis. It has previously been reported that miRNA-96 (miR-96) is overexpressed in human colorectal cancer (CRC). However, the underlying mechanism of miR-96 regulation in CRC remains to be elucidated. In the present study, miR-96 was confirmed to be upregulated in CRC tissues by reverse transcription quantitative polymerase chain reaction. MTT assay, colony formation assay and cell cycle analysis revealed that miR-96 overexpression led to increased tumor cell viability, colony formation ability and cell cycle progression. By contrast, inhibition of miR-96 resulted in the suppression of cell proliferation. It was also demonstrated that miR-96 reduced the messenger RNA and protein expression levels of tumor protein p53 inducible nuclear protein 1 (TP53INP1), forkhead box protein O1 (FOXO1) and FOXO3a, which are closely associated with cell proliferation. A luciferase reporter assay indicated that miR-96 inhibited luciferase intensity controlled by the 3'UTRs of TP53INP1, FOXO1 and FOXO3a. In conclusion, the results of the present study demonstrated that miR-96 contributed to CRC cell growth and that TP53INP1, FOXO1 and FOXO3a were direct targets of miR-96, suggesting that miR-96 may have the potential to be used in the development of miRNA‑based therapies for CRC patients.

  20. Nuclear lamina in plant cells

    Institute of Scientific and Technical Information of China (English)

    汪健; 杨澄; 翟中和

    1996-01-01

    By using selective extraction and diethylene glycol distearate (DGD) embedment and embedment-free electron microscopy, the nuclear lamina was demonstrated in carrot and Ginkgo male generative cells. Western blotting revealed that the nuclear lamina was composed of A-type and B-type lamins which contained at least 66-ku and 84-ku or 66-ku and 86-ku polypeptides, respectively. These lamin proteins were localized at the nudear periphery as shown by immunogold-labelling. In situ hybridization for light microscope and electron microscope showed that plant cells have the homologous sequences of animal lamin cDNA. The sorting site of lamin mRNA is mainly distributed in the cytoplasm near the nudear envelope. The data have verified that there indeed exists nudear lamina in plant cells.

  1. Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    BOU; ShorGan

    2009-01-01

    In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO- Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex- pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans- genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and

  2. Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    GUO XuDong; YANG DongShan; Ao XuDong; WU Xia; LI GuangPeng; WANG LingLing; BAO MingTao; XUE Lian; BOU ShorGan

    2009-01-01

    In the present study, cashmere goat fetal flbroblasta were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1(IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasta cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h.Parthenogenetic ooctyes were used as a model to Investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% va 83.9%, P>0.05 and 23.1% vs 17.2%, P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05).After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex-pressing red fluorescence. Two of the red fluorescent blastocysta were randomly selected to identify transgene by polymeraee chain reaction. Both were positive. These results showed that: (i) RFP and Neo genes were correctly expressed indicating that transgenlc somatic cell lines and positive trans-genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and

  3. Nuclear export of proteins and drug resistance in cancer

    OpenAIRE

    Turner, Joel G.; Dawson, Jana; Sullivan, Daniel M.

    2011-01-01

    The intracellular location of a protein is crucial to its normal functioning in a cell. Cancer cells utilize the normal processes of nuclear-cytoplasmic transport through the nuclear pore complex of a cell to effectively evade anti-neoplastic mechanisms. CRM1-mediated export is increased in various cancers. Proteins that are exported in cancer include tumor-suppressive proteins such as retinoblastoma, APC, p53, BRAC1, FOXO proteins, INI1/hSNF5, galectin-3, Bok, nucleophosmin, RASSF2, Merlin, ...

  4. Spag16, an axonemal central apparatus gene, encodes a male germ cell nuclear speckle protein that regulates SPAG16 mRNA expression.

    Directory of Open Access Journals (Sweden)

    David R Nagarkatti-Gude

    Full Text Available Spag16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the "9+2" axoneme. In Chlamydomonas, the PF20 gene encodes a single protein present in the central pair of the axoneme. Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis. Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. SPAG16S expression precedes expression of SPAG16L. Mice homozygous for a knockout of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. Mice chimeric for a mutation deleting the transcripts for both SPAG16L and SPAG16S have a profound defect in spermatogenesis. We show here that transduction of SPAG16S into cultured dispersed mouse male germ cells and BEAS-2B human bronchial epithelial cells increases SPAG16L expression, but has no effect on the expression of several other axoneme components. We also demonstrate that the Spag16L promoter shows increased activity in the presence of SPAG16S. The distinct nuclear localization of SPAG16S and its ability to modulate Spag16L mRNA expression suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells. This is a unique example of a highly conserved axonemal protein gene that encodes two protein products with different functions.

  5. Higher order nuclear organization in growth arrest of humanmammary epithelial cells: A novel role for telomere-associated proteinTIN2

    Energy Technology Data Exchange (ETDEWEB)

    Kaminker, Patrick; Plachot, Cedric; Kim, Sahn-Ho; Chung, Peter; Crippen, Danielle; Petersen, Ole W.; Bissell, Mina J.; Campisi, Judith; Lelievre, Sophie A.

    2004-12-15

    Nuclear organization, such as the formation of specific nuclear subdomains, is generally thought to be involved in the control of cellular phenotype; however, there are relatively few specific examples of how mammalian nuclei organize during radical changes in phenotype, such as those which occur during differentiation and growth arrest. Using human mammary epithelial cells (HMECs) in which growth arrest is essential for morphological differentiation, we show that the arrest of cell proliferation is accompanied by a reorganization of the telomere-associated protein, TIN2, into one to three large nuclear subdomains. The large TIN2 domains do not contain telomeres and occur concomitant with the continued presence of TIN2 at telomeres. The TIN2 domains were sensitive to DNAse, but not RNAse, occurred frequently, but not exclusively near nucleoli, and overlapped often with dense domains containing heterochromatin protein l{gamma}. Expression of truncated forms of TIN2 simultaneously prevented the formation of TIN2 domains and relaxed the stringent morphogenesis-induced growth arrest in HMECs. Our findings reveal a novel extra-telomeric organization of TIN2 associated with the control of cell proliferation and identify TIN2 as an important regulator of mammary epithelial differentiation.

  6. IRE1 KNOCKDOWN MODIFIES THE GLUTAMINE AND GLUCOSE DEPRIVATION EFFECT ON THE EXPRESSION OF NUCLEAR GENES ENCODING MITOCHONDRIAL PROTEINS IN U87 GLIOMA CELLS

    Directory of Open Access Journals (Sweden)

    O. O.

    2016-04-01

    Full Text Available We have studied the glucose and glutamine deprivation effect on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells in relation to inhibition of inositol requiring enzyme-1 (IRE1. It was shown that glutamine deprivation down-regulated the expression of mitochondrial (NADP+-dependent isocitrate dehydrogenase 2 (IDH2, malic enzyme 2 (ME2, mitochondrial aspartate aminotransferase (GOT2, and subunit B of succinate dehydrogenase (SDHB genes in control glioma cells in gene specific manner. At the same time, the expression level of malate dehydrogenase 2 (MDH2 and subunit D of succinate dehydrogenase (SDHD genes in these cells was not changed upon glutamine deprivation. It was also shown that inhibition of ІRE1 signaling enzyme function in U87 glioma cells modified the glutamine deprivation effect on the expression of all studied genes. Furthermore, the expression of the majority of studied genes was resistant to glucose deprivation, except IDH2 and SDHB genes, which expression levels were slightly down-regulated. Inhibition of IRE1 modified the effect of glucose deprivation on ME2, SDHB, SDHD, and GOT2 genes expression. Therefore, glucose and glutamine deprivation affected the expression level of the majority of nuclear genes encoding mitochondrial proteins in relation to the functional activity of IRE1 enzyme, which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth.

  7. Retinoic acid differentially affects in vitro proliferation, differentiation and mineralization of two fish bone-derived cell lines: different gene expression of nuclear receptors and ECM proteins.

    Science.gov (United States)

    Fernández, Ignacio; Tiago, Daniel M; Laizé, Vincent; Leonor Cancela, M; Gisbert, Enric

    2014-03-01

    Retinoic acid (RA), the main active metabolite of vitamin A, regulates vertebrate morphogenesis through signaling pathways not yet fully understood. Such process involves the specific activation of retinoic acid and retinoid X receptors (RARs and RXRs), which are nuclear receptors of the steroid/thyroid hormone receptor superfamily. Teleost fish are suitable models to study vertebrate development, such as skeletogenesis. Cell systems capable of in vitro mineralization have been developed for several fish species and may provide new insights into the specific cellular and molecular events related to vitamin A activity in bone, complementary to in vivo studies. This work aims at investigating the in vitro effects of RA (0.5 and 12.5 μM) on proliferation, differentiation and extracellular matrix (ECM) mineralization of two gilthead seabream bone-derived cell lines (VSa13 and VSa16), and at identifying molecular targets of its action through gene expression analysis. RA induced phenotypic changes and cellular proliferation was inhibited in both cell lines in a cell type-dependent manner (36-59% in VSa13 and 17-46% in VSa16 cells). While RA stimulated mineral deposition in VSa13 cell cultures (50-62% stimulation), it inhibited the mineralization of extracellular matrix in VSa16 cells (11-57% inhibition). Expression of hormone receptor genes (rars and rxrs), and extracellular matrix-related genes such as matrix and bone Gla proteins (mgp and bglap), osteopontin (spp1) and type I collagen (col1a1) were differentially regulated upon exposure to RA in proliferating, differentiating and mineralizing cultures of VSa13 and VSa16 cells. Altogether, our results show: (i) RA affects proliferative and mineralogenic activities in two fish skeletal cell types and (ii) that during phenotype transitions, specific RA nuclear receptors and bone-related genes are differentially expressed in a cell type-dependent manner. PMID:24291400

  8. Nuclear multidrug-resistance related protein 1 contributes to multidrug-resistance of mucoepidermoid carcinoma mainly via regulating multidrug-resistance protein 1: a human mucoepidermoid carcinoma cells model and Spearman's rank correlation analysis.

    Directory of Open Access Journals (Sweden)

    Bolei Cai

    Full Text Available BACKGROUND: Multidrug resistance-related protein 1 (MRP1/ABCC1 and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1 are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR of mucoepidermoid carcinoma (MEC. The present study investigated how MRP1 contributes to MDR in the nuclei of MEC cells. METHODS: Western blot and RT-PCR was carried out to investigate the change of multidrug-resistance protein 1 (MDR1 in MC3/5FU cells after MRP1 was downregulated through RNA interference (RNAi. Immunohistochemistry (IHC staining of 127 cases of MEC tissues was scored with the expression index (EI. The EI of MDR1 and MRP1 (or nuclear MRP1 was analyzed with Spearman's rank correlation analysis. Using multiple tumor tissue assays, the location of MRP1 in other tissues was checked by HIC. Luciferase reporter assays of MDR1 promoter was carried out to check the connection between MRP1 and MDR1 promoter. RESULTS: MRP1 downregulation led to a decreased MDR1 expression in MC3/5FU cells which was caused by decreased activity of MDR1 promoter. IHC study of 127 cases of MEC tissues demonstrated a strong positive correlation between nuclear MRP1 expression and MDR1 expression. Furthermore, IHC study of multiple tumor tissue array sections showed that although nuclear MRP1 widely existed in MEC tissues, it was not found in normal tissues or other tumor tissues. CONCLUSIONS: Our findings indicate that nuclear MRP1 contributes to MDR mainly through regulating MDR1 expression in MEC. And the unique location of MRP1 made it an available target in identifying MEC from other tumors.

  9. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

    International Nuclear Information System (INIS)

    Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3Cpro induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5′ non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3Cpro.

  10. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, Paul; Schafer, Elizabeth A.; Rieder, Elizabeth, E-mail: elizabeth.rieder@ars.usda.gov

    2012-03-30

    Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3C{sup pro} induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5 Prime non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3C{sup pro}.

  11. Active Nuclear Import of Membrane Proteins Revisited

    NARCIS (Netherlands)

    Laba, Justyna K; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the m

  12. Nuclear DNA sensor IFI16 as circulating protein in autoimmune diseases is a signal of damage that impairs endothelial cells through high-affinity membrane binding.

    Directory of Open Access Journals (Sweden)

    Francesca Gugliesi

    Full Text Available IFI16, a nuclear pathogenic DNA sensor induced by several pro-inflammatory cytokines, is a multifaceted protein with various functions. It is also a target for autoantibodies as specific antibodies have been demonstrated in the sera of patients affected by systemic autoimmune diseases. Following transfection of virus-derived DNA, or treatment with UVB, IFI16 delocalizes from the nucleus to the cytoplasm and is then eventually released into the extracellular milieu. In this study, using an in-house capture enzyme-linked immunsorbent assay we demonstrate that significant levels of IFI16 protein can also exist as circulating form in the sera of autoimmune patients. We also show that the rIFI16 protein, when added in-vitro to endothelial cells, does not affect cell viability, but severely limits their tubulogenesis and transwell migration activities. These inhibitory effects are fully reversed in the presence of anti-IFI16 N-terminal antibodies, indicating that its extracellular activity resides within the N-terminus. It was further demonstrated that endogenous IFI16 released by apoptotic cells bind neighboring cells in a co-culture. Immunofluorescence assays revealed existence of high-affinity binding sites on the plasma membrane of endothelial cells. Free recombinant IFI16 binds these sites on HUVEC with dissociation constant of 2.7 nM, radioiodinated and unlabeled IFI16 compete for binding sites, with inhibition constant (Ki of 14.43 nM and half maximal inhibitory concentration (IC50 of 67.88 nM; these data allow us to estimate the presence of 250,000 to 450,000 specific binding sites per cell. Corroborating the results from functional assays, this binding could be completely inhibited using anti-IFI16 N-terminal antibody, but not with an antibody raised against the IFI16 C-terminal. Altogether, these data demonstrate that IFI16 may exist as circulating protein in the sera of autoimmune patients which binds endothelial cells causing damage

  13. Nuclear envelope proteins Nesprin2 and LaminA regulate proliferation and apoptosis of vascular endothelial cells in response to shear stress.

    Science.gov (United States)

    Han, Yue; Wang, Lu; Yao, Qing-Ping; Zhang, Ping; Liu, Bo; Wang, Guo-Liang; Shen, Bao-Rong; Cheng, Binbin; Wang, Yingxiao; Jiang, Zong-Lai; Qi, Ying-Xin

    2015-05-01

    The dysfunction of vascular endothelial cells (ECs) influenced by flow shear stress is crucial for vascular remodeling. However, the roles of nuclear envelope (NE) proteins in shear stress-induced EC dysfunction are still unknown. Our results indicated that, compared with normal shear stress (NSS), low shear stress (LowSS) suppressed the expression of two types of NE proteins, Nesprin2 and LaminA, and increased the proliferation and apoptosis of ECs. Targeted small interfering RNA (siRNA) and gene overexpression plasmid transfection revealed that Nesprin2 and LaminA participate in the regulation of EC proliferation and apoptosis. A protein/DNA array was further used to detect the activation of transcription factors in ECs following transfection with target siRNAs and overexpression plasmids. The regulation of AP-2 and TFIID mediated by Nesprin2 and the activation of Stat-1, Stat-3, Stat-5 and Stat-6 by LaminA were verified under shear stress. Furthermore, using Ingenuity Pathway Analysis software and real-time RT-PCR, the effects of Nesprin2 or LaminA on the downstream target genes of AP-2, TFIID, and Stat-1, Stat-3, Stat-5 and Stat-6, respectively, were investigated under LowSS. Our study has revealed that NE proteins are novel mechano-sensitive molecules in ECs. LowSS suppresses the expression of Nesprin2 and LaminA, which may subsequently modulate the activation of important transcription factors and eventually lead to EC dysfunction.

  14. Distinct Roles for Key Karyogamy Proteins during Yeast Nuclear Fusion

    OpenAIRE

    Melloy, Patricia; Shen, Shu; White, Erin; Rose, Mark D.

    2009-01-01

    During yeast mating, cell fusion is followed by the congression and fusion of the two nuclei. Proteins required for nuclear fusion are found at the surface (Prm3p) and within the lumen (Kar2p, Kar5p, and Kar8p) of the nuclear envelope (NE). Electron tomography (ET) of zygotes revealed that mutations in these proteins block nuclear fusion with different morphologies, suggesting that they act in different steps of fusion. Specifically, prm3 zygotes were blocked before formation of membrane brid...

  15. Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-06-11

    Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

  16. Protein dynamics from nuclear magnetic relaxation.

    Science.gov (United States)

    Charlier, Cyril; Cousin, Samuel F; Ferrage, Fabien

    2016-05-01

    Nuclear magnetic resonance is a ubiquitous spectroscopic tool to explore molecules with atomic resolution. Nuclear magnetic relaxation is intimately connected to molecular motions. Many methods and models have been developed to measure and interpret the characteristic rates of nuclear magnetic relaxation in proteins. These approaches shed light on a rich and diverse range of motions covering timescales from picoseconds to seconds. Here, we introduce some of the basic concepts upon which these approaches are built and provide a series of illustrations.

  17. Brd4-Mediated Nuclear Retention of the Papillomavirus E2 Protein Contributes to Its Stabilization in Host Cells

    OpenAIRE

    Jing Li; Qing Li; Jason Diaz; Jianxin You

    2014-01-01

    Papillomavirus E2 is a multifunctional viral protein that regulates many aspects of the viral life cycle including viral episome maintenance, transcriptional activation, and repression. E2 is degraded by the ubiquitin-proteasome pathway. Cellular bromodomain protein Brd4 has been implicated in the stabilization of the E2 protein. E2 normally shuttles between the cytoplasm and the nucleus. In this study, we demonstrate that E2 ubiquitylation mostly occurs in the cytoplasm. We also find that th...

  18. EXPRESSIONS OF P53, PROLIFERATING CELL NUCLEAR ANITIGEN, BCL-2 PROTEIN AND THEIR SIGNIFICANCE IN SALIVARY ADENOID CYSTIC CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To study the effects of P53, PCNA, Bcl-2 protein and their relationship in salivary adenoid cystic carcinoma(SACC). Methods These proteins were examined by immunohistochemistry. Results Overexpressions of P53 and PCNA were revealed in ACC samples, they were higher than those in (polymorphous adenomas) PA, but expression of Bcl-2 protein was not different between ACC and PA. In 3 subtypes of ACC, expressions of 3 proteins were different. Conclusion Mutations of P53, Bcl-2 may be involed in the occurrence of SACC, expression of PCNA and mutation of P53 may coexist in the development of the SACC.

  19. High frequency of tumor cells with nuclear Egr-1 protein expression in human bladder cancer is associated with disease progression

    DEFF Research Database (Denmark)

    Egerod, Frederikke N S Lihme; Bartels, Annette; Fristrup, Niels;

    2009-01-01

    BACKGROUND: Egr-1 (early growth response-1 transcription factor) has been proposed to be involved in invasion and metastasis processes of human bladder cancer, but Egr-1 protein expression levels in human bladder cancer have not been investigated. In the present study we investigated the expressi...

  20. Phosphorylation and biosynthesis of high molecular weight proteins of tumor nuclear meatrix

    Institute of Scientific and Technical Information of China (English)

    BAZARNOVATM; TVBULDYAEVA; 等

    1998-01-01

    Our previous studies showed a predominance of high molecular weight protein group in tumor nuclear matrices.Contrary to normal cells,proteins of this group are preferentially phosphorylated.Phosphoproteins of hepatoma nuclear matrix are selectively subjected to rapid proteolysis.By alkali treatment and a monoclonal antibody against phosphotyrosyl residue the presence of two high molecular weight bands of phosphotyrosyl-containing proteins was detected in nuclear matrices of tumor but not of normal liver cells.High molecular weight protein group of tumor nuclear matrices revealed also a rapid turnover and preferential incorporation of labeled amino acids selectively inhibited by chloramphenicol.

  1. Pro-recombination role of Srs2 protein requires SUMO (small ubiquitin-like modifier) but is independent of PCNA (proliferating cell nuclear antigen) interaction

    DEFF Research Database (Denmark)

    Kolesar, Peter; Altmannova, Veronika; Pinela da Silva, Sonia Cristina;

    2016-01-01

    -interacting motif (SIM) of Srs2 is important for the interaction with several recombination factors. Lack of SIM, but not proliferating cell nuclear antigen (PCNA)-interacting motif (PIM), leads to increased cell death under circumstances requiring homologous recombination for DNA repair. Simultaneous mutation...

  2. Identification and characterization of proteins involved in nuclear organization using Drosophila GFP protein trap lines.

    Directory of Open Access Journals (Sweden)

    Margaret Rohrbaugh

    Full Text Available BACKGROUND: Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31 gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl, a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology. CONCLUSIONS/SIGNIFICANCE: These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these

  3. Two nuclear oncogenic proteins, P135gag-myb-ets and p61/63myc, cooperate to induce transformation of chicken neuroretina cells.

    OpenAIRE

    Amouyel, P; Laudet, V; P. Martin; Li, R P; Quatannens, B; Stéhelin, D; Saule, S.

    1989-01-01

    Several studies have shown that full transformation of primary rodent fibroblasts can be achieved in vitro through the cooperation of two oncogenes (usually one nuclear and one cytoplasmic) classified on the basis of different complementation groups. We have shown previously that cooperation between v-mil (cytoplasmic, serine-threonine kinase product), and v-myc (nuclear, DNA-binding product) is required to transform 7-day-old chicken neuroretina cells, which in usual culture medium do not ra...

  4. Rapid flow cytometric measurement of protein inclusions and nuclear trafficking

    Science.gov (United States)

    Whiten, D. R.; San Gil, R.; McAlary, L.; Yerbury, J. J.; Ecroyd, H.; Wilson, M. R.

    2016-01-01

    Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules. PMID:27516358

  5. High levels of nuclear MYC protein predict the presence of MYC rearrangement in diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Green, Tina Marie; Nielsen, Ole; de Stricker, Karin;

    2012-01-01

    Determining the presence of MYC gene rearrangements is becoming an increasingly important part of the diagnostic workup in aggressive lymphoma. Cytogenetic MYC alterations aid in differentiating diffuse large B-cell lymphoma (DLBCL) from Burkitt lymphoma. In addition, MYC aberrations are associated...... with poor prognosis in DLBCL. Fluorescence in situ hybridization and karyotyping are standard tests for detecting MYC aberrations, but these techniques are laborious and expensive. Here, we studied MYC status of 219 DLBCLs and Burkitt lymphomas using fluorescence in situ hybridization, immunohistochemistry......, and quantitative real-time polymerase chain reaction (QRT-PCR). Overall, 15% of the cases had an MYC break. QRT-PCR analysis of MYC expression showed that 72% of DLBCLs with an MYC break had aberrantly high or low levels of MYC transcript. Excluding the cases with aberrantly low MYC expression, we found...

  6. Proliferating cell nuclear antigen in neutrophil fate.

    Science.gov (United States)

    Witko-Sarsat, Véronique; Ohayon, Delphine

    2016-09-01

    The life span of a neutrophil is a tightly regulated process as extended survival is beneficial for pathogen elimination and cell death necessary to prevent cytotoxic content release from activated neutrophils at the inflammatory site. Therefore, the control between survival and death must be a dynamic process. We have previously described that proliferating cell nuclear antigen (PCNA) which is known as a nuclear protein pivotal in DNA synthesis, is a key element in controlling neutrophil survival through its association with procaspases. Contrary to the dogma which asserted that PCNA has a strictly nuclear function, in mature neutrophils, PCNA is present exclusively within the cytosol due to its nuclear export at the end of the granulocytic differentiation. More recent studies are consistent with the notion that the cytosolic scaffold of PCNA is aimed at modulating neutrophil fate rather than simply preventing death. Ultimately, targeting neutrophil survival might have important applications not just in the field of immunology and inflammation, but also in hematology and transfusion. The neutrophil emerges as a unique and powerful cellular model to unravel the basic mechanisms governing the cell cycle-independent functions of PCNA and should be considered as a leader of the pack. PMID:27558345

  7. Nucleomorphin. A novel, acidic, nuclear calmodulin-binding protein from dictyostelium that regulates nuclear number.

    Science.gov (United States)

    Myre, Michael A; O'Day, Danton H

    2002-05-31

    Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using (35)S-recombinant calmodulin (CaM) as a probe has revealed approximately three-dozen Ca(2+)-dependent calmodulin binding proteins. Here, we report the molecular cloning, expression, and subcellular localization of a gene encoding a novel calmodulin-binding protein (CaMBP); we have called nucleomorphin, from D. discoideum. A lambdaZAP cDNA expression library of cells from multicellular development was screened using a recombinant calmodulin probe ((35)S-VU1-CaM). The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular mass of 38.7 kDa and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca(2+)-dependent manner. Amino-terminal fusion to the green fluorescence protein (GFP) showed that GFP-NumA localized to the nucleus as distinct arc-like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery, but the number of nuclei in these cells was increased markedly compared with control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, these data present the first identification of a member of the nucleoplasmin family as a calmodulin-binding protein and suggest nucleomorphin has a role in nuclear structure in Dictyostelium. PMID:11919178

  8. Several novel nuclear envelope transmembrane proteins identified in skeletal muscle have cytoskeletal associations.

    Science.gov (United States)

    Wilkie, Gavin S; Korfali, Nadia; Swanson, Selene K; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R W; Florens, Laurence; Schirmer, Eric C

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  9. In vitro nuclear interactome of the HIV-1 Tat protein.

    LENUS (Irish Health Repository)

    Gautier, Virginie W

    2009-01-01

    BACKGROUND: One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86-101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. RESULTS: Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. CONCLUSION: We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will

  10. Early localization of NPA58, a rat nuclear pore-associated protein, to the reforming nuclear envelope during mitosis

    Indian Academy of Sciences (India)

    Radhika Ganeshan; Nandini Rangaraj; Veena K Parnaik

    2001-03-01

    We have studied the mitotic reassembly of the nuclear envelope, using antibodies to nuclear marker proteins and NPA58 in F-111 rat fibroblast cells. In earlier studies we have proposed that NPA58, a 58 kDa rat nuclear protein, is involved in nuclear protein import. In this report, NPA58 is shown to be localized on the cytoplasmic face of the envelope in interphase cells, in close association with nuclear pores. In mitotic cells NPA58 is dispersed in the cytoplasm till anaphase. The targeting of NPA58 to the reforming nuclear envelope in early telophase coincides with the recruitment of a well-characterized class of nuclear pore proteins recognized by the antibody mAb 414, and occurs prior to the incorporation of lamin B1 into the envelope. Significant protein import activity is detectable only after localization of NPA58 in the newly-formed envelope. The early targeting of NPA58 is consistent with its proposed role in nuclear transport.

  11. Estrogen promotes fat mass and obesity-associated protein nuclear localization and enhances endometrial cancer cell proliferation via the mTOR signaling pathway.

    Science.gov (United States)

    Zhu, Yaping; Shen, Jiaqi; Gao, Liyan; Feng, Youji

    2016-04-01

    Extensive exposure to estrogen is generally acknowledged as a risk factor for endometrial cancer. Given that the accumulation of adipocytes also contributes to the increased production of estrogen, in the present study, we evaluated the expression of the fat mass and obesity-associated (FTO) gene in endometrial tumor tissues and further explored the mechanism of how estrogen facilitates FTO nuclear localization and promotes endometrial cancer cell proliferation. Immunohistochemical (IHC) staining assay was used to detect the FTO expression in endometrial tumor samples. Western blotting was performed to investigate the mechanism of estrogen-induced FTO nuclear localization. siRNA was used to knock down ERα and further explore its role in FTO nuclear localization. MTT assay was carried out to determine cell proliferation. We found that FTO was overexpressed in endometrial carcinoma tissues and served as a poor prognostic marker. Additionally, estrogen induced FTO nuclear accumulation via the mTOR signaling pathway and the nuclear localization was ERα-dependent, which contributed to enhanced proliferative activity. Therefore, the present study provides new insight into the mechanisms of estrogen-induced proliferation, implying the possibility of using FTO as a potential therapeutic target for the treatment of endometrial cancer.

  12. Proteins present in tannin cenocyte mother cells in Sambucus racemosa L.

    OpenAIRE

    Zobel, A M

    2015-01-01

    It has been demonstrated that protein content and concentration are higher in mononucleate tannin-cells than in the parenchyma cells of Sambucus racemosa. Cytoplasmic and nuclear free acid proteins markedly prevail here. It is believed that they may be enzymatic proteins. Increase of acid proteins content within the nucleus of tannin cells causes an increase of the nucleus size. The content of nuclear bound basic proteins in tannin cells, may be lower than in the neighbouring parenchymal cells.

  13. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death.

    Science.gov (United States)

    Raab, M; Gentili, M; de Belly, H; Thiam, H R; Vargas, P; Jimenez, A J; Lautenschlaeger, F; Voituriez, Raphaël; Lennon-Duménil, A M; Manel, N; Piel, M

    2016-04-15

    In eukaryotic cells, the nuclear envelope separates the genomic DNA from the cytoplasmic space and regulates protein trafficking between the two compartments. This barrier is only transiently dissolved during mitosis. Here, we found that it also opened at high frequency in migrating mammalian cells during interphase, which allowed nuclear proteins to leak out and cytoplasmic proteins to leak in. This transient opening was caused by nuclear deformation and was rapidly repaired in an ESCRT (endosomal sorting complexes required for transport)-dependent manner. DNA double-strand breaks coincided with nuclear envelope opening events. As a consequence, survival of cells migrating through confining environments depended on efficient nuclear envelope and DNA repair machineries. Nuclear envelope opening in migrating leukocytes could have potentially important consequences for normal and pathological immune responses. PMID:27013426

  14. Kaposi sarcoma herpes virus latency associated nuclear antigen protein release the G2/M cell cycle blocks by modulating ATM/ATR mediated checkpoint pathway.

    Directory of Open Access Journals (Sweden)

    Amit Kumar

    Full Text Available The Kaposi's sarcoma-associated herpesvirus infects the human population and maintains latency stage of viral life cycle in a variety of cell types including cells of epithelial, mesenchymal and endothelial origin. The establishment of latent infection by KSHV requires the expression of an unique repertoire of genes among which latency associated nuclear antigen (LANA plays a critical role in the replication of the viral genome. LANA regulates the transcription of a number of viral and cellular genes essential for the survival of the virus in the host cell. The present study demonstrates the disruption of the host G2/M cell cycle checkpoint regulation as an associated function of LANA. DNA profile of LANA expressing human B-cells demonstrated the ability of this nuclear antigen in relieving the drug (Nocodazole induced G2/M checkpoint arrest. Caffeine suppressed nocodazole induced G2/M arrest indicating involvement of the ATM/ATR. Notably, we have also shown the direct interaction of LANA with Chk2, the ATM/ATR signalling effector and is responsible for the release of the G2/M cell cycle block.

  15. Nuclear Multidrug-Resistance Related Protein 1 Contributes to Multidrug-Resistance of Mucoepidermoid Carcinoma Mainly via Regulating Multidrug-Resistance Protein 1: A Human Mucoepidermoid Carcinoma Cells Model and Spearman's Rank Correlation Analysis

    OpenAIRE

    Bolei Cai; Ye Miao; Yuan Liu; Xiaofang Xu; Sumin Guan; Junzheng Wu; Yanpu Liu

    2013-01-01

    BACKGROUND: Multidrug resistance-related protein 1 (MRP1/ABCC1) and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1) are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH) and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR) of...

  16. Andrographolide Enhances Nuclear Factor-κB Subunit p65 Ser536 Dephosphorylation through Activation of Protein Phosphatase 2A in Vascular Smooth Muscle Cells*

    OpenAIRE

    Hsieh, Cheng Y.; Hsu, Ming J.; Hsiao, George; Wang, Yi H.; Huang, Chi W.; Chen, Shiuan W.; Jayakumar, Thanasekaran; Chiu, Pei T.; Chiu, Yi H.; Sheu, Joen R

    2010-01-01

    Recent studies have demonstrated that transcription factor nuclear factor (NF)-κB inhibition may contribute to the protective anti-inflammatory actions of andrographolide, an abundant component of plants of the genus Andrographis. However, the precise mechanism by which andrographolide inhibits NF-κB signaling remains unclear. We thus investigated the mechanism involved in andrographolide suppression of NF-κB signaling in rat vascular smooth muscle cells (VSMCs) exposed to proinflammatory sti...

  17. Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein--localization of a targeting domain.

    Science.gov (United States)

    Söderqvist, H; Imreh, G; Kihlmark, M; Linnman, C; Ringertz, N; Hallberg, E

    1997-12-15

    The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear pores of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticulum/nuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121. PMID:9461306

  18. Analysis of the cellular uptake and nuclear delivery of insulin-like growth factor binding protein-3 in human osteosarcoma cells

    OpenAIRE

    Laich, A; Matscheski, A.; Mück, C.; Ferrando-May, E.; H. Pircher; Ebner, H L; Micutkova, L.; Huber, L A; M. Hermann; Offerdinger, M.; Hess, M. W.; Jansen-Dürr, P; Zwerschke, W.

    2010-01-01

    Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is an important regulator of cell proliferation and survival, which plays an important role in a variety of epithelial cancers, including prostate cancer, cervical cancer and breast cancer. IGFBP-3 was described as a tumor suppressor in the prostate and identified as a functional cellular target for the E7 oncoprotein of human papillomaviruses. IGFBP-3 interacts with IGF-I outside the cell; however, IGF-independent actions of IGFBP...

  19. Proximity Utilizing Biotinylation of Nuclear Proteins in vivo

    Directory of Open Access Journals (Sweden)

    Arman Kulyyassov

    2015-06-01

    Full Text Available Introduction. The human genome consists of roughly 30,000 genes coding for over 500,000 different proteins, of which more than 10,000 proteins can be produced by the cell at any given time (the cellular “proteome”. It has been estimated that over 80% of proteins do not operate alone, but in complexes. These protein-protein interactions (PPI are regulated by several mechanisms. For example, post-translational modifications (methylation, acetylation, phosphorylation, or ubiquitination or metal-binding can lead to conformational changes that alter the affinity and kinetic parameters of the interaction. Many PPIs are part of larger cellular networks of interactions or interactomes. Indeed, these interactions are at the core of the entire interactomics system of any living cell, and so, aberrant PPIs are the basis of multiple diseases, such as neurodegenerative diseases and cancer. The objective of this study was to develop a method of monitoring protein-protein interactions and proximity dependence in vivo.Methods. The biotin ligase BirA was fused to the protein of interest, and the Biotin Acceptor Peptide (BAP was fused to an interacting partner to make the detection of its biotinylation possible by western blot or mass spectrometry.Results. Using several experimental systems (BirA.A + BAP.B, we showed that the biotinylation is interaction/proximity dependent. Here, A and B are the next nuclear proteins used in the experiments – 3 paralogues of heterochromatin protein HP1a (CBX5, HP1b (CBX1, HP1g (CBX3, wild type and transcription mutant factor Kap1, translesion DNA polymerase PolH and E3, ubiquitin ligase RAD18, Proliferative Cell Nuclear Antigen (PCNA, ubiquitin Ub, SUMO-2/3, different types and isoforms of histones H2A, H2Az, H3.1, H3.3, CenpA, H2A.BBD, and macroH2A. The variant of this approach is termed PUB-NChIP (Proximity Utilizing Biotinylation with Native Chromatin Immuno-precipitation and is designed to purify and study the protein

  20. Identifying Protein-Protein Associations at the Nuclear Envelope with BioID.

    Science.gov (United States)

    Kim, Dae In; Jensen, Samuel C; Roux, Kyle J

    2016-01-01

    The nuclear envelope (NE) is a critical cellular structure whose constituents and roles in a myriad of cellular processes seem ever expanding. To determine the underlying mechanisms by which the NE constituents participate in various cellular events, it is necessary to understand the nature of their protein-protein associations. BioID (proximity-dependent biotin identification) is a recently established method to generate a history of protein-protein associations as they occur over time in living cells. BioID is based on fusion of a bait protein to a promiscuous biotin ligase. Expression of the BioID fusion protein in a relevant cellular environment enables biotinylation of vicinal and interacting proteins of the bait protein, permitting isolation and identification by conventional biotin-affinity capture and mass-spec analysis. In this way, BioID provides unique capabilities to identify protein-protein associations at the NE. In this chapter we provide a detailed protocol for the application of BioID to the study of NE proteins.

  1. Hepatitis B Virus X Protein Up-Regulates AKR1C1 Expression Through Nuclear Factor-Y in Human Hepatocarcinoma Cells

    OpenAIRE

    Kai LI; Ding, Shijia; Chen, Ke; Qin, Dongdong; Qu, Jialin; Wang, Sen; Sheng, Yanrui; Zou, Chengcheng; Chen, Limin; Tang, Hua

    2013-01-01

    Background The hepatitis B virus X (HBx) protein has long been recognized as an important transcriptional transactivator of several genes. Human aldo-keto reductase family 1, member C1 (AKR1C1), a member of the family of AKR1CS, is significantly increased in HBx-expressed cells. Objectives This study aimed to investigate the possible mechanism of HBx in regulating AKR1C1 expression in HepG2.2.15 cells and the role of AKR1C1 for HBV-induced HCC. Materials and Methods RT-PCR was performed to de...

  2. Nuclear envelope rupture and repair during cancer cell migration

    Science.gov (United States)

    Denais, Celine M.; Gilbert, Rachel M.; Isermann, Philipp; McGregor, Alexandra L.; te Lindert, Mariska; Weigelin, Bettina; Davidson, Patricia M.; Friedl, Peter; Wolf, Katarina; Lammerding, Jan

    2016-01-01

    During cancer metastasis, tumor cells penetrate tissues through tight interstitial spaces, requiring extensive deformation of the cell and its nucleus. Here, we investigated tumor cell migration in confining microenvironments in vitro and in vivo. Nuclear deformation caused localized loss of nuclear envelope (NE) integrity, which led to the uncontrolled exchange of nucleo-cytoplasmic content, herniation of chromatin across the NE, and DNA damage. The incidence of NE rupture increased with cell confinement and with depletion of nuclear lamins, NE proteins that structurally support the nucleus. Cells restored NE integrity using components of the endosomal sorting complexes required for transport-III (ESCRT-III) machinery. Our findings indicate that cell migration incurs substantial physical stress on the NE and its content, requiring efficient NE and DNA damage repair for survival. PMID:27013428

  3. PARP activation promotes nuclear AID accumulation in lymphoma cells.

    Science.gov (United States)

    Tepper, Sandra; Jeschke, Julia; Böttcher, Katrin; Schmidt, Angelika; Davari, Kathrin; Müller, Peter; Kremmer, Elisabeth; Hemmerich, Peter; Pfeil, Ines; Jungnickel, Berit

    2016-03-15

    Activation-induced cytidine deaminase (AID) initiates immunoglobulin diversification in germinal center B cells by targeted introduction of DNA damage. As aberrant nuclear AID action contributes to the generation of B cell lymphoma, the protein's activity is tightly regulated, e.g. by nuclear/cytoplasmic shuttling and nuclear degradation. In the present study, we asked whether DNA damage may affect regulation of the AID protein. We show that exogenous DNA damage that mainly activates base excision repair leads to prevention of proteasomal degradation of AID and hence its nuclear accumulation. Inhibitor as well as knockout studies indicate that activation of poly (ADP-ribose) polymerase (PARP) by DNA damaging agents promotes both phenomena. These findings suggest that PARP inhibitors influence DNA damage dependent AID regulation, with interesting implications for the regulation of AID function and chemotherapy of lymphoma.

  4. Fishing Fish Stem Cells and Nuclear Transplants

    OpenAIRE

    Yunhan Hong

    2011-01-01

    Fish has been the subject of various research fields, ranging from ecology, evolution, physiology and toxicology to aquaculture. In the past decades fish has attracted considerable attention for functional genomics, cancer biology and developmental genetics, in particular nuclear transfer for understanding of cytoplasmic-nuclear relationship. This special issue reports on recent progress made in fish stem cells and nuclear transfer.

  5. Preparation of the Human Cytomegalovirus Nuclear Egress Complex and Associated Proteins.

    Science.gov (United States)

    Sharma, Mayuri; Kamil, Jeremy P; Coen, Donald M

    2016-01-01

    Herpesviruses, like most DNA viruses, replicate their genomes in the host cell nucleus. Their DNA is then packaged and assembled into viral nucleocapsids, which, in most cases, are too large to pass through the nuclear pore complex. Instead, herpesviruses use a complex multistep pathway, termed nuclear egress, to exit the nucleus. Key players in this process include two conserved viral proteins that form the nuclear egress complex (NEC). In human cytomegalovirus, these NEC proteins are UL50, embedded in the inner nuclear membrane, and its nucleoplasmic partner UL53. Both are essential for viral nuclear egress. However, other viral components as well as host nuclear envelope proteins may also participate in nuclear egress. Identifying these viral and cellular factors may provide important insight into the herpesvirus lifecycle and its relationship to the underlying, yet still-mysterious, host nuclear egress pathway. We developed an immunoprecipitation-based protocol, described herein, to identify protein-protein interactions involving the NEC from the nuclear fraction of infected cells that express an epitope-tagged version of NEC subunit UL53.

  6. Nuclear substructure reorganization during late stageerythropoiesis is selective and does not involve caspase cleavage ofmajor nuclear substructural proteins

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Sharon Wald; Lo, Annie J.; Short, Sarah A.; Koury, MarkJ.; Mohandas, Narla; Chasis, Joel Anne

    2005-04-06

    Enucleation, a rare feature of mammalian differentiation, occurs in three cell types: erythroblasts, lens epithelium and keratinocytes. Previous investigations suggest that caspase activation functions in lens epithelial and keratinocyte enucleation, as well as in early erythropoiesis encompassing BFU-E differentiation to proerythroblast. To determine whether caspase activation contributes to later erythropoiesis and whether nuclear substructures other than chromatin reorganize, we analyzed distributions of nuclear subcompartment proteins and assayed for caspase-induced cleavage of subcompartmental target proteins in mouse erythroblasts. We found that patterns of lamin B in the filamentous network interacting with both the nuclear envelope and DNA, nuclear matrix protein NuMA, and splicing factors Sm and SC35 persisted during nuclear condensation, consistent with effective transcription of genes expressed late in differentiation. Thus nuclear reorganization prior to enucleation is selective, allowing maintenance of critical transcriptional processes independent of extensive chromosomal reorganization. Consistent with these data, we found no evidence for caspase-induced cleavage of major nuclear subcompartment proteins during late erythropoiesis, in contrast to what has been observed in early erythropoiesis and in lens epithelial and keratinocyte differentiation. These findings imply that nuclear condensation and extrusion during terminal erythroid differentiation involve novel mechanisms that do not entail major activation of apoptotic machinery.

  7. Tandem affinity purification to identify cytosolic and nuclear gβγ-interacting proteins.

    Science.gov (United States)

    Campden, Rhiannon; Pétrin, Darlaine; Robitaille, Mélanie; Audet, Nicolas; Gora, Sarah; Angers, Stéphane; Hébert, Terence E

    2015-01-01

    It has become clear in recent years that the Gβγ subunits of heterotrimeric proteins serve broad roles in the regulation of cellular activity and interact with many proteins in different subcellular locations including the nucleus. Protein affinity purification is a common method to identify and confirm protein interactions. When used in conjugation with mass spectrometry it can be used to identify novel protein interactions with a given bait protein. The tandem affinity purification (TAP) technique identifies partner proteins bound to tagged protein bait. Combined with protocols to enrich the nuclear fraction of whole cell lysate through sucrose cushions, TAP allows for purification of interacting proteins found specifically in the nucleus. Here we describe the use of the TAP technique on cytosolic and nuclear lysates to identify candidate proteins, through mass spectrometry, that bind to Gβ1 subunits.

  8. The BRCA1-binding protein BRAP2 can act as a cytoplasmic retention factor for nuclear and nuclear envelope-localizing testicular proteins.

    Science.gov (United States)

    Davies, Rebecca G; Wagstaff, Kylie M; McLaughlin, Eileen A; Loveland, Kate L; Jans, David A

    2013-12-01

    Regulation of nuclear protein import is central to many cellular processes such as development, with a key mechanism being factors that retain cargoes in the cytoplasm that normally localize in the nucleus. The breast cancer antigen BRCA1-binding protein BRAP2 has been reported as a novel negative regulator of nuclear import of various nuclear localization signal (NLS)-containing viral and cellular proteins, but although implicated in differentiation pathways and highly expressed in tissues including testis, the gamut of targets for BRAP2 action in a developmental context is unknown. As a first step towards defining the BRAP2 interactome, we performed a yeast-2-hybrid screen to identify binding partners of BRAP2 in human testis. Here we report characterization for the first time of three of these: the high mobility group (HMG)-box-domain-containing chromatin component HMG20A, nuclear mitotic apparatus protein NuMA1 and synaptic nuclear envelope protein SYNE2. Co-immunoprecipitation experiments indicate association of BRAP2 with HMG20A, NuMA1, and SYNE2 in testis, underlining the physiological relevance of the interactions, with immunohistochemistry showing that where BRAP2 is co-expressed with HMG20A and NuMA1, both are present in the cytoplasm, in contrast to their nuclear localization in other testicular cell types. Importantly, quantitative confocal microscopic analysis of cultured cells indicates that ectopic expression of BRAP2 inhibits nuclear localization of HMG20A and NuMA1, and prevents nuclear envelope accumulation of SYNE2, the first report of BRAP2 altering localization of a non-nuclear protein. These results imply for the first time that BRAP2 may have an important role in modulating subcellular localization during testicular development. PMID:23707952

  9. Joint modeling of cell and nuclear shape variation.

    Science.gov (United States)

    Johnson, Gregory R; Buck, Taraz E; Sullivan, Devin P; Rohde, Gustavo K; Murphy, Robert F

    2015-11-01

    Modeling cell shape variation is critical to our understanding of cell biology. Previous work has demonstrated the utility of nonrigid image registration methods for the construction of nonparametric nuclear shape models in which pairwise deformation distances are measured between all shapes and are embedded into a low-dimensional shape space. Using these methods, we explore the relationship between cell shape and nuclear shape. We find that these are frequently dependent on each other and use this as the motivation for the development of combined cell and nuclear shape space models, extending nonparametric cell representations to multiple-component three-dimensional cellular shapes and identifying modes of joint shape variation. We learn a first-order dynamics model to predict cell and nuclear shapes, given shapes at a previous time point. We use this to determine the effects of endogenous protein tags or drugs on the shape dynamics of cell lines and show that tagged C1QBP reduces the correlation between cell and nuclear shape. To reduce the computational cost of learning these models, we demonstrate the ability to reconstruct shape spaces using a fraction of computed pairwise distances. The open-source tools provide a powerful basis for future studies of the molecular basis of cell organization. PMID:26354424

  10. Expressão nuclear do P53 em carcinoma de células transicionais da bexiga Nuclear expression of P53 protein in transitional cell carcinoma of the bladder

    Directory of Open Access Journals (Sweden)

    José Anastácio Dias Neto

    2002-01-01

    Full Text Available OBJETIVO: Investigar a expressão imuno-histoquímica da p53 com fator de risco em carcinoma de células transicionais da bexiga (CCT. MÉTODOS: Foram estudados restrospectivamente 90 pacientes com CCT com idade média de 71 anos: G1 - 45, G2 - 29, G3 - 16, pTa-1 - 62 e pT2-4 - 28. Entre os pacientes com tumores não invasivos houve recidiva vesical em 35 casos (55,5%. Os tumores superficiais foram tratados por ressecção trans-uretral associados ao BCG (>G1, e os invasivos por cistectomia radical. O tempo médio de seguimento dos pacientes foi de 55 meses e 25 deles faleceram da doença. A expressão imuno-histoquímica foi estudada em peças preservadas em formol 10% e blocos de parafina pelo método da avidina-biotina-imunoperoxidase. Considerou-se p53 positivo o tumor com índice de marcação nuclear superior a 10%. RESULTADOS: A expressão da p53 mostrou associação com o grau do tumor e com o estádio da lesão primária (p=0,01, mas não com o tamanho do tumor vesical (p=0,25 ou com a taxa de recidiva dos tumores superficiais (p=0,81. Houve forte correlação entre o padrão de marcação da p53 com metástases (p=0,002 e com a sobrevida dos pacientes (p=0,003. CONCLUSÃO: A expressão da p53 mostrou valor preditivo para grau tumoral, estádio, incidência de metástases e sobrevida dos pacientes, mas não para recidiva vesical dos tumores superficiais.OBJECTIVE: To investigate the immunoexpression of p53 protein as a risk factor in transitional cell carcinoma of the bladder (TCC. METHODS: We analyzed retrospectively 90 patients with TCC and mean age of 71 years: G1 - 45, G1 - 29, G3 - 16, pTa-1 - 62 and pT2-4 - 28. The superficial TCC were treated TUR plus intravesical BCG (>G1, and the invasives by radical cystectomy and urinary diversion. The mean time of followup was 55 months and 25 patients died of the disease. The rate or reccurence in superficial tumors was 55.5%. The p53 immunoexpression was determined in formalin fixed

  11. A simple polymeric model describes cell nuclear mechanical response

    Science.gov (United States)

    Banigan, Edward; Stephens, Andrew; Marko, John

    The cell nucleus must continually resist inter- and intracellular mechanical forces, and proper mechanical response is essential to basic cell biological functions as diverse as migration, differentiation, and gene regulation. Experiments probing nuclear mechanics reveal that the nucleus stiffens under strain, leading to two characteristic regimes of force response. This behavior depends sensitively on the intermediate filament protein lamin A, which comprises the outer layer of the nucleus, and the properties of the chromatin interior. To understand these mechanics, we study a simulation model of a polymeric shell encapsulating a semiflexible polymer. This minimalistic model qualitatively captures the typical experimental nuclear force-extension relation and observed nuclear morphologies. Using a Flory-like theory, we explain the simulation results and mathematically estimate the force-extension relation. The model and experiments suggest that chromatin organization is a dominant contributor to nuclear mechanics, while the lamina protects cell nuclei from large deformations.

  12. Polymorphic membrane protein (PMP) 20 and PMP 21 of Chlamydia pneumoniae induce proinflammatory mediators in human endothelial cells in vitro by activation of the nuclear factor-kappaB pathway.

    Science.gov (United States)

    Niessner, Alexander; Kaun, Christoph; Zorn, Gerlinde; Speidl, Walter; Türel, Zeynep; Christiansen, Gunna; Pedersen, Anna-Sofie; Birkelund, Svend; Simon, Susan; Georgopoulos, Apostolos; Graninger, Wolfgang; de Martin, Rainer; Lipp, Joachim; Binder, Bernd R; Maurer, Gerald; Huber, Kurt; Wojta, Johann

    2003-07-01

    We tested whether polymorphic membrane proteins (PMPs) of Chlamydia pneumoniae might play a role in triggering an inflammatory response in human endothelial cells. Of 15 purified, recombinant chlamydial PMPs tested, 2 (PMP 20 and PMP 21) dose-dependently increased the production of the inflammatory mediators interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1), in cultured human endothelial cells; production of IL-8 was also increased. When endothelial cells were infected by live C. pneumoniae, an increase in the production of IL-6, IL-8, and MCP-1 was seen. We used adenovirus-induced overexpression of IkappaBalpha-an inhibitor of nuclear factor (NF)-kappaB-to demonstrate that PMP 20 and PMP 21 increase the production of IL-6 and MCP-1 in human endothelial cells by activation of the NF-kappaB pathway, because, in cells overexpressing IkappaBalpha, treatment with the respective PMP did not result in increased production of IL-6 and MCP-1. Thus, C. pneumoniae could, by interactions of its PMPs with the endothelium, contribute to the process of vascular injury during the development and progression of atherosclerotic lesions.

  13. Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles.

    OpenAIRE

    Krämer, A

    1987-01-01

    Upon chromatographic fractionation of HeLa cell nuclear extracts, small RNAs of 145 and 66/65 nucleotides, respectively, were detected that are distinct from the abundant small RNAs present in the extract. These RNAs are precipitated by antibodies directed against the trimethylguanosine cap structure, characteristic for small nuclear RNAs (snRNAs) of the U type. The RNAs of 145 and 66/65 nucleotides appear to be associated with at least one of the proteins common to the major small nuclear ri...

  14. Nuclear translocation of the 1,25D{sub 3}-MARRS (membrane associated rapid response to steroids) receptor protein and NF{kappa}B in differentiating NB4 leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wenqing; Beilhartz, Greg; Roy, Yvette; Richard, Cynthia L.; Curtin, Maureen; Brown, Lauren; Cadieux, Danielle [Departments of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Coppolino, Marc [Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Farach-Carson, Mary C. [Department of Biological Sciences, University of Delaware, Newark, DE 19716 (United States); Nemere, Ilka [Department of Nutrition and Food Sciences, Center for Integrated BioSystems, Utah State University, Logan, UT 84322 8700 (United States); Meckling, Kelly A., E-mail: kmecklin@uoguelph.ca [Departments of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada)

    2010-04-15

    1,25 Dihydroxyvitamin D{sub 3} (1,25D{sub 3}) primes NB4 promyelocytic leukemia cells to differentiate along the monocyte/macrophage lineage through a non-genomic mechanism. Here we show that NB4 cells express high levels of the recently identified membrane receptor for 1,25D{sub 3}, which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D{sub 3}-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NF{kappa}B). In unstimulated cells, 1,25D{sub 3}-MARRS can be co-immunoprecipitated with antibodies directed at NF{kappa}B, and NF{kappa}B is co-precipitated when antibodies against 1,25D{sub 3}-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D{sub 3}-MARRS and NF{kappa}B begin translocating to the nucleus within minutes of co-stimulation with 1,25D{sub 3} and phorbol ester. The predominant nuclear localization of both proteins precedes the expression of the monocyte/macrophage phenotype and suggests that this event may be critical to the differentiation pathway. This suggests a role for 1,25D{sub 3}-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NF{kappa}B and other factors with which it may be interacting.

  15. Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida;

    2011-01-01

    -cell stage embryos were processed at different points in time post activation (2 hpa, 4 hpa, 8 hpa, and 12 hpa) for detailed nuclear and nucleolar analysis by TEM, and immunofluorescence for visualization of nucleolar proteins related to transcription (UBF) and processing (fibrillarin). Bovine and porcine...

  16. Molecular structure and biological function of proliferating cell nuclear antigen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Proliferating cell nuclear antigen (PCNA) is the core component of replication complex in eukaryote.As a processive factor of DNA polymerase delta, PCNA coordinates the replication process by interacting with various replication proteins. PCNA appears to play an essential role in many cell events, such as DNA damage repair, cell cycle regulation, and apoptosis, through the coordination or organization of different partners. PCNA is an essential factor in cell proliferation, and has clinical significance in tumor research. In this article we review the functional structure of PCNA, which acts as a function switch in different cell events.

  17. The conserved domain CR2 of Epstein-Barr virus nuclear antigen leader protein is responsible not only for nuclear matrix association but also for nuclear localization.

    Science.gov (United States)

    Yokoyama, A; Kawaguchi, Y; Kitabayashi, I; Ohki, M; Hirai, K

    2001-01-20

    There is a growing body of evidence for the importance of the nuclear matrix in various nuclear events including gene expression and DNA replication. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a nuclear matrix-associated protein that has been suggested to play an important role in EBV-induced transformation. To define the biological significance of the association of EBNA-LP with the nuclear matrix, we mapped the domain of EBNA-LP responsible for nuclear matrix association and investigated the functions of the EBNA-LP mutant mutagenized by substitution of alanines for the cluster of arginine residues in the mapped region. The results of the present study were as follows. (i) Transiently expressed EBNA-LP in COS-7 or BOSC23 cells was associated with the nuclear matrix, similarly to that in EBV-infected B cells. (ii) Mutational analysis of EBNA-LP revealed that a 10-amino acid segment of EBNA-LP is critical for nuclear matrix association of the protein. Interestingly, the identified region overlapped with the region CR2 of EBNA-LP conserved among a subset of primate gammaherpesviruses. The identified segment is referred to as EBNA-LP NMTS (nuclear matrix targeting signal). (iii) The EBNA-LP mutant with the arginine to alanine substitutions in NMTS was no longer localized not only to the nuclear matrix but also to the nucleus. (iv) The EBNA-LP mutant lacked its ability to coactivate EBNA-2-dependent transactivation. These results indicated that EBNA-LP needs to be localized in the nucleus and/or associated with the nuclear matrix through CR2 to elicit its function such as the coactivation of the EBNA-2-dependent transcriptional activation. PMID:11162796

  18. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek;

    2009-01-01

    , somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...

  19. Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion.

    Science.gov (United States)

    Krull, Sandra; Dörries, Julia; Boysen, Björn; Reidenbach, Sonja; Magnius, Lars; Norder, Helene; Thyberg, Johan; Cordes, Volker C

    2010-05-19

    Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel- and cone-like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC-associated protein Tpr and its large coiled coil-forming domain. RNAi-mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub-compartment and delimiting heterochromatin distribution.

  20. Cytomegalovirus Assembly Protein Precursor and Proteinase Precursor Contain Two Nuclear Localization Signals That Mediate Their Own Nuclear Translocation and That of the Major Capsid Protein

    OpenAIRE

    Plafker, Scott M.; Gibson, Wade

    1998-01-01

    The cytomegalovirus (CMV) assembly protein precursor (pAP) interacts with the major capsid protein (MCP), and this interaction is required for nuclear translocation of the MCP, which otherwise remains in the cytoplasm of transfected cells (L. J. Wood et al., J. Virol. 71:179–190, 1997). We have interpreted this finding to indicate that the CMV MCP lacks its own nuclear localization signal (NLS) and utilizes the pAP as an NLS-bearing escort into the nucleus. The CMV pAP amino acid sequence has...

  1. Nuclear translocation of EGF receptor regulated by Epstein-Barr virus encoded latent membrane protein 1

    Institute of Scientific and Technical Information of China (English)

    TAO; Yongguang; SONG; Xin; TAN; Yunnian; LIN; Xiaofeng; ZH

    2004-01-01

    Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV encoded proteins, and also it has always been the core of the oncogenic mechanism of EBV. Traditional receptor theory demonstrates that cell surface receptors exert biological functions on the membrane, which neither enter into the nucleus nor directly affect the transcription of the target genes. But, advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly developed our knowledge of the biological function of cell surface receptors. In this study, we used Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1 integrated NPC cell line and the expression of LMP1 in which could be regulated by Tet system. We found that LMP1 could regulate the nuclear translocation of EGFR in a dose-dependent manner from both quantitative and qualitative levels through the Western blot analysis and the immunofluorescent analysis with a laser scanning confocal microscope. We further demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation, and the nuclear accumulation of EGFR regulated by LMP1 was in a ligand-independent manner. These findings provide a novel view that the regulation of LMP1 on the nuclear translocation of EGFR is critical for the process of nasopharyngeal carcinoma.

  2. Heat shock protein 70 protects against seizure-induced neuronal cell death in the hippocampus following experimental status epilepticus via inhibition of nuclear factor-κB activation-induced nitric oxide synthase II expression.

    Science.gov (United States)

    Chang, Chiung-Chih; Chen, Shang-Der; Lin, Tsu-Kung; Chang, Wen-Neng; Liou, Chia-Wei; Chang, Alice Y W; Chan, Samuel H H; Chuang, Yao-Chung

    2014-02-01

    Status epilepticus induces subcellular changes that may eventually lead to neuronal cell death in the hippocampus. Based on an animal model of status epilepticus, our laboratory showed previously that sustained hippocampal seizure activity activates nuclear factor-κB (NF-κB) and upregulates nitric oxide synthase (NOS) II gene expression, leading to apoptotic neuronal cell death in the hippocampus. The present study examined the potential modulatory role of heat shock protein 70 (HSP70) on NF-κB signaling in the hippocampus following experimental status epilepticus. In Sprague-Dawley rats, kainic acid (KA) was microinjected unilaterally into the hippocampal CA3 subfield to induce prolonged bilateral seizure activity. Expression of HSP70 was elevated as early as 1h after the elicitation of sustained seizure activity, followed by a progressive elevation that peaked at 24h. Pretreatment with an antisense oligonucleotide against hsp70 decreased the HSP70 expression, and significantly augmented IκB kinase (IKK) activity and phosphorylation of IκBα, alongside enhanced nuclear translocation and DNA binding activity of NF-κB in the hippocampal CA3 neurons and glial cells. These cellular events were followed by enhanced upregulation of NOS II and peroxynitrite expression 3h after sustained seizure activity that led to an increase of caspase-3 and DNA fragmentation in the hippocampal CA3 neurons 7days after experimental status epilepticus. We concluded that HSP70 protects against apoptotic cell death induced by NF-κB activation and NOS II-peroxynitrite signaling cascade in the hippocampal CA3 and glial cells following experimental status epilepticus via suppression of IKK activity and deactivation of IκBα.

  3. Tus, an E. coli protein, contains mammalian nuclear targeting and exporting signals.

    Directory of Open Access Journals (Sweden)

    Stanislaw J Kaczmarczyk

    Full Text Available Shuttling of proteins between nucleus and cytoplasm in mammalian cells is facilitated by the presence of nuclear localization signals (NLS and nuclear export signals (NES, respectively. However, we have found that Tus, an E. coli replication fork arresting protein, contains separate sequences that function efficiently as NLS and NES in mammalian cell lines, as judged by cellular location of GFP-fusion proteins. The NLS was localized to a short stretch of 9 amino acids in the carboxy-terminus of Tus protein. Alterations of any of these basic amino acids almost completely abolished the nuclear targeting. The NES comprises a cluster of leucine/hydrophobic residues located within 21 amino acids at the amino terminus of Tus. Finally, we have shown that purified GFP-Tus fusion protein or GFP-Tus NLS fusion protein, when added to the culture media, was internalized very efficiently into mammalian cells. Thus, Tus is perhaps the first reported bacterial protein to possess both NLS and NES, and has the capability to transduce protein into mammalian cells.

  4. Manassantin B isolated from Saururus chinensis inhibits cyclooxygenase-2-dependent prostaglandin D2 generation by blocking Fyn-mediated nuclear factor-kappaB and mitogen activated protein kinase pathways in bone marrow derived-mast cells.

    Science.gov (United States)

    Lu, Yue; Hwang, Seung-Lark; Son, Jong Keun; Chang, Hyeun Wook

    2013-01-01

    The authors investigated the effect of manassantin B (Man B) isolated from Saururus chinensis (S. chinensis) on cyclooxygenase-2 (COX-2)-dependent prostaglandin D2 (PGD2) generation in mouse bone marrow derived-mast cells (BMMCs). Man B inhibited the generation of PGD2 dose-dependently by inhibiting COX-2 expression in immunoglobulin E (IgE)/Ag-stimulated BMMCs. To elucidate the mechanism responsible for the inhibition of COX-2 expression by Man B, the effects of Man B on the activation of nuclear factor-kappaB (NF-κB), a transcription factor essential and mitogen-activated protein kinases (MAPKs) for COX-2 induction, were examined. Man B attenuated the nuclear translocation of NF-κB p65 and its DNA-binding activity by inhibiting inhibitors of kappa Bα (IκBα) degradation and concomitantly suppressing IκB kinase (IKK) phosphorylation. In addition, Man B suppressed phosphorylation of MAPKs including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38. It was also found that Man B suppressed Fyn kinase activation and consequent downstream signaling processes, including those involving Syk, Gab2, and Akt. Taken together, the present results suggest that Man B suppresses COX-2 dependent PGD2 generation by primarily inhibiting Fyn kinase in FcεRI-mediated mast cells.

  5. Association of bovine papillomavirus E2 protein with nuclear structures in vivo.

    Science.gov (United States)

    Kurg, Reet; Sild, Kristiina; Ilves, Aigi; Sepp, Mari; Ustav, Mart

    2005-08-01

    Papillomaviruses are small DNA viruses which have the capacity to establish a persistent infection in mammalian epithelial cells. The papillomavirus E2 protein is a central coordinator of viral gene expression, genome replication, and maintenance. We have investigated the distribution of bovine papillomavirus E2 protein in nuclei of proliferating cells and found that E2 is associated with cellular chromatin. This distribution does not change during the entire cell cycle. The N-terminal transactivation domain, but not the C-terminal DNA-binding domain, of the E2 protein is responsible for this association. The majority of the full-length E2 protein can only be detected in chromatin-enriched fractions but not as a free protein in the nucleus. Limited micrococcal nuclease digestion revealed that the E2 protein partitioned to different chromatin regions. A fraction of the E2 protein was located at nuclear sites that are resistant against nuclease attack, whereas the remaining E2 resided on compact chromatin accessible to micrococcal nuclease. These data suggest that there are two pools of E2 in the cell nucleus: one that localizes on transcriptionally inactive compact chromatin and the other, which compartmentalizes to transcriptionally active nuclear structures of the cell. Our data also suggest that E2 associates with chromatin through cellular protein(s), which in turn is released from chromatin at 0.4 M salt. PMID:16051845

  6. RanBP3 influences interactions between CRM1 and its nuclear protein export substrates

    OpenAIRE

    Englmeier, Ludwig; Fornerod, Maarten; Bischoff, F. Ralf; Petosa, Carlo; Mattaj, Iain W.; Kutay, Ulrike

    2001-01-01

    We investigated the role of RanBP3, a nuclear member of the Ran-binding protein 1 family, in CRM1-mediated protein export in higher eukaryotes. RanBP3 interacts directly with CRM1 and also forms a trimeric complex with CRM1 and RanGTP. However, RanBP3 does not bind to CRM1 like an export substrate. Instead, it can stabilize CRM1–export substrate interaction. Nuclear RanBP3 stimulates CRM1-dependent protein export in permeabilized cells. These data indicate that RanBP3 functions by a novel mec...

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

    Directory of Open Access Journals (Sweden)

    Andrea Cerutti

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

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

    International Nuclear Information System (INIS)

    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-NLSSV40) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLSSV40 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-NLSSV40 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-NLSSV40 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-NLSSV40 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

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

    Energy Technology Data Exchange (ETDEWEB)

    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 biogenesis takes place. GFP-NLS{sup SV40} in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS{sup SV40} formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS{sup SV40} likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS{sup SV40} can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus.

  10. Andrographolide stimulates p38 mitogen-activated protein kinase-nuclear factor erythroid-2-related factor 2-heme oxygenase 1 signaling in primary cerebral endothelial cells for definite protection against ischemic stroke in rats.

    Science.gov (United States)

    Yen, Ting-Lin; Chen, Ray-Jade; Jayakumar, Thanasekaran; Lu, Wan-Jung; Hsieh, Cheng-Ying; Hsu, Ming-Jen; Yang, Chih-Hao; Chang, Chao-Chien; Lin, Yen-Kuang; Lin, Kuan-Hung; Sheu, Joen-Rong

    2016-04-01

    Stroke pathogenesis involves complex oxidative stress-related pathways. The nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) pathways have been considered molecular targets in pharmacologic intervention for ischemic diseases. Andrographolide, a labdane diterpene, has received increasing attention in recent years because of its various pharmacologic activities. We determined that andrographolide modulates the mitogen-activated protein kinase (MAPK)-Nrf2-HO-1 signaling cascade in primary cerebral endothelial cells (CECs) to provide positive protection against middle cerebral artery occlusion (MCAO)-induced ischemic stroke in rats. In the present study, andrographolide (10 μM) increased HO-1 protein and messenger RNA expressions, Nrf2 phosphorylation, and nuclear translocation in CECs, and these activities were disrupted by a p38 MAPK inhibitor, SB203580, but not by the extracellular signal-regulated kinase inhibitor PD98059 or c-Jun amino-terminal kinase inhibitor SP600125. Similar results were observed in confocal microscopy analysis. Moreover, andrographolide-induced Nrf2 and HO-1 protein expressions were significantly inhibited by Nrf2 small interfering RNA. Moreover, HO-1 knockdown attenuated the protective effect of andrographolide against oxygen-glucose deprivation-induced CEC death. Andrographolide (0.1 mg/kg) significantly suppressed free radical formation, blood-brain barrier disruption, and brain infarction in MCAO-insulted rats, and these effects were reversed by the HO-1 inhibitor zinc protoporphyrin IX. The mechanism is attributable to HO-1 activation, as directly evidenced by andrographolide-induced pronounced HO-1 expression in brain tissues, which was highly localized in the cerebral capillary. In conclusion, andrographolide increased Nrf2-HO-1 expression through p38 MAPK regulation, confirming that it provides protection against MCAO-induced brain injury. These findings provide strong evidence that andrographolide could

  11. Nuclear T-STAR protein expression correlates with HER2 status, hormone receptor negativity and prolonged recurrence free survival in primary breast cancer and decreased cancer cell growth in vitro.

    Directory of Open Access Journals (Sweden)

    Sandra Sernbo

    Full Text Available T-STAR (testis-signal transduction and activation of RNA is an RNA binding protein, containing an SH3-binding domain and thus potentially playing a role in integration of cell signaling and RNA metabolism. The specific function of T-STAR is unknown and its implication in cancer is poorly characterized. Expression of T-STAR has been reported in human testis, muscle and brain tissues, and is associated with a growth-inhibitory role in immortalized fibroblasts. The aim of this paper was to investigate the functional role of T-STAR through (i survival analysis of patients with primary invasive breast cancer and (ii experimental evaluation of the effect of T-STAR on breast cancer cell growth. T-STAR protein expression was analysed by immunohistochemistry (IHC in tissue microarrays with tumors from 289 patients with primary invasive breast cancer, and correlations to clinicopathological characteristics, recurrence-free and overall survival (RFS and OS and established tumor markers such as HER2 and ER status were evaluated. In addition, the function of T-STAR was investigated using siRNA-mediated knock-down and overexpression of the gene in six breast cancer cell lines. Of the tumors analysed, 86% showed nuclear T-STAR expression, which was significantly associated with an improved RFS and strongly associated with positive HER2 status and negative hormone receptor status. Furthermore, experimental data showed that overexpression of T-STAR decreased cellular growth while knock-down increased it, as shown both by thymidine incorporation and metabolic activity. In summary, we demonstrate that T-STAR protein expression correlates with an improved RFS in primary breast cancer. This is supported by functional data, indicating that T-STAR regulation is of importance both for breast cancer biology and clinical outcome but future studies are needed to determine a potential role in patient stratification.

  12. Characterization of a 65 kDa NIF in the nuclear matrix of the monocot Allium cepa that interacts with nuclear spectrin-like proteins.

    Science.gov (United States)

    Pérez-Munive, Clara; Blumenthal, Sonal S D; de la Espina, Susana Moreno Díaz

    2012-01-01

    Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled-coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross-react with anti-intermediate filament and anti-lamin antibodies, form filaments 6-12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin-like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin-like proteins by co-immunoprecipitation and co-localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with Mr and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin-like proteins. Its similarities with some of the proteins described as onion lamin-like proteins suggest that they are highly related or perhaps the same proteins.

  13. Tau protein function in living cells

    OpenAIRE

    1986-01-01

    Tau protein from mammalian brain promotes microtubule polymerization in vitro and is induced during nerve cell differentiation. However, the effects of tau or any other microtubule-associated protein on tubulin assembly within cells are presently unknown. We have tested tau protein activity in vivo by microinjection into a cell type that has no endogenous tau protein. Immunofluorescence shows that tau protein microinjected into fibroblast cells associates specifically with microtubules. The i...

  14. 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.

    Science.gov (United States)

    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 (171EDVSRFIKGKLLQKQQKIYKDLERF195) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues 48KKSYQDPEIIAHSRPRK64 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 48EF49 abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the 48EF49 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. PMID:15896312

  15. Reprogramming cells with synthetic proteins

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Yang

    2015-06-01

    Full Text Available Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

  16. PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

    Science.gov (United States)

    Corbeil-Girard, Louis-Philippe; Klein, Arnaud F; Sasseville, A Marie-Josée; Lavoie, Hugo; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Duranceau, André; Codère, François; Bouchard, Jean-Pierre; Karpati, George; Rouleau, Guy A; Massie, Bernard; Langelier, Yves; Brais, Bernard

    2005-04-01

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function. PMID:15755682

  17. PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

    Science.gov (United States)

    Corbeil-Girard, Louis-Philippe; Klein, Arnaud F; Sasseville, A Marie-Josée; Lavoie, Hugo; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Duranceau, André; Codère, François; Bouchard, Jean-Pierre; Karpati, George; Rouleau, Guy A; Massie, Bernard; Langelier, Yves; Brais, Bernard

    2005-04-01

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.

  18. Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Jennifer A Mitchell

    Full Text Available In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.

  19. Porcine circovirus-2 capsid protein induces cell death in PK15 cells

    Energy Technology Data Exchange (ETDEWEB)

    Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark; Czub, Markus

    2014-11-15

    Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.

  20. Neuroprotection Signaling of Nuclear Akt in Neuronal Cells

    OpenAIRE

    Ahn, Jee-Yin

    2014-01-01

    Akt is one of the central kinases that perform a pivotal function in mediating survival signaling in a wide range of neuronal cell types in response to growth factor stimulation. The recent findings of a number of targets for Akt suggest that it prohibits neuronal death by both impinging on the cytoplasmic cell death machinery and by regulating nuclear proteins. The presence of active Akt in the nuclei of mammalian cells is no longer debatable, and this has been corroborated by the finding of...

  1. Alterations in the nuclear proteome of HIV-1 infected T-cells

    Energy Technology Data Exchange (ETDEWEB)

    DeBoer, Jason [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Jagadish, Teena; Haverland, Nicole A. [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Ciborowski, Pawel [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States)

    2014-11-15

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.

  2. Heterogeneity in the kinetics of nuclear proteins and trajectories of substructures associated with heterochromatin

    Directory of Open Access Journals (Sweden)

    Stixová Lenka

    2011-03-01

    Full Text Available Abstract Background Protein exchange kinetics correlate with the level of chromatin condensation and, in many cases, with the level of transcription. We used fluorescence recovery after photobleaching (FRAP to analyse the kinetics of 18 proteins and determine the relationships between nuclear arrangement, protein molecular weight, global transcription level, and recovery kinetics. In particular, we studied heterochromatin-specific heterochromatin protein 1β (HP1β B lymphoma Mo-MLV insertion region 1 (BMI1, and telomeric-repeat binding factor 1 (TRF1 proteins, and nucleolus-related proteins, upstream binding factor (UBF and RNA polymerase I large subunit (RPA194. We considered whether the trajectories and kinetics of particular proteins change in response to histone hyperacetylation by histone deacetylase (HDAC inhibitors or after suppression of transcription by actinomycin D. Results We show that protein dynamics are influenced by many factors and events, including nuclear pattern and transcription activity. A slower recovery after photobleaching was found when proteins, such as HP1β, BMI1, TRF1, and others accumulated at specific foci. In identical cells, proteins that were evenly dispersed throughout the nucleoplasm recovered more rapidly. Distinct trajectories for HP1β, BMI1, and TRF1 were observed after hyperacetylation or suppression of transcription. The relationship between protein trajectory and transcription level was confirmed for telomeric protein TRF1, but not for HP1β or BMI1 proteins. Moreover, heterogeneity of foci movement was especially observed when we made distinctions between centrally and peripherally positioned foci. Conclusion Based on our results, we propose that protein kinetics are likely influenced by several factors, including chromatin condensation, differentiation, local protein density, protein binding efficiency, and nuclear pattern. These factors and events likely cooperate to dictate the mobility of

  3. Nuclear domain 10-associated proteins recognize and segregate intranuclear DNA/protein complexes to negate gene expression

    Directory of Open Access Journals (Sweden)

    Rivera-Molina Yisel A

    2012-09-01

    Full Text Available Abstract Background DNA viruses, such as herpes simplex virus type 1 (HSV-1, Simian virus 40 (SV40, and Cytomegaloviruses (CMV, start their replicative processes and transcription at specific nuclear domains known as ND10 (nuclear domain 10, also called PML bodies. It has been previously determined that for HSV-1 and SV40, a short DNA sequence and its binding protein are required and sufficient for cell localization of viral DNA replication and gene transcription. Results Our recent observations provide evidence that a foreign (not endogenous DNA/protein complex in the nucleus recruits ND10 proteins. First, the complexes formed from the bacterial lac operator DNA and its binding protein (lac repressor, or from HPV11 (human papillomavirus 11 origin DNA and its binding protein (E2, co-localized with different ND10 proteins. Second, the HSV-1 amplicon without inserted lac operator DNA repeats distributed in the nucleus randomly, whereas the amplicon with lac operator DNA repeats associated with ND10, suggesting that DNA-binding proteins are required to localize at ND10. The cellular intrinsic DNA/protein complex (as detected for U2 DNA showed no association with ND10. Furthermore, our examination of PML−/−, Daxx−/−, and Sp100-negative cells led to our discovering that DNA/protein complexes recruit ND10 protein independently. Using the GFP-LacI/Operator system, we were able to direct the transfected DNA to ND10 and found that gene expression was significantly repressed when the transfected DNA was directed to ND10. Conclusion Taken together, the results suggest that cells recognize DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins.

  4. Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing

    Energy Technology Data Exchange (ETDEWEB)

    Yan Chunlan; Wu Wei [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Li Haiyan [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Huzhou Maternity and Child Care Hospital, Huzhou, Zhejiang 313000 (China); Zhang Guanglin [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Zhejiang-California International Nanosystems Institute, Hangzhou, Zhejiang 310029 (China)

    2010-04-01

    Benzo[a]pyrene (BaP) is a potent pro-carcinogen generated from the combustion of fossil fuel and cigarette smoke. Previously, using a proteomic approach, we have shown that BaP can induce changes in the expression of many cellular proteins, including transcription regulators. In the present study, using a similar approach, we examined the nuclear protein response to BaP in HeLa cells and found that BaP treatment caused expression changes in many nuclear proteins. Twenty-four of these proteins were successfully identified, several of which are involved in the alternative splicing of mRNA, DNA replication, recombination, and repair. The changed expression levels were further confirmed by immunoblot analysis using specific antibodies for two proteins, Lamin A and mitotic checkpoint protein Bub3. The nuclear localization of these two proteins was also confirmed by confocal microscopy. To determine whether alternative splicing was activated following BaP treatment, we examined Fas and CD44, two genes previously shown to be targets of alternative splicing in respond to DNA damage. While no significant activation of alternative splicing was observed for Fas, CD44 splicing variants were found after BaP treatment. Together, these data show that DNA damage induces dramatic changes in nuclear protein expression, and that alternative splicing might be involved in the cellular response to DNA damage.

  5. Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Lloyd, Richard E., E-mail: rlloyd@bcm.edu

    2015-05-15

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.

  6. Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

    International Nuclear Information System (INIS)

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication

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

    Energy Technology Data Exchange (ETDEWEB)

    Siyam, Arwa [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); Wang, Suzhen; Qin, Chunlin; Mues, Gabriele [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Stevens, Roy [Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); D' Souza, Rena N. [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Lu, Yongbo, E-mail: ylu@bcd.tamhsc.edu [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)

    2012-08-03

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

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

    International Nuclear Information System (INIS)

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

  9. Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Tonatiuh Barrios-García

    2016-06-01

    Full Text Available Tristetraprolin (TTP is a 34-kDa, zinc finger-containing factor that in mammalian cells acts as a tumor suppressor protein through two different mechanisms. In the cytoplasm TTP promotes the decay of hundreds of mRNAs encoding cell factors involved in inflammation, tissue invasion, and metastasis. In the cell nucleus TTP has been identified as a transcriptional corepressor of the estrogen receptor alpha (ERα, which has been associated to the development and progression of the majority of breast cancer tumors. In this work we report that nuclear TTP modulates the transactivation activity of progesterone receptor (PR, glucocorticoid receptor (GR and androgen receptor (AR. In recent years these steroid nuclear receptors have been shown to be of clinical and therapeutical relevance in breast cancer. The functional association between TTP and steroid nuclear receptors is supported by the finding that TTP physically interacts with ERα, PR, GR and AR in vivo. We also show that TTP overexpression attenuates the transactivation of all the steroid nuclear receptors tested. In contrast, siRNA-mediated reduction of endogenous TTP expression in MCF-7 cells produced an increase in the transcriptional activities of ERα, PR, GR and AR. Taken together, these results suggest that the function of nuclear TTP in breast cancer cells is to act as a corepressor of ERα, PR, GR and AR. We propose that the reduction of TTP expression observed in different types of breast cancer tumors may contribute to the development of this disease by producing a dysregulation of the transactivation activity of multiple steroid nuclear receptors.

  10. Discovery and characterization of a new cell-penetrating protein.

    Science.gov (United States)

    Simeon, Rudo L; Chamoun, Ana Maria; McMillin, Thomas; Chen, Zhilei

    2013-12-20

    We describe a new cell-penetrating protein, B1, capable of delivering conjugated proteins and nucleic acids into mammalian cells. B1 is a 244-amino-acid product of a single-base frameshift in the gene encoding enhanced green fluorescent protein (eGFP). The molecule has a net positive charge of 43 and a very high charge-to-mass ratio of 1.5. eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein when exposed to concentrations as low as 1 μM. The protein was found to cluster in the paranuclear region of TZM-bl cells. Most importantly, we show that B1 not only facilitates cellular uptake but allows biomolecular cargo to reach sites of biological relevance. For example, baby hamster kidney cells underwent DNA recombination when exposed to B1-tagged Cre recombinase at protein concentrations as low as 2.5 μM, indicating potent nuclear delivery of functional protein cargos. Additionally, B1 delivers noncovalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites accessible to the cellular translation and transcription machinery, as gauged by detection of encoded reporter functions, with efficiency comparable to commercially available cationic lipid reagents. B1 appears to utilize cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells. These studies provide both a new tool for intracellular delivery of biomolecules and insights that could aid in the design of more effective cell penetrating proteins.

  11. Control of Toll-like receptor-mediated T cell-independent type 1 antibody responses by the inducible nuclear protein IκB-ζ.

    Science.gov (United States)

    Hanihara-Tatsuzawa, Fumito; Miura, Hanae; Kobayashi, Shuhei; Isagawa, Takayuki; Okuma, Atsushi; Manabe, Ichiro; MaruYama, Takashi

    2014-11-01

    Antibody responses have been classified as being either T cell-dependent or T cell-independent (TI). TI antibody responses are further classified as being either type 1 (TI-1) or type 2 (TI-2), depending on their requirement for B cell-mediated antigen receptor signaling. Although the mechanistic basis of antibody responses has been studied extensively, it remains unclear whether different antibody responses share similarities in their transcriptional regulation. Here, we show that mice deficient in IκB-ζ, specifically in their B cells, have impaired TI-1 antibody responses but normal T cell-dependent and TI-2 antibody responses. The absence of IκB-ζ in B cells also impaired proliferation triggered by Toll-like receptor (TLR) activation, plasma cell differentiation, and class switch recombination (CSR). Mechanistically, IκB-ζ-deficient B cells could not induce TLR-mediated induction of activation-induced cytidine deaminase (AID), a class-switch DNA recombinase. Retroviral transduction of AID in IκB-ζ-deficient B cells restored CSR activity. Furthermore, acetylation of histone H3 in the vicinity of the transcription start site of the gene that encodes AID was reduced in IκB-ζ-deficient B cells relative to IκB-ζ-expressing B cells. These results indicate that IκB-ζ regulates TLR-mediated CSR by inducing AID. Moreover, IκB-ζ defines differences in the transcriptional regulation of different antibody responses. PMID:25124037

  12. Identification of potential nuclear reprogramming and differentiation factors by a novel selection method for cloning chromatin-binding proteins

    Institute of Scientific and Technical Information of China (English)

    LiuWang; AihuaZheng; LingYi; ChongrenXu; MingxiaoDing; HongkuiDeng

    2005-01-01

    Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation.

  13. Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response

    DEFF Research Database (Denmark)

    Bennetzen, Martin; Andersen, J.S.; Lasen, D.H.;

    2013-01-01

    Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time...... to assess lysine acetylation status and thereby validate the mass spectrometry data. We thus present evidence that nuclear proteins, including those known to regulate cellular functions via epigenetic modifications of histones, are regulated by (de)acetylation in a timely manner upon cell's exposure...

  14. Genistein suppresses tumor necrosis factor α-induced inflammation via modulating reactive oxygen species/Akt/nuclear factor ΚB and adenosine monophosphate-activated protein kinase signal pathways in human synoviocyte MH7A cells

    Directory of Open Access Journals (Sweden)

    Li J

    2014-03-01

    Full Text Available Jinchao Li,1,* Jun Li,2,* Ye Yue,1 Yiping Hu,1 Wenxiang Cheng,1 Ruoxi Liu,3 Xiaohua Pan,4 Peng Zhang1 1Center for Translational Medicine Research and Development, Shenzhen Institute of Advanced Technology, Chinese Academy of Science, Shenzhen, 2Emergency Surgery Department, Shaanxi Provincial People’s Hospital, Xi’an, 3Department of Orthopedics, Shandong University of Traditional Chinese Medicine, Jinan, 4Department of Orthopedics, Second Clinical Medical College, Jinan University, Shenzhen, People’ Republic of China *These authors contributed equally to this work Aims: Genistein, an isoflavone derivative found in soy, is known as a promising treatment for rheumatoid arthritis (RA. However, the detailed molecular mechanism of genistein in suppression of proinflammatory cytokine production remains ambiguous. The aim of this work was to evaluate the signal pathway by which genistein modulates inflammatory cytokine expression. Materials and methods: MH7A cells were stimulated with tumor necrosis factor (TNF-α and incubated with genistein, and interleukin (IL-1β, IL-6, and IL-8 production was measured by enzyme-linked immunosorbent assay. Nuclear translocation of nuclear factor (NF- ΚB was measured by a confocal fluorescence microscopy. The intracellular accumulation of reactive oxygen species (ROS was monitored using the fluorescent probe 5-6-chloromethyl-2´,7´-dichlorodihydrofluorescein diacetate. Signal-transduction protein expression was measured by Western blot. Results: Genistein decreased the secretion of IL-1β, IL-6, and IL-8 from TNF-α-stimulated MH7A cells in a dose-dependent manner. Genistein prevented TNF-α -induced NF-ΚB translocation as well as phosphorylation of IΚB kinase-α/β and IΚBα, and also suppressed TNF-α-induced AMPK inhibition. The production of IL-1β, IL-6, and IL-8 induced by TNF-α was decreased by the phosphatidylinositol-3 kinase inhibitor LY294002, suggesting that inhibition of Akt activation might

  15. NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Heyu [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Ma, Xi [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); State Key Lab of Animal Nutrition, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193 (China); Shi, Taiping [Chinese National Human Genome Center, Beijing. 3-707 North YongChang Road BDA, Beijing 100176 (China); Song, Quansheng [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Zhao, Hongshan, E-mail: hongshan@bjmu.edu.cn [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Ma, Dalong [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China)

    2010-01-01

    NSA2 (Nop seven-associated 2) was previously identified in a high throughput screen of novel human genes associated with cell proliferation, and the NSA2 protein is evolutionarily conserved across different species. In this study, we revealed that NSA2 is broadly expressed in human tissues and cultured cell lines, and located in the nucleolus of the cell. Both of the putative nuclear localization signals (NLSs) of NSA2, also overlapped with nucleolar localization signals (NoLSs), are capable of directing nucleolar accumulation. Moreover, over-expression of the NSA2 protein promoted cell growth in different cell lines and regulated the G1/S transition in the cell cycle. SiRNA silencing of the NSA2 transcript attenuated the cell growth and dramatically blocked the cell cycle in G1/S transition. Our results demonstrated that NSA2 is a nucleolar protein involved in cell proliferation and cell cycle regulation.

  16. SRY interacts with ribosomal proteins S7 and L13a in nuclear speckles.

    Science.gov (United States)

    Sato, Youichi; Yano, Shojiro; Ewis, Ashraf A; Nakahori, Yutaka

    2011-05-01

    The SRY (sex-determining region on the Y chromosome) is essential for male development; however, the molecular mechanism by which the SRY induces testis development is still unclear. To elucidate the mechanism of testis development, we identified SRY-interacting proteins using a yeast two-hybrid system. We found two ribosomal proteins, RPS7 (ribosomal protein S7) and RPL13a (ribosomal protein L13a) that interact with the HMG (high-mobility group) box domain of SRY. Furthermore, we confirmed the intracellular distributions of RPS7, RPL13a and SRY and found that the three proteins were co-expressed in COS1 cells. SRY, RPS7 and RPL13a were co-localized in nuclear speckles. These findings suggest that SRY plays an important role in activities associated with nuclear speckles via an unknown mechanism.

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

    International Nuclear Information System (INIS)

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

  18. Characterization of the Nuclear Localization Signal of High Risk HPV16 E2 Protein

    OpenAIRE

    Klucevsek, Kristin; Wertz, Mary; Lucchi, John; Leszczynski, Anna; Moroianu, Junona

    2006-01-01

    The E2 protein of high risk human papillomavirus type 16 (HPV16) contains an amino-terminal (N) domain, a hinge (H) region and a carboxyl-terminal (C) DNA binding domain. Using enhanced green fluorescent protein (EGFP) fusions with full length E2 and E2 domains in transfection assays in HeLa cells we found that the C domain is responsible for the nuclear localization of E2 in vivo, whereas the N and H domains do not contain additional nuclear localization signals (NLSs). Deletion analysis of ...

  19. CHANGES OF NUCLEAR MATRIX PROTEIN AND ITS RELATIONSHIP WITH c-erbB-2 IN HUMAN COLON ADENOCARCINOMA

    Institute of Scientific and Technical Information of China (English)

    WANG Ya-lan; GAO Jing; LI Yuan-yuan

    2005-01-01

    Objective: Nuclear matrix protein is tissue, cell-type specific, and tumor-relative. It plays an important role in the regulation of intranuclear processes. Some researches also showed that a c-erbB-2 promoter-specific DNA-binding nuclear matrix protein is present only in malignant human breast tissues and induces mitogenesis and cell surface expression of the c-erbB-2 protein in resting NIH/3T3 cells. But it is not clear that how it in colon adenocarcinomas. Methods:Two-dimensional gel electrophoretic method was used for NMP identification and immunohistochemistry was used for c-erbB-2 detection in 12 cases of colon adenocarcinomas and matched adjacent normal colon tissues. Results: 5 different nuclear matrix proteins (named C1-C5) were identified in 12 colon adenocarcinoma specimens, but not in the matched adjacent normal colon tissues; 3 nuclear matrix proteins (named N1-N3) were identified in all 12 matched adjacent normal colon tissues, but not in colon adenocarcinoma specimens. A nuclear matrix protein (named N4) was detected in all of 9moderated-well differentiated adenocarcinomas and all 12 matched adjacent normal colon tissues, but not in 3poor-differentiated adenocarcinomas. All of the 10 colon adenocarcinomas which had the nuclear matrix protein C4 were c-erbB-2 expression positive. Conclusion: The data suggest that there are specific nuclear matrix proteins in colon adenocarcinomas and its subtypes, which maybe valuable to serve as markers of colon adenocarcinomas in future. Nuclear matrix protein C4 probably is a c-erbB-2 promotor-specific nuclear matrix protein in colon adenocarcinomas, and may induce the expression of c-erbB-2.

  20. Metformin inhibits nuclear factor-κB activation and inflammatory cytokines expression induced by high glucose via adenosine monophosphate-activated protein kinase activation in rat glomerular mesangial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Gu Junfei; Ye Shandong; Wang Shan; Sun Wenjia; Hu Yuanyuan

    2014-01-01

    Background The renoprotective mechanisms of adenosine monophosphate (AMP)-activated protein kinase (AMPK) agonist-metformin have not been stated clearly.We hypothesized that metformin may ameliorate inflammation via AMPK interaction with critical inflammatory cytokines The aim of this study was to observe the effects of metformin on expression of nuclear factor-κB (NF-κB),monocyte chemoattractant protein-1 (MCP-1),intercellular adhesion molecule-1 (ICAM-1) and transforming growth factor-beta 1 (TGF-β1) induced by high glucose (HG) in cultured rat glomerular mesangial cells (MCs).Methods MCs were cultured in the medium with normal concentration glucose (group NG,5.6 mmol/L),high concentration glucose (group HG,25 mmol/L) and different concentrations of metformin (group M1,M2,M3).After 48-hour exposure,the supernatants and MCs were collected.The expression of NF-κB,MCP-1,ICAM-1,and TGF-β1 mRNA was analyzed by real time polymerase chain reaction.Westem blotting was used to detect the expression of AMPK,phospho-Thr-172 AMPK (p-AMPK),NF-κB p65,MCP-1,ICAM-1,and TGF-β1 protein.Results After stimulated by HG,the expression of NF-κB,MCP-1,ICAM-1,TGF-β1 mRNA and protein of MCs in group HG increased significantly compared with group NG (P <0.05).Both genes and protein expression of NF-κB,MCP-1,ICAM-1,TGF-β1 of MCs induced by high glucose were markedly reduced after metformin treatment in a dose-dependent manner (P <0.05).The expression of p-AMPK increased with the rising of metformin concentration,presenting the opposite trend,while the level of total-AMPK protein was unchanged with exposure to HG or metformin.Conlusion Metformin can suppress the expression of NF-κB,MCP-1,ICAM-1 and TGF-β1 of glomerular MCs induced by high glucose via AMPK activation,which may partlv contribute to its reno-protection.

  1. Nuclear myosin I regulates cell membrane tension

    Science.gov (United States)

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  2. Aldosterone stimulates nuclear factor-kappa B activity and transcription of intercellular adhesion molecule-1 and connective tissue growth factor in rat mesangial cells via serum- and glucocorticoid-inducible protein kinase-1.

    Science.gov (United States)

    Terada, Yoshio; Ueda, Satoko; Hamada, Kazu; Shimamura, Yoshiko; Ogata, Koji; Inoue, Kosuke; Taniguchi, Yoshinori; Kagawa, Toru; Horino, Taro; Takao, Toshihiro

    2012-02-01

    Several clinical and experimental data support the hypothesis that aldosterone contributes to the progression of renal injury. To determine the signaling pathway of aldosterone in relation to fibrosis and inflammation in mesangial cells, we investigated the effects of aldosterone on expression and activation of serum- and glucocorticoid-inducible protein kinase-1 (SGK1), the activation of nuclear factor-kappa B (NF-κB activation, and the expressions of intercellular adhesion molecule-1 (ICAM-1) and connective tissue growth factor (CTGF). Aldosterone stimulated SGK1 expression, phosphorylation (Ser-256), and kinase activity. The increments of phosphorylation and expression of SGK1 induced by aldosterone were inhibited by mineralocorticoid receptor (MR) inhibitor (eplerenone). Aldosterone stimulated NF-κB activity measured by NF-κB responsive elements, luciferase assay, and the levels of inhibitor of kappa B (IκB) phosphorylation. This aldosterone-induced activation of NF-κB was inhibited by the transfection of dominant-negative SGK1. Furthermore, aldosterone augmented the promoter activities and protein expressions of ICAM-1 and CTGF. The effects of aldosterone on ICAM-1 and CTGF promoter activities and protein expressions were inhibited by the transfection of dominant-negative SGK1 and dominant-negative IκBα. We also found that the MR antagonist significantly ameliorated the glomerular injury and enhancements in SGK1, ICAM-1, and CTGF expressions induced by 1% sodium chloride and aldosterone in vivo. In conclusion, our findings suggest that aldosterone stimulates ICAM-1 and CTGF transcription via activation of SGK1 and NF-κB, which may be involved in the progression of aldosterone-induced mesangial fibrosis and inflammation. MR antagonists may serve as useful therapeutic targets for the treatment of glomerular inflammatory disease.

  3. Morin suppresses inflammatory cytokine expression by downregulation of nuclear factor-κB and mitogen-activated protein kinase (MAPK) signaling pathways in lipopolysaccharide-stimulated primary bovine mammary epithelial cells.

    Science.gov (United States)

    Wang, Jingjing; Guo, Changming; Wei, Zhengkai; He, Xuexiu; Kou, Jinhua; Zhou, Ershun; Yang, Zhengtao; Fu, Yunhe

    2016-04-01

    Morin, a flavonoid isolated from Chinese herbs of the Moraceae family, has been reported to possess antiinflammatory activity. However, the effects of morin on mastitis have not been investigated. The present study was conducted to elucidate the antiinflammatory properties of morin on lipopolysaccharide (LPS)-stimulated primary bovine mammary epithelial cells (bMEC). The viability of bMEC was analyzed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay. Subsequently, bMEC were stimulated with LPS in the presence or absence of morin. Gene expression of proinflammatory cytokines was determined by quantitative real-time PCR (qRT-PCR). Nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα) protein, extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) were detected by Western blotting. The results showed that cell viability was not affected by morin. Moreover, morin inhibited the gene expression of tumor necrosis factor-α (TNF-α), IL-6, and IL-1β in LPS-stimulated bMEC in a dose-dependent manner. Western blot analysis showed that morin suppressed the phosphorylation of IκBα, NF-κB unit p65, ERK, p38, and JNK in LPS-stimulated bMEC. In conclusion, the protective effects of morin on LPS-induced inflammatory response in bMEC may be due to its ability to suppress NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. These findings suggest that morin may be used as antiinflammatory drug for mastitis.

  4. Nuclear (DNA, RNA, histone and non-histone protein) and nucleolar changes during growth and senescence of may apple leaves.

    Science.gov (United States)

    Bhattacharya, P K; Pappelis, A J; Lee, S C; BeMiller, J N; Karagiannis, C S

    1996-12-20

    Quantitative interference microscopy was used to determine changes in nuclear and nucleolar indices (dry mass and cross-sectional area) in upper and lower epidermal cells and adjacent leaf-margin hair cells of the May apple (Podophyllum peltatum L.) leaves over a 42-day period (after leaves emerged above the ground litter). These indices decreased in a highly correlated manner. A ploidy variation may exist between epidermal cells and leaf-margin hair cells. Using the leaf-margin hair cells model, six nuclear macromolecule indices (total nucleic acid, DNA, RNA, total nuclear protein, histone and non-histone protein), nuclear volume, nucleolar volume and perinucleolar volume (measured using quantitative epifluorescence-phase contrast microscopy) all declined with age (42-day study) in a highly correlated manner. The degeneration of the nucleus and nucleolus in the three leaf locations studied followed the patterns observed for programmed cellular senescence and death (necrosis) in epidermal cells of onion leaf bases (stored tissue; leaf bases did not contain chlorophyll) and human epithelial cells (buccal; cervical). We conclude that the epidermal cells and leaf-margin hair cells from green leaves of the May Apple are ideal for the study of programmed cell senescence and death in plants, especially for the partitioning of this process into the study of: the point-of-no-return (solubilization of the karyoskeleton and loss of non-histone proteins and RNA associated with the karyoskeleton from the nucleus); nuclear pycnosis (loss of nuclear dry mass and volume and loss of nuclear internal support structure); chromatin condensation, margination along the inner nuclear envelope; and DNA-histone degeneration; degeneration of the nucleolus and loss of the perinucleolar zone of exclusion. The characterization of chlorenchyma cells during the 42-day period should now be undertaken (leaf senescence as indicated by the beginning of yellowing about 35 days after emergence) to

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

    Energy Technology Data Exchange (ETDEWEB)

    Barbarin, Alice; Séité, Paule [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Godet, Julie [Laboratoire d’anatomie et de cytologie pathologiques, CHU de Poitiers, 2 rue de la Milétrie, 86000 Poitiers (France); Bensalma, Souheyla; Muller, Jean-Marc [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Chadéneau, Corinne, E-mail: corinne.chadeneau@univ-poitiers.fr [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France)

    2014-11-28

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

  6. Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA

    OpenAIRE

    Orzalli, Megan H.; Conwell, Sara E.; Berrios, Christian; DeCaprio, James A.; Knipe, David M.

    2013-01-01

    Cells have evolved mechanisms to silence foreign DNA to prevent the expression of foreign genes within them. In mammalian cells, this involves the assembly of heterochromatin on foreign DNAs such as viral or transfected DNA. Herpesviruses have evolved strategies to counteract these host mechanisms to express their own genes. Herein we demonstrate that the nuclear DNA sensor IFN-inducible protein 16 (IFI16) is involved in the host silencing response to foreign DNA. IFI16 promotes the assembly ...

  7. Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly.

    Science.gov (United States)

    Chaffee, Blake R; Shang, Fu; Chang, Min-Lee; Clement, Tracy M; Eddy, Edward M; Wagner, Brad D; Nakahara, Masaki; Nagata, Shigekazu; Robinson, Michael L; Taylor, Allen

    2014-09-01

    Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIβ (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation.

  8. Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage

    OpenAIRE

    Rolletschek Alexandra; Solozobova Valeriya; Blattner Christine

    2009-01-01

    Abstract Background P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. Results In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells...

  9. Active Degradation Explains the Distribution of Nuclear Proteins during Cellular Senescence.

    Directory of Open Access Journals (Sweden)

    Enrico Giampieri

    Full Text Available The amount of cellular proteins is a crucial parameter that is known to vary between cells as a function of the replicative passages, and can be important during physiological aging. The process of protein degradation is known to be performed by a series of enzymatic reactions, ranging from an initial step of protein ubiquitination to their final fragmentation by the proteasome. In this paper we propose a stochastic dynamical model of nuclear proteins concentration resulting from a balance between a constant production of proteins and their degradation by a cooperative enzymatic reaction. The predictions of this model are compared with experimental data obtained by fluorescence measurements of the amount of nuclear proteins in murine tail fibroblast (MTF undergoing cellular senescence. Our model provides a three-parameter stationary distribution that is in good agreement with the experimental data even during the transition to the senescent state, where the nuclear protein concentration changes abruptly. The estimation of three parameters (cooperativity, saturation threshold, and maximal velocity of the reaction, and their evolution during replicative passages shows that only the maximal velocity varies significantly. Based on our modeling we speculate the reduction of functionality of the protein degradation mechanism as a possible competitive inhibition of the proteasome.

  10. Proneural proteins Achaete and Scute associate with nuclear actin to promote formation of external sensory organs.

    Science.gov (United States)

    Hsiao, Yun-Ling; Chen, Yu-Ju; Chang, Yi-Jie; Yeh, Hsiao-Fong; Huang, Yi-Chun; Pi, Haiwei

    2014-01-01

    Basic helix-loop-helix (bHLH) proneural proteins promote neurogenesis through transcriptional regulation. Although much is known about the tissue-specific regulation of proneural gene expression, how proneural proteins interact with transcriptional machinery to activate downstream target genes is less clear. Drosophila proneural proteins Achaete (Ac) and Scute (Sc) induce external sensory organ formation by activating neural precursor gene expression. Through co-immunoprecipitation and mass spectrometric analyses, we found that nuclear but not cytoplasmic actin associated with the Ac and Sc proteins in Drosophila S2 cells. Daughterless (Da), the common heterodimeric partner of Drosophila bHLH proteins, was observed to associate with nuclear actin through proneural proteins. A yeast two-hybrid assay revealed that the binding specificity between actin and Ac or Sc was conserved in yeast nuclei without the presence of additional Drosophila factors. We further show that actin is required in external sensory organ formation. Reduction in actin gene activity impaired proneural-protein-dependent expression of the neural precursor genes, as well as formation of neural precursors. Furthermore, increased nuclear actin levels, obtained by expression of nucleus-localized actin, elevated Ac-Da-dependent gene transcription as well as Ac-mediated external sensory organ formation. Taken together, our in vivo and in vitro observations suggest a novel link for actin in proneural-protein-mediated transcriptional activation and neural precursor differentiation.

  11. Prm3p Is a Pheromone-induced Peripheral Nuclear Envelope Protein Required for Yeast Nuclear Fusion

    OpenAIRE

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

    2009-01-01

    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 pheromon...

  12. Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair

    International Nuclear Information System (INIS)

    The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 430C and 450C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 450C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells were incubated at 370C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 410C a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks. (author)

  13. Ozone induces a proinflammatory response in primary human bronchial epithelial cells through mitogen-activated protein kinase activation without nuclear factor-κB activation.

    Science.gov (United States)

    McCullough, Shaun D; Duncan, Kelly E; Swanton, Samantha M; Dailey, Lisa A; Diaz-Sanchez, David; Devlin, Robert B

    2014-09-01

    Ground-level ozone (O3) is a ubiquitous environmental air pollutant that is a potent inducer of airway inflammation and has been linked with respiratory and cardiovascular morbidity and mortality. Some studies using transformed or immortalized cells have attributed O3-mediated expression of inflammatory cytokines with activation of the canonical NF-κB pathway. In this study, we sought to characterize the O3-mediated activation of cellular signaling pathways using primary human bronchial epithelial cells obtained from a panel of donors. We demonstrate that the O3-induced expression of proinflammatory cytokines requires the activation of the epidermal growth factor receptor/MEK/ERK and MKK4/p38 mitogen-activated signaling pathways but does not appear to involve activation of canonical NF-κB signaling. In addition to providing a novel mechanistic model for the O3-mediated induction of proinflammatory cytokines, these findings highlight the importance of using primary cells over cell lines in mechanistic studies.

  14. Ozone Induces a Proinflammatory Response in Primary Human Bronchial Epithelial Cells Through Mitogen-Activated Protein Kinase Activation Without Nuclear Factor-kB Activation

    Science.gov (United States)

    Ground-level ozone (O3) is a ubiquitous environmental air pollutant that is a potent inducer of airway inflammation and has been linked with both respiratory and cardiovascular morbidity and mortality. Some studies using transformed or immortalized cells have attributed O3-medi...

  15. Detecting protein-protein interactions in living cells

    DEFF Research Database (Denmark)

    Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche;

    2009-01-01

    to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C...

  16. The nuclear architectural protein HMGA1a triggers receptor-mediated endocytosis.

    Science.gov (United States)

    Wu, Wuwei; Wan, Wei; Li, Alexander D Q

    2009-11-01

    High mobility group proteins A (HMGA), nuclear architectural factors, locate in the cell nuclei and mostly execute gene-regulation function. However, our results reveal that a HMGA member (HMGA1a) has a unique plasma membrane receptor; this receptor specifically binds to HMGA-decorated species, effectively mediates endocytosis, and internalizes extracellular HMGA-functionalized cargoes. Indeed, dyes or nanoparticles labeled with HMGA1a protein readily enter Hela cells. Using a stratagem chemical cross-linker, we covalently bonded the HMGA receptor to the HMGA1a-GFP fusion protein, thus capturing the plasma membrane receptor. Subsequent Western blots and SDS-PAGE gel revealed that the HMGA receptor is a 26-kDa protein. Confocal live-cell microscopic imaging was used to monitor the whole endocytic process, in which the internalized HMGA1a-decorated species are transported by motor proteins on microtubules and eventually arrive at the late endosomes/lysosomes. Cell viability assays also suggested that extracellular HMGA1a protein directly influences the survival ability of Hela cells in a dose-dependent manner, implying versatility of HMGA1a protein and its potent role to suppress cancer cell survivability and to regulate growth. PMID:19739099

  17. Bacterial cell division proteins as antibiotic targets

    NARCIS (Netherlands)

    T. den Blaauwen; J.M. Andreu; O. Monasterio

    2014-01-01

    Proteins involved in bacterial cell division often do not have a counterpart in eukaryotic cells and they are essential for the survival of the bacteria. The genetic accessibility of many bacterial species in combination with the Green Fluorescence Protein revolution to study localization of protein

  18. Identification of a functional nuclear export signal in the green fluorescent protein asFP499

    International Nuclear Information System (INIS)

    The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified 194LRMEKLNI201 as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is First report of a GFP that contains a functional NES

  19. The tight junction protein Z O-2 has several functional nuclear export signals

    International Nuclear Information System (INIS)

    The tight junction (TJ) protein ZO-2 changes its subcellular distribution according to the state of confluency of the culture. Thus in confluent monolayers, it localizes at the TJ region whereas in sparse cultures it concentrates at the nucleus. The canine sequence of ZO-2 displays four putative nuclear export signals (NES), two at the second PDZ domain (NES-0 and NES-1) and the rest at the GK region (NES-2 and NES-3). The functionality of NES-0 and NES-3 was unknown, hence here we have explored it with a nuclear export assay, injecting into the nucleus of MDCK cells peptides corresponding to the ZO-2 NES sequences chemically coupled to ovalbumin. We show that both NES-0 and NES-3 are functional and sensitive to leptomycin B. We also demonstrate that NES-1, previously characterized as a non functional NES, is rendered capable of nuclear export upon the acquisition of a negative charge at its Ser369 residue. Experiments performed injecting at the nucleus WT and mutated ZO-2-GST fusion proteins revealed the need of both NES-0 and NES-1, and NES-2 and NES-3 for attaining an efficient nuclear exit of the respective amino and middle segments of ZO-2. Moreover, the transfection of MDCK cells with full-length ZO-2 revealed that the mutation of any of the NES present in the molecule was sufficient to induce nuclear accumulation of the protein

  20. Nuclear localization signal in a cancer-related transcriptional regulator protein NAC1.

    Science.gov (United States)

    Okazaki, Kosuke; Nakayama, Naomi; Nariai, Yuko; Nakayama, Kentaro; Miyazaki, Kohji; Maruyama, Riruke; Kato, Hiroaki; Kosugi, Shunichi; Urano, Takeshi; Sakashita, Gyosuke

    2012-10-01

    Nucleus accumbens-associated protein 1 (NAC1) might have potential oncogenic properties and participate in regulatory networks for pluripotency. Although NAC1 is described as a transcriptional regulator, the nuclear import machinery of NAC1 remains unclear. We found, using a point mutant, that dimer formation was not committed to the nuclear localization of NAC1 and, using deletion mutants, that the amino-terminal half of NAC1 harbored a potential nuclear localization signal (NLS). Wild type, but not mutants of this region, alone was sufficient to drive the importation of green fluorescent protein (GFP) into the nucleus. Bimax1, a synthetic peptide that blocks the importin α/β pathway, impaired nuclear localization of NAC1 in cells. We also used the binding properties of importin to demonstrate that this region is an NLS. Furthermore, the transcriptional regulator function of NAC1 was dependent on its nuclear localization activity in cells. Taken together, these results show that the region with a bipartite motif constitutes a functional nuclear import sequence in NAC1 that is independent of NAC1 dimer formation. The identification of an NAC1 NLS thus clarifies the mechanism through which NAC1 translocates to the nucleus to regulate the transcription of genes involved in oncogenicity and pluripotency.

  1. Nuclear actions of insulin-like growth factor binding protein-3.

    Science.gov (United States)

    Baxter, Robert C

    2015-09-10

    In addition to its actions outside the cell, cellular uptake and nuclear import of insulin-like growth factor binding protein-3 (IGFBP-3) has been recognized for almost two decades, but knowledge of its nuclear actions has been slow to emerge. IGFBP-3 has a functional nuclear localization signal and interacts with the nuclear transport protein importin-β. Within the nucleus IGFBP-3 appears to have a role in transcriptional regulation. It can bind to the nuclear receptor, retinoid X receptor-α and several of its dimerization partners, including retinoic acid receptor, vitamin D receptor (VDR), and peroxisome proliferator-activated receptor-γ (PPARγ). These interactions modulate the functions of these receptors, for example inhibiting VDR-dependent transcription in osteoblasts and PPARγ-dependent transcription in adipocytes. Nuclear IGFBP-3 can be detected by immunohistochemistry in cancer and other tissues, and its presence in the nucleus has been shown in many cell culture studies to be necessary for its pro-apoptotic effect, which may also involve interaction with the nuclear receptor Nur77, and export from the nucleus. IGFBP-3 is p53-inducible and in response to DNA damage, forms a complex with the epidermal growth factor receptor (EGFR), translocating to the nucleus to interact with DNA-dependent protein kinase. Inhibition of EGFR kinase activity or downregulation of IGFBP-3 can inhibit DNA double strand-break repair by nonhomologous end joining. IGFBP-3 thus has the ability to influence many cell functions through its interactions with intranuclear pathways, but the importance of these interactions in vivo, and their potential to be targeted for therapeutic benefit, require further investigation. PMID:26074086

  2. XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells.

    Science.gov (United States)

    Taylor-Kashton, Cheryl; Lichtensztejn, Daniel; Baloglu, Erkan; Senapedis, William; Shacham, Sharon; Kauffman, Michael G; Kotb, Rami; Mai, Sabine

    2016-12-01

    Previous work has shown that the three-dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT-185, KPT-330/selinexor, and KPT-8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment-naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non-lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711-2719, 2016. © 2016 Wiley Periodicals, Inc. PMID:26991404

  3. An improved genetic system for detection and analysis of protein nuclear import signals

    Directory of Open Access Journals (Sweden)

    Derbyshire Stephanie

    2007-01-01

    Full Text Available Abstract Background Nuclear import of proteins is typically mediated by their physical interaction with soluble cytosolic receptor proteins via a nuclear localization signal (NLS. A simple genetic assay to detect active NLSs based on their function in the yeast Saccharomyces cerevisiae has been previously described. In that system, a chimera consisting of a modified bacterial LexA DNA binding domain and the transcriptional activation domain of the yeast Gal4 protein is fused to a candidate NLS. A functional NLS will redirect the chimeric fusion to the yeast cell nucleus and activate transcription of a reporter gene. Results We have reengineered this nuclear import system to expand its utility and tested it using known NLS sequences from adenovirus E1A. Firstly, the vector has been reconstructed to reduce the level of chimera expression. Secondly, an irrelevant "stuffer" sequence from the E. coli maltose binding protein was used to increase the size of the chimera above the passive diffusion limit of the nuclear pore complex. The improved vector also contains an expanded multiple cloning site and a hemagglutinin epitope tag to allow confirmation of expression. Conclusion The alterations in expression level and composition of the fusions used in this nuclear import system greatly reduce background activity in β-galactosidase assays, improving sensitivity and allowing more quantitative analysis of NLS bearing sequences.

  4. Sumoylation of Human Translationally Controlled Tumor Protein Is Important for Its Nuclear Transport

    Directory of Open Access Journals (Sweden)

    Gnanasekar Munirathinam

    2012-01-01

    Full Text Available Translationally controlled tumor protein (TCTP lacks nuclear bipartite localization signal sequence; yet TCTP is present abundantly in the nucleus. At present it is not known how TCTP gets transported to the nucleus. Sequence analyses showed that all TCTPs described to date have putative small ubiquitin-like modifier (SUMO motifs. Since SUMO modification plays an important role in the nuclear transport of proteins, we evaluated whether SUMO motifs are important for transport of TCTP into the nucleus. We show that TCTP exists in sumoylated form in cytoplasm and nucleus of mammalian cells. Point mutation of lysine residue in the SUMO motif compromised the ability of TCTP to get sumoylated in vitro. When cells were transfected with FLAG-tagged mutated TCTP, nuclear transport of TCTP was inhibited confirming that sumoylation is critical for the nuclear transport of TCTP. Our previous studies demonstrated that TCTP can function as an antioxidant protein in the nucleus. When we mutated TCTP at the SUMO motif the antioxidant function of TCTP was compromised. Results presented in this study thus show that sumoylation plays an important role in the transport of TCTP into the nucleus where they function as antioxidant protein.

  5. Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Pei-Hong Jiang; Yoshiharu Motoo; Stéphane Garcia; Juan Lucio Iovanna; Marie-Josèphe Pébusque; Norio Sawabu

    2006-01-01

    AIM: Overexpression of tumor protein p53-induced nuclear protein 1 (TP53INP1) induces G1 cell cycle arrest and increases p53-mediated apoptosis. To clarify the clinical importance of TP53INP1, we analyzed TP53INP1and p53 expression in gastric cancer.METHODS: TP53INP1 and p53 expression were examined using immunohistochemistry in 142 cases of gastric cancer. The apoptosis of gastric cancer cells was analyzed using the TUNEL method. The relationship between the expression of TP53INP1 and clinicopathological factors was statistically analyzed.RESULTS: TP53INP1 was expressed in 98% (139/142cases) of non-cancerous gastric tissues and was downexpressed in 64% (91/142 cases) of gastric cancer lesions from the same patients. TP53INP1 expression was significantly decreased (43.9%) in poorly differentiated adenocarcinoma compared with well or moderately differentiated adenocarcinoma (81.6%).Cancers invading the submucosa or deeper showed lower positively (59.1%) compared with mucosal cancers (85.2%). Decrease or loss of TP53INP1 expression was significantly correlated with lymphatic invasion (54.3%vs 82.0% without lymphatic invasion) and node-positive patients (31.3% vs 68.3% in node-negative patients).P53 was expressed in 68 (47.9%) patients of gastric cancer, whereas it was absent in normal gastric tissues.A significant association was also observed between TP53INP1 status and the level of apoptosis in tumor cells: the apoptotic index in TP53INP1-positive tissues was significantly higher than that in TP53INP1-negative portions. Finally, when survival data were analyzed,loss of TP53INP1 expression had a significant effect in predicting a poor prognosis (P= 0.0006).CONCLUSION: TP53INP1-positive rate decreases with the progression of gastric cancer. TP53INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TP53INP1is related to the apoptosis of gastric cancer cells. The decreased expression of the TP53INP1 protein may

  6. Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains.

    Science.gov (United States)

    Boyer, Patrick D; Ganesh, Sairaam; Qin, Zhao; Holt, Brian D; Buehler, Markus J; Islam, Mohammad F; Dahl, Kris Noel

    2016-02-10

    Single-walled carbon nanotubes (SWCNTs) have great potential for cell-based therapies due to their unique intrinsic optical and physical characteristics. Consequently, broad classes of dispersants have been identified that individually suspend SWCNTs in water and cell media in addition to reducing nanotube toxicity to cells. Unambiguous control and verification of the localization and distribution of SWCNTs within cells, particularly to the nucleus, is needed to advance subcellular technologies utilizing nanotubes. Here we report delivery of SWCNTs to the nucleus by noncovalently attaching the tail domain of the nuclear protein lamin B1 (LB1), which we engineer from the full-length LMNB1 cDNA. More than half of this low molecular weight globular protein is intrinsically disordered but has an immunoglobulin-fold composed of a central hydrophobic core, which is highly suitable for associating with SWCNTs, stably suspending SWCNTs in water and cell media. In addition, LB1 has an exposed nuclear localization sequence to promote active nuclear import of SWCNTs. These SWCNTs-LB1 dispersions in water and cell media display near-infrared (NIR) absorption spectra with sharp van Hove peaks and an NIR fluorescence spectra, suggesting that LB1 individually disperses nanotubes. The dispersing capability of SWCNTs by LB1 is similar to that by albumin proteins. The SWCNTs-LB1 dispersions with concentrations ≥150 μg/mL (≥30 μg/mL) in water (cell media) remain stable for ≥75 days (≥3 days) at 4 °C (37 °C). Further, molecular dynamics modeling of association of LB1 with SWCNTs reveal that the exposure of the nuclear localization sequence is independent of LB1 binding conformation. Measurements from confocal Raman spectroscopy and microscopy, NIR fluorescence imaging of SWCNTs, and fluorescence lifetime imaging microscopy show that millions of these SWCNTs-LB1 complexes enter HeLa cells, localize to the nucleus of cells, and interact with DNA. We postulate that the

  7. Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains.

    Science.gov (United States)

    Boyer, Patrick D; Ganesh, Sairaam; Qin, Zhao; Holt, Brian D; Buehler, Markus J; Islam, Mohammad F; Dahl, Kris Noel

    2016-02-10

    Single-walled carbon nanotubes (SWCNTs) have great potential for cell-based therapies due to their unique intrinsic optical and physical characteristics. Consequently, broad classes of dispersants have been identified that individually suspend SWCNTs in water and cell media in addition to reducing nanotube toxicity to cells. Unambiguous control and verification of the localization and distribution of SWCNTs within cells, particularly to the nucleus, is needed to advance subcellular technologies utilizing nanotubes. Here we report delivery of SWCNTs to the nucleus by noncovalently attaching the tail domain of the nuclear protein lamin B1 (LB1), which we engineer from the full-length LMNB1 cDNA. More than half of this low molecular weight globular protein is intrinsically disordered but has an immunoglobulin-fold composed of a central hydrophobic core, which is highly suitable for associating with SWCNTs, stably suspending SWCNTs in water and cell media. In addition, LB1 has an exposed nuclear localization sequence to promote active nuclear import of SWCNTs. These SWCNTs-LB1 dispersions in water and cell media display near-infrared (NIR) absorption spectra with sharp van Hove peaks and an NIR fluorescence spectra, suggesting that LB1 individually disperses nanotubes. The dispersing capability of SWCNTs by LB1 is similar to that by albumin proteins. The SWCNTs-LB1 dispersions with concentrations ≥150 μg/mL (≥30 μg/mL) in water (cell media) remain stable for ≥75 days (≥3 days) at 4 °C (37 °C). Further, molecular dynamics modeling of association of LB1 with SWCNTs reveal that the exposure of the nuclear localization sequence is independent of LB1 binding conformation. Measurements from confocal Raman spectroscopy and microscopy, NIR fluorescence imaging of SWCNTs, and fluorescence lifetime imaging microscopy show that millions of these SWCNTs-LB1 complexes enter HeLa cells, localize to the nucleus of cells, and interact with DNA. We postulate that the

  8. The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein.

    OpenAIRE

    Michael, W M; Eder, P S; Dreyfuss, G

    1997-01-01

    Protein import into the nucleus and export from the nucleus are signal-mediated processes that require energy. The nuclear transport process about which the most information is currently available is classical nuclear localization signal (NLS)-mediated nuclear import. However, details concerning the signal-mediated export of proteins and RNAs as well as alternative nuclear import pathways are beginning to emerge. An example of this is the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 pro...

  9. Regulation of Nuclear Localization of Signaling Proteins by Cytokinin

    Energy Technology Data Exchange (ETDEWEB)

    Kieber, J.J.

    2010-05-01

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

  10. Versatile protein tagging in cells with split fluorescent protein

    OpenAIRE

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A.; Ishikawa, Hiroaki; Leonetti, Manuel D.; Marshall, Wallace F.; Jonathan S Weissman; Huang, Bo

    2016-01-01

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respec...

  11. Kinesin-like proteins are involved in postmitotic nuclear migration of the unicellular green alga Micrasterias denticulata.

    Science.gov (United States)

    Holzinger, Andreas; Lütz-Meindl, Ursula

    2002-01-01

    The unicellular green alga Micrasterias denticulata performs a two-directional postmitotic nuclear migration during development, a passive migration into the growing semicell, and a microtubule mediated backward migration towards the cell centre. The present study provides first evidence for force generation by motor proteins of the kinesin family in this process. The new kinesin specific inhibitor adociasulfate-2 causes abnormal nuclear displacement at 18 microM. AMP-PNP, a non hydrolyseable ATP analogue or the general ATPase inhibitors calyculin A and sodium orthovanadate also disturb nuclear migration. In addition kinesin-like proteins are detected by means of immunoblotting using antibodies against brain kinesin, plant derived antibodies to kinesin-like proteins and a calmodulin binding kinesin-like protein. Immunoelectron microscopy suggests a correlation of conventional kinesin-like proteins, but not of the calmodulin binding kinesin-like protein to the microtubule apparatus associated with the migrating nucleus. PMID:12175672

  12. GAGE cancer-germline antigens are recruited to the nuclear envelope by germ cell-less (GCL)

    DEFF Research Database (Denmark)

    Gjerstorff, Morten F; Rösner, Heike I; Pedersen, Christina B;

    2012-01-01

    metazoan transcriptional regulator, Germ cell-less (GCL), as an interaction partner of GAGE12I. GCL directly binds LEM-domain proteins (LAP2β, emerin, MAN1) at the nuclear envelope, and we found that GAGE proteins were recruited to the nuclear envelope inner membrane by GCL. Based on yeast two...

  13. Distinct effects of nuclear membrane localization on gene transcription silencing in Drosophila S2 cells and germ cells

    Institute of Scientific and Technical Information of China (English)

    Lu Sui; Yanhong Yang

    2011-01-01

    Nuclear envelope proteins have important roles in chromatin organization and signal-dependent transcriptional regulation. A previous study reported that the inner nuclear membrane protein, Otefin (Ote), was essential for germline stem cell (GSC) maintenance via interaction with Smad complex. The interaction of Otc with the Smad complex recruits the bam locus to the nuclear periphery and subsequently results in bam transcriptional silencing, revealing that nuclear peripheral localization is essential for bam gene regulation. However, it remains unknown whether the nuclear peripheral localization is sufficient for bam silencing. To address this issue, we have established a tethering system, in which the Gal4 DNA binding domain (DBD) of the Flag:Gal4 DBD:Ote △ LEM fusion protein physically interacts with the Gal4 binding sites upstream of bamP-gfp to artificially recruit the reporter gene gfp to the nuclear membrane. Our data demonstrated that the nuclear peripheral localization seemed to affect the expression of the target naked gene in S2 cells. By contrast, in Drosophila germ cells, the nuclear membrane localization was not sufficient for gene silencing.

  14. The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain

    International Nuclear Information System (INIS)

    The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter-enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization

  15. Nuclear vasohibin-2 promotes cell proliferation by inducing G0/G1 to S phase progression.

    Science.gov (United States)

    Ge, Qianqian; Zhou, Jia; Tu, Min; Xue, Xiaofeng; Li, Zhanjun; Lu, Zipeng; Wei, Jishu; Song, Guoxin; Chen, Jianmin; Guo, Feng; Jiang, Kuirong; Miao, Yi; Gao, Wentao

    2015-09-01

    As a member of the vasohibin (VASH2) family, VASH2 is localized intracellularly as a nuclear and cytoplasmic type. Cytoplasmic VASH2 is associated with carcinoma angiogenesis and malignant transformation and promotes cancer growth. However, the function of nuclear VASH2 has yet to be investigated. The aim of the present study was to detect the nuclear VASH2 expression profile in human organs and tissues by protein microarray technique. To examine the function of nuclear VASH2, we analyzed the relationship between nuclear VASH2 and Ki-67, and stably constructed VASH2 overexpression and knockdown in LO2 and HepG2 cell lines, based on a previous study in hepatic cells. The study was conducted using bromodeoxyuridine, immunofluorescent staining, western blot analysis and flow cytometry. Nuclear VASH2 was highly expressed in actively dividing cells in normal and cancer tissues. There was a significant positive correlation between nuclear VASH2 and Ki-67, indicating that nuclear VASH2 positively correlated with cell proliferation in normal and cancer tissues. The bromodeoxyuridine (BrdU) proliferation test showed that nuclear VASH2 increased the S-phase population and promoted cell proliferation, while VASH2 knockdown reduced BrdU absorbance. Cell cycle analysis revealed that nuclear VASH2 overexpression increased the S-phase population in LO2 and HepG2 cells, while nuclear VASH2 knockdown reduced the S-phase population and increased the G0/G1 population. The findings of this study challenge the classic view of VASH2, which was previously reported as an angiogenesis factor. Furthermore, to the best of our knowledge, these results are the first clinical data indicating that nuclear VASH2, but not cytoplasmic VASH2, promotes cell proliferation by driving the cell cycle from the G0/G1 to S phase.

  16. Nuclear microscopy of rat colon epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Ren, M., E-mail: phyrenmq@nus.edu.sg [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Rajendran, Reshmi [Lab of Molecular Imaging, Singapore Bioimaging Consotium, 11 Biopolis Way, 02-02 Helios, Singapore 138667 (Singapore); Ng, Mary [Department of Pharmacology, National University of Singapore (Singapore); Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Jenner, Andrew Michael [Illawara Health and Medical Research Institute (IHMRI), University of Wollongong, NSW 2522 (Australia)

    2011-10-15

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  17. The Inner Nuclear Membrane Protein Nemp1 Is a New Type of RanGTP-Binding Protein in Eukaryotes.

    Directory of Open Access Journals (Sweden)

    Takashi Shibano

    Full Text Available The inner nuclear membrane (INM protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein.

  18. The Inner Nuclear Membrane Protein Nemp1 Is a New Type of RanGTP-Binding Protein in Eukaryotes.

    Science.gov (United States)

    Shibano, Takashi; Mamada, Hiroshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Taira, Masanori

    2015-01-01

    The inner nuclear membrane (INM) protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs) and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein.

  19. Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ostlund, Cecilia [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Guan, Tinglu [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Figlewicz, Denise A. [Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (United States); Hays, Arthur P. [Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Worman, Howard J. [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Gerace, Larry [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Schirmer, Eric C., E-mail: e.schirmer@ed.ac.uk [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR (United Kingdom)

    2009-11-13

    Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

  20. STUDY ON NUCLEAR MATRIX PROTEINS FROM HUMAN BREAST CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    HE Qian; ZHANG Shu-qun; CHU Yong-lie; JIA Xiao-li; JIANG Jian-tao

    2009-01-01

    Objective To investigate the marker protein of human breast carcinoma from nuclear matrix proteins (NMPs).Methods NMPs were injected subcutaneously into rabbit to get antiserum, which was used to detect the NMPs specificity for breast carcinoma.Results There was an apparent positive band (100kD) in the NMPs of breast carcinoma, which did not exist in normal breast and other tumors that were detected.Conclusion One or one group of 100kD NMPs were found to be related to human breast carcinoma, which may be involved in the carcinogenesis and development of human breast carcinoma and valuable for breast carcinoma diagnosis.

  1. CCAAT/Enhancer-binding Protein α (C/EBPα) and Hepatocyte Nuclear Factor 4α (HNF4α) Synergistically Cooperate with Constitutive Androstane Receptor to Transactivate the Human Cytochrome P450 2B6 (CYP2B6) Gene: APPLICATION TO THE DEVELOPMENT OF A METABOLICALLY COMPETENT HUMAN HEPATIC CELL MODEL*

    OpenAIRE

    Benet, Marta; Lahoz, Agustín; Guzmán, Carla; Castell, José V; Jover, Ramiro

    2010-01-01

    The transcription of tissue-specific and inducible genes is usually subject to the dynamic control of multiple activators. Dedifferentiated hepatic cell lines lose the expression of tissue-specific activators and many characteristic hepatic genes, such as drug-metabolizing cytochrome P450. Here we demonstrate that by combining adenoviral vectors for CCAAT/enhancer-binding protein α (C/EBPα), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor, the CYP2B6 expression and ...

  2. Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

    International Nuclear Information System (INIS)

    The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requires permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells

  3. Characterization of a family of novel cysteine- serine-rich nuclear proteins (CSRNP.

    Directory of Open Access Journals (Sweden)

    Sébastien Gingras

    Full Text Available Gene array analysis has been widely used to identify genes induced during T cell activation. Our studies identified an immediate early gene that is strongly induced in response to IL-2 in mouse T cells which we named cysteine- serine-rich nuclear protein-1 (CSRNP-1. The human ortholog was previously identified as an AXIN1 induced gene (AXUD1. The protein does not contain sequence defined domains or motifs annotated in public databases, however the gene is a member of a family of three mammalian genes that share conserved regions, including cysteine- and serine-rich regions and a basic domain, they encode nuclear proteins, possess transcriptional activation domain and bind the sequence AGAGTG. Consequently we propose the nomenclature of CSRNP-1, -2 and -3 for the family. To elucidate the physiological functions of CSRNP-1, -2 and -3, we generated mice deficient for each of these genes by homologous recombination in embryonic stem cells. Although the CSRNP proteins have the hallmark of transcription factors and CSRNP-1 expression is highly induced by IL-2, deletion of the individual genes had no obvious consequences on normal mouse development, hematopoiesis or T cell functions. However, combined deficiencies cause partial neonatal lethality suggesting that the genes have redundant functions.

  4. Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage

    Directory of Open Access Journals (Sweden)

    Rolletschek Alexandra

    2009-06-01

    Full Text Available Abstract Background P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. Results In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. Conclusion In embryonic stem cells where (anti-proliferative p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

  5. Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein–protein interfaces

    Energy Technology Data Exchange (ETDEWEB)

    Wylie, Benjamin J. [Columbia University, Department of Chemistry (United States); Dzikovski, Boris G. [Cornell University, National Biomedical Center for Advanced ESR Technology, Department of Chemistry and Chemical Biology (United States); Pawsey, Shane; Caporini, Marc; Rosay, Melanie [Bruker BioSpin Corporation (United States); Freed, Jack H. [Cornell University, National Biomedical Center for Advanced ESR Technology, Department of Chemistry and Chemical Biology (United States); McDermott, Ann E., E-mail: aem5@columbia.edu [Columbia University, Department of Chemistry (United States)

    2015-04-15

    We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.

  6. Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells.

    Directory of Open Access Journals (Sweden)

    Sarita Raghunayakula

    Full Text Available Annulate lamellae are cytoplasmic organelles containing stacked sheets of membranes embedded with pore complexes. These cytoplasmic pore complexes at annulate lamellae are morphologically similar to nuclear pore complexes at the nuclear envelope. Although annulate lamellae has been observed in nearly all types of cells, their biological functions are still largely unknown. Here we show that SUMO1-modification of the Ran GTPase-activating protein RanGAP1 not only target RanGAP1 to its known sites at nuclear pore complexes but also to annulate lamellae pore complexes through interactions with the Ran-binding protein RanBP2 and the SUMO-conjugating enzyme Ubc9 in mammalian cells. Furthermore, upregulation of annulate lamellae, which decreases the number of nuclear pore complexes and concurrently increases that of annulate lamellae pore complexes, causes a redistribution of nuclear transport receptors including importin α/β and the exportin CRM1 from nuclear pore complexes to annulate lamellae pore complexes and also reduces the rates of nuclear import and export. Moreover, our results reveal that importin α/β-mediated import complexes initially accumulate at annulate lamellae pore complexes upon the activation of nuclear import and subsequently disassociate for nuclear import through nuclear pore complexes in cells with upregulation of annulate lamellae. Lastly, CRM1-mediated export complexes are concentrated at both nuclear pore complexes and annulate lamellae pore complexes when the disassembly of these export complexes is inhibited by transient expression of a Ran GTPase mutant arrested in its GTP-bound form, suggesting that RanGAP1/RanBP2-activated RanGTP hydrolysis at these pore complexes is required for the dissociation of the export complexes. Hence, our findings provide a foundation for further investigation of how upregulation of annulate lamellae decreases the rates of nuclear transport and also for elucidation of the biological

  7. A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor.

    Science.gov (United States)

    Ibarra-Coronado, Elizabeth Guadalupe; Escobedo, Galileo; Nava-Castro, Karen; Jesús Ramses, Chávez-Rios; Hernández-Bello, Romel; García-Varela, Martìn; Ambrosio, Javier R; Reynoso-Ducoing, Olivia; Fonseca-Liñán, Rocío; Ortega-Pierres, Guadalupe; Pavón, Lenin; Hernández, María Eugenia; Morales-Montor, Jorge

    2011-01-01

    The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is described in the estradiol-dependent reproduction of the helminth parasite Taenia crassiceps. Previous results have shown that 17-β-estradiol induces a concentration-dependent increase in bud number of in vitro cultured cysticerci. This effect is inhibited when parasites are also incubated in the presence of an ER binding-inhibitor (tamoxifen). RT-PCR assays using specific oligonucleotides of the most conserved ER sequences, showed expression by the parasite of a mRNA band of molecular weight and sequence corresponding to an ER. Western blot assays revealed reactivity with a 66 kDa protein corresponding to the parasite ER protein. Tamoxifen treatment strongly reduced the production of the T. crassiceps ER-like protein. Antibody specificity was demonstrated by immunoprecipitating the total parasite protein extract with anti-ER-antibodies. Cross-contamination by host cells was discarded by flow cytometry analysis. ER was specifically detected on cells expressing paramyosin, a specific helminth cell marker. Parasite cells expressing the ER-like protein were located by confocal microscopy in the subtegumental tissue exclusively. Analysis of the ER-like protein by bidimensional electrophoresis and immunoblot identified a specific protein of molecular weight and isoelectric point similar to a vertebrates ER. Sequencing of the spot produced a small fragment of protein similar to the mammalian nuclear ER. Together these results show that T. crassiceps expresses an ER-like protein which activates the budding of T. crassiceps cysticerci in vitro. To the best of our knowledge, this is the first report of an ER-like protein in parasites. This finding may have strong implications in the fields of host-parasite co-evolution as well as in sex-associated susceptibility to this infection, and could be an important target for the design of new drugs.

  8. Critical Role for the Protons in FRTL-5 Thyroid Cells: Nuclear Sphingomyelinase Induced-Damage

    Directory of Open Access Journals (Sweden)

    Elisabetta Albi

    2014-06-01

    Full Text Available Proliferating thyroid cells are more sensitive to UV-C radiations than quiescent cells. The effect is mediated by nuclear phosphatidylcholine and sphingomyelin metabolism. It was demonstrated that proton beams arrest cell growth and stimulate apoptosis but until now there have been no indications in the literature about their possible mechanism of action. Here we studied the effect of protons on FRTL-5 cells in culture. We showed that proton beams stimulate slightly nuclear neutral sphingomyelinase activity and inhibit nuclear sphingomyelin-synthase activity in quiescent cells whereas stimulate strongly nuclear neutral sphingomyelinase activity and do not change nuclear sphingomyelin-synthase activity in proliferating cells. The study of neutral sphingomyelinase/sphingomyelin-synthase ratio, a marker of functional state of the cells, indicated that proton beams induce FRTL-5 cells in a proapoptotic state if the cells are quiescent and in an initial apoptotic state if the cells are proliferating. The changes of cell life are accompanied by a decrease of nuclear sphingomyelin and increase of bax protein.

  9. Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane.

    Science.gov (United States)

    Vijayaraghavan, Balaje; Jafferali, Mohammed Hakim; Figueroa, Ricardo A; Hallberg, Einar

    2016-07-01

    Samp1 is a transmembrane protein of the inner nuclear membrane (INM), which interacts with the nuclear lamina and the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in interphase and during mitosis, it localizes to the mitotic spindle. Samp1 was recently found to coprecipitate a protein complex containing Ran, a GTPase with fundamental regulatory functions both in interphase and in mitosis. To investigate the interaction between Samp1 and Ran in further detail, we have designed and expressed recombinant fusion proteins of the Chaetomium thermophilum homolog of Samp1 (Ct.Samp1) and human Ran. Pulldown experiments show that Samp1 binds directly to Ran and that Samp1 binds better to RanGTP compared to RanGDP. Samp1 also preferred RanGTP over RanGDP in living tsBN2 cells. We also show that the Ran binding domain is located between amino acids 75-135 in the nucleoplasmically exposed N-terminal tail of Samp1. This domain is unique for Samp1, without homology in any other proteins in fungi or metazoa. Samp1 is the first known transmembrane protein that binds to Ran and could provide a unique local binding site for RanGTP in the INM. Samp1 overexpression resulted in increased Ran concentrations in the nuclear periphery supporting this idea. PMID:27541860

  10. MitoDrome: a database of Drosophila melanogaster nuclear genes encoding proteins targeted to the mitochondrion

    Science.gov (United States)

    Sardiello, Marco; Licciulli, Flavio; Catalano, Domenico; Attimonelli, Marcella; Caggese, Corrado

    2003-01-01

    Mitochondria are organelles present in the cytoplasm of most eukaryotic cells; although they have their own DNA, the majority of the proteins necessary for a functional mitochondrion are coded by the nuclear DNA and only after transcription and translation they are imported in the mitochondrion as proteins. The primary role of the mitochondrion is electron transport and oxidative phosphorylation. Although it has been studied for a long time, the interest of researchers in mitochondria is still alive thanks to the discovery of mitochondrial role in apoptosis, aging and cancer. Aim of the MitoDrome database is to annotate the Drosophila melanogaster nuclear genes coding for mitochondrial proteins in order to contribute to the functional characterization of nuclear genes coding for mitochondrial proteins and to knowledge of gene diseases related to mitochondrial dysfunctions. Indeed D. melanogaster is one of the most studied organisms and a model for the Human genome. Data are derived from the comparison of Human mitochondrial proteins versus the Drosophila genome, ESTs and cDNA sequence data available in the FlyBase database. Links from the MitoDrome entries to the related homologous entries available in MitoNuC will be soon imple-mented. The MitoDrome database is available at http://bighost.area.ba.cnr.it/BIG/MitoDrome. Data are organised in a flat-file format and can be retrieved using the SRS system. PMID:12520013

  11. Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

    Energy Technology Data Exchange (ETDEWEB)

    Saad, Fawzy A. [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Torres, Marie [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Wang, Hao [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Graham, Lila, E-mail: lilagraham@cs.com [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

  12. FGFR2 protein expression in breast cancer: nuclear localisation and correlation with patient genotype

    Directory of Open Access Journals (Sweden)

    Thompson Alastair M

    2011-03-01

    Full Text Available Abstract Background Single Nucleotide Polymorphisms (SNPs in intron 2 of the Fibroblast Growth Factor Receptor Type 2 (FGFR2 gene, including rs2981582, contribute to multifactorial breast cancer susceptibility. The high risk polymorphism haplotype in the FGFR2 gene has been associated with increased mRNA transcription and altered transcription factor binding but the effect on FGFR2 protein expression is unknown. 40 breast tumours were identified from individuals with known rs2981582 genotype. Tumour sections were stained for FGFR2 protein expression, and scored for nuclear and cytoplasmic staining in tumour and surrounding normal tissue. Findings FGFR2 immunohistochemistry demonstrated variable nuclear staining in normal tissue and tumour tissue, as well as consistent cytoplasmic staining. We did not find an association between nuclear staining for FGFR2 and genotype, and there was no association between FGFR2 staining and estrogen or progestogen receptor status. There was an association between presence of nuclear staining for FGFR2 in normal tissue and presence of nuclear staining in the adjacent tumour (Fishers exact test, p = 0.002. Conclusions Variable nuclear staining for FGFR2 in breast cancer, but an absence of correlation with rs2981582 genotype suggests that the mechanism of action of polymorphisms at the FGFR2 locus may be more complex than a direct effect on mRNA expression levels in the final cancer. The effect may relate to FGFR2 function or localisation during breast development or tumourigenesis. Nuclear localisation of FGFR2 suggests an important additional role for this protein in breast development and breast cancer, in addition to its function as a classical cell surface receptor.

  13. Protein Kinase C-δ mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction

    International Nuclear Information System (INIS)

    We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta (ΔPKC-δ). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the ΔPKC-δ, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that ΔPKC-δ mediated the down-regulation of hnRNP K protein during apoptosis: PKC-δ inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-δ-deficient apoptotic KG1a cells; conditional induction of ΔPKC-δ in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of ΔPKC-δ. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-δ down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

  14. Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81

    Directory of Open Access Journals (Sweden)

    Petros Batsios

    2016-03-01

    Full Text Available The nuclear envelope (NE consists of the outer and inner nuclear membrane (INM, whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 are localized to the NE during the entire cell cycle. Immuno-electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src11–646, a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture.

  15. Thermodynamics of protein destabilization in live cells.

    Science.gov (United States)

    Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, François-Xavier; Bekei, Beata; Logan, Derek T; Selenko, Philipp; Wennerström, Håkan; Oliveberg, Mikael

    2015-10-01

    Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

  16. Discovering the World of Plant Nuclear Proteins (Chapter 2)

    OpenAIRE

    Petrovská, B.; Šebela, M.; Doležel, J. (Jaroslav)

    2016-01-01

    Despite the separation of evolutionary lineages many hundred million years ago,/ncells of all eukaryotic organisms are structurally similar. Their control centre – the/nnucleus – contains most of the DNA of the cell and regulates the majority of cellular/nprocesses. DNA is packed in a small volume of the nucleus after interacting with/nnuclear proteins. These proteins facilitate DNA folding into a small space; participate/nin DNA replication, repair and transcription; and help to separate it ...

  17. Functional dynamics of cell surface membrane proteins

    Science.gov (United States)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

  18. Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number.

    Science.gov (United States)

    Myre, Michael A; O'Day, Danton H

    2004-09-17

    Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin-binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins. Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells. We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait. Here we show that nucleomorphin interacts with calcium-binding protein 4a (CBP4a) in a Ca(2+)-dependent manner. Further deletion analysis suggests this interaction requires residues found within the DEED domain. NumA and CBP4a mRNAs are expressed at the same stages of development. CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined. Since the interaction of CBP4a with nucleomorphin requires the DEED domain, this suggests that CBP4a may respond to Ca(2+)-signalling through modulating factors that might function in concert to regulate nuclear number. PMID:15325281

  19. Mapping of nuclear import signal and importin {alpha}3 binding regions of 52K protein of bovine adenovirus-3

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, Carolyn P.; Ayalew, Lisanework E. [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); Tikoo, Suresh K., E-mail: suresh.tik@usask.ca [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); School of Public Health, University of Saskatchewan, Saskatoon, SK S7N 5E5 Canada (Canada)

    2012-10-10

    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 acid 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.

  20. Somatic Cell Nuclear Transfer in the Mouse

    Science.gov (United States)

    Kishigami, Satoshi; Wakayama, Teruhiko

    Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

  1. Protein variants in human cells: enumeration by protein indexing

    International Nuclear Information System (INIS)

    In any attempt to construct a catalog of the proteins of a given species, the genetic heterogeneity of natural plant and animal populations makes it necessary to consider variants of each protein. Thus in compiling a Human Protein Index using high resolution two-dimensional electrophoresis as a separating technique, protein variants differing from the wild type by charge or by polypeptide length will be recognized as different and must be accounted for. Results of several recent investigations have argued for average heterozygosities of approximately 1% for human cellular proteins examined by two-dimensional electrophoresis, while the results of classical biochemical genetics lead to higher results. The ability to observe genetic variation in large numbers of proteins can be valuable in several contexts. More than 2000 human genetic diseases have been identified, the vast majority of which have not as yet been associated with a defect in a particular protein. The present study of 63 human fibroblast cell lines was initiated in order to determine the level of genetic variation at many loci and to see whether known genetic diseases were associated with any obvious variant proteins that might be expressed in fibroblasts in culture. The main result is a group of ten new putative variants available in permanent cell lines

  2. Protein carbonylation, protein aggregation and neuronal cell death in a murine model of multiple sclerosis

    Science.gov (United States)

    Dasgupta, Anushka

    Many studies have suggested that oxidative stress plays an important role in the pathophysiology of both multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Yet, the mechanism by which oxidative stress leads to tissue damage in these disorders is unclear. Recent work from our laboratory has revealed that protein carbonylation, a major oxidative modification caused by severe and/or chronic oxidative stress conditions, is elevated in MS and EAE. Furthermore, protein carbonylation has been shown to alter protein structure leading to misfolding/aggregation. These findings prompted me to hypothesize that carbonylated proteins, formed as a consequence of oxidative stress and/or decreased proteasomal activity, promote protein aggregation to mediate neuronal apoptosis in vitro and in EAE. To test this novel hypothesis, I first characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of myelin-oligodendrocyte glycoprotein (MOG)35-55 peptide-induced EAE in C57BL/6 mice [Chapter 2]. The results show that carbonylated proteins accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. I discovered not only that there is a temporal correlation between protein carbonylation and apoptosis but also that carbonyl levels are significantly higher in apoptotic cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are also present during the course of EAE, which seems to be due to reduced autophagy. In chapter 3, I show that when gluthathione levels are reduced to those in EAE spinal cord, both neuron-like PC12 (nPC12) cells and primary neuronal cultures accumulate carbonylated proteins and undergo cell death (both by necrosis and apoptosis). Immunocytochemical and biochemical studies also revealed a temporal

  3. Transcriptional activation of NAD{sup +}-dependent protein deacetylase SIRT1 by nuclear receptor TLX

    Energy Technology Data Exchange (ETDEWEB)

    Iwahara, Naotoshi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Hisahara, Shin; Hayashi, Takashi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Department of Neurology, Sapporo Medical University, Sapporo 060-8556 (Japan); Horio, Yoshiyuki, E-mail: horio@sapmed.ac.jp [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan)

    2009-09-04

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD{sup +}-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

  4. Production of transgenic calves by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    GONG Guochun; WAN Rong; HUANG Yinghua; LI Ning; DAI Yunping; FAN Baoliang; ZHU Huabing; WANG Lili; WANG Haiping; TANG Bo; LIU Ying; LI Rong

    2004-01-01

    Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector (pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was carried out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves, and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves. PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.

  5. Characterization of Adapter Protein NRBP as a Negative Regulator of T Cell Activation

    Institute of Scientific and Technical Information of China (English)

    WANG Hui; LIN Zhi-xin; WU Jun

    2008-01-01

    Adapter proteins can regulate the gene transcriptions in disparate signaling pathway by interacting with multiple signaling molecules, including T cell activation signaling. Nuclear receptor binding protein (NRBP), a novel adapter protein, represents a small family of evolutionarily conserved proteins with homologs in Caenorhabditis elegans (C. elegans), Drosophila melanogaster (D.melanogaster), mouse and human. Here, we demonstrated that overexpression of NRBP in Jurkat TAg cells specifically impairs T cell receptor (TCR) or phorbol myristate acetate (PMA)/ionomycin-mediated signaling leading to nuclear factor of activated T cells (NFAT) promoter activation. Furthermore, the N-terminal of NRBP is necessary for its regulation of NFAT activation. Finally, we showed that NRBP has minimal effect on both TCR- and PMA-induced CD69 up-regulation in Jurkat TAg cells, which suggests that NRBP may function downstream of protein kinase C (PKC)/Ras pathway.

  6. Novel nuclear targeting coiled-coil protein of Helicobacter pylori showing Ca(2+)-independent, Mg(2+)-dependent DNase I activity.

    Science.gov (United States)

    Kwon, Young Chul; Kim, Sinil; Lee, Yong Seok; Lee, Je Chul; Cho, Myung-Je; Lee, Woo-Kon; Kang, Hyung-Lyun; Song, Jae-Young; Baik, Seung Chul; Ro, Hyeon Su

    2016-05-01

    HP0059, an uncharacterized gene of Helicobacter pylori, encodes a 284-aa-long protein containing a nuclear localization sequence (NLS) and multiple leucine-rich heptad repeats. Effects of HP0059 proteins in human stomach cells were assessed by incubation of recombinant HP0059 proteins with the AGS human gastric carcinoma cell line. Wild-type HP0059 proteins showed cytotoxicity in AGS cells in a concentration-dependent manner, whereas NLS mutant protein showed no effect, suggesting that the cytotoxicity is attributed to host nuclear localization. AGS cells transfected with pEGFP-HP0059 plasmid showed strong GFP signal merged to the chromosomal DNA region. The chromosome was fragmented into multiple distinct dots merged with the GFP signal after 12 h of incubation. The chromosome fragmentation was further explored by incubation of AGS chromosomal DNA with recombinant HP0059 proteins, which leaded to complete degradation of the chromosomal DNA. HP0059 protein also degraded circular plasmid DNA without consensus, being an indication of DNase I activity. The DNase was activated by MgCl2, but not by CaCl2. The activity was completely blocked by EDTA. The optimal pH and temperature for DNase activity were 7.0-8.0 and 55°C, respectively. These results indicate that HP0059 possesses a novel DNase I activity along with a role in the genomic instability of human gastric cells, which may result in the transformation of gastric cells. PMID:27095458

  7. Proteomic profiling of nuclear fractions from native renal inner medullary collecting duct cells.

    Science.gov (United States)

    Pickering, Christina M; Grady, Cameron; Medvar, Barbara; Emamian, Milad; Sandoval, Pablo C; Zhao, Yue; Yang, Chin-Rang; Jung, Hyun Jun; Chou, Chung-Lin; Knepper, Mark A

    2016-02-01

    The control of renal water excretion occurs in part by regulation of transcription in response to vasopressin in cells of the collecting duct. A systems biology-based approach to understanding transcriptional control in renal collecting duct cells depends on knowledge of what transcription factors and other regulatory proteins are present in the cells' nuclei. The goal of this article is to report comprehensive proteomic profiling of cellular fractions enriched in nuclear proteins from native inner medullary collecting duct (IMCD) cells of the rat. Multidimensional separation procedures and state-of-the art protein mass spectrometry produced 18 GB of spectral data that allowed the high-stringency identification of 5,048 proteins in nuclear pellet (NP) and nuclear extract (NE) fractions of biochemically isolated rat IMCD cells (URL: https://helixweb.nih.gov/ESBL/Database/IMCD_Nucleus/). The analysis identified 369 transcription factor proteins out of the 1,371 transcription factors coded by the rat genome. The analysis added 1,511 proteins to the recognized proteome of rat IMCD cells, now amounting to 8,290 unique proteins. Analysis of samples treated with the vasopressin analog dDAVP (1 nM for 30 min) or its vehicle revealed 99 proteins in the NP fraction and 88 proteins in the NE fraction with significant changes in spectral counts (Fisher exact test, P < 0.005). Among those altered by vasopressin were seven distinct histone proteins, all of which showed decreased abundance in the NP fraction, consistent with a possible effect of vasopressin to induce chromatin remodeling. The results provide a data resource for future studies of vasopressin-mediated transcriptional regulation in the renal collecting duct.

  8. The 5‘—flanking cis—acting elements of the human ε—globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

    Institute of Scientific and Technical Information of China (English)

    YANZHIJIANG; RUOLANQIAN

    1998-01-01

    The nuclear matrix attachment regions(MARs) and the binding nuclear matrix proteins in the 5'-flanking cisacting elements of the human ε-globin gene have been examined.Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII,-446bp- -419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells,indicating that ε-PREII may be an erythroidspecific facultative MAR.In gel mobility shift assay and Southwestern blotting assay,an erythroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (ε-PREII).Furthermore,we demonstrated that the silencer (-392bp- -177bp) upstream of the human ε-globin gene could associate with the nuclear matrices from K562,HEL and Raji cells.In addition,the nuclear matrix proteins prepared from these three cell lines could also bind to this silencer,suggesting that this silencer element might be a constitutive nuclear matrix attachment region(constitutive MAR).Our results demonstrated that the nuclear matrix and nuclear matrix proteins might play an important role in the regulation of the human ε-globin gene expression.

  9. Tumor protein 53-induced nuclear protein 1 (TP53INP1 enhances p53 function and represses tumorigenesis

    Directory of Open Access Journals (Sweden)

    Jeyran eShahbazi

    2013-05-01

    Full Text Available Tumor protein 53-induced nuclear protein 1 (TP53INP1 is a stress-induced p53 target gene whose expression is modulated by transcription factors such as p53, p73 and E2F1. TP53INP1 gene encodes two isoforms of TP53INP1 proteins, TP53INP1α and TP53INP1β, both of which appear to be key elements in p53 function. When associated with homeodomain-interacting protein kinase-2 (HIPK2, TP53INP1 phosphorylates p53 protein at Serine 46, enhances p53 protein stability and its transcriptional activity, leading to transcriptional activation of p53 target genes such as p21, PIG-3 and MDM2, cell growth arrest and apoptosis upon DNA damage stress. The anti-proliferative and pro-apoptotic activities of TP53INP1 indicate that TP53INP1 has an important role in cellular homeostasis and DNA damage response. Deficiency in TP53INP1 expression results in increased tumorigenesis; while TP53INP1 expression is repressed during early stages of cancer by factors such as miR-155. This review aims to summarize the roles of TP53INP1 in blocking tumor progression through p53-dependant and p53-independent pathways, as well as the elements which repress TP53INP1 expression, hence highlighting its potential as a therapeutic target in cancer treatment.

  10. Generation of embryonic stem cells from mouse adipose-tissue derived cells via somatic cell nuclear transfer.

    Science.gov (United States)

    Qin, Yiren; Qin, Jilong; Zhou, Chikai; Li, Jinsong; Gao, Wei-Qiang

    2015-01-01

    Somatic cells can be reprogrammed into embryonic stem cells (ESCs) by nuclear transfer (NT-ESCs), or into induced pluripotent stem cells (iPSCs) by the "Yamanaka method." However, recent studies have indicated that mouse and human iPSCs are prone to epigenetic and transcriptional aberrations, and that NT-ESCs correspond more closely to ESCs derived from in vitro fertilized embryos than iPSCs. In addition, the procedure of NT-ESCs does not involve gene modification. Demonstration of generation of NT-ESCs using an easily-accessible source of adult cell types would be very important. Adipose tissue is a source of readily accessible donor cells and can be isolated from both males and females at different ages. Here we report that NT-ESCs can be generated from adipose tissue-derived cells (ADCs). At morphological, mRNA and protein levels, these NT-ESCs show classic ESC colonies, exhibit alkaline phosphatase (AP) activity, and display normal diploid karyotypes. Importantly, these cells express pluripotent markers including Oct4, Sox2, Nanog and SSEA-1. Furthermore, they can differentiate in vivo into various types of cells from 3 germinal layers by teratoma formation assays. This study demonstrates for the first time that ESCs can be generated from the adipose tissue by somatic cell nuclear transfer (SCNT) and suggests that ADCs can be a new donor-cell type for potential therapeutic cloning.

  11. Characterization of Aes nuclear foci in colorectal cancer cells.

    Science.gov (United States)

    Itatani, Yoshiro; Sonoshita, Masahiro; Kakizaki, Fumihiko; Okawa, Katsuya; Stifani, Stefano; Itoh, Hideaki; Sakai, Yoshiharu; Taketo, M Mark

    2016-01-01

    Amino-terminal enhancer of split (Aes) is a member of Groucho/Transducin-like enhancer (TLE) family. Aes is a recently found metastasis suppressor of colorectal cancer (CRC) that inhibits Notch signalling, and forms nuclear foci together with TLE1. Although some Notch-associated proteins are known to form subnuclear bodies, little is known regarding the dynamics or functions of these structures. Here, we show that Aes nuclear foci in CRC observed under an electron microscope are in a rather amorphous structure, lacking surrounding membrane. Investigation of their behaviour during the cell cycle by time-lapse cinematography showed that Aes nuclear foci dissolve during mitosis and reassemble after completion of cytokinesis. We have also found that heat shock cognate 70 (HSC70) is an essential component of Aes foci. Pharmacological inhibition of the HSC70 ATPase activity with VER155008 reduces Aes focus formation. These results provide insight into the understanding of Aes-mediated inhibition of Notch signalling. PMID:26229111

  12. Nuclear trafficking of EGFR by Vps34 represses Arf expression to promote lung tumor cell survival.

    Science.gov (United States)

    Dayde, D; Guerard, M; Perron, P; Hatat, A-S; Barrial, C; Eymin, B; Gazzeri, S

    2016-07-28

    Epidermal growth factor receptor (EGFR) is a cell surface receptor that has an essential role in cell proliferation and survival, and overexpression of EGFR is a common feature of human cancers. In Non-small-cell lung cancer (NSCLC), activating mutations of EGFR have also been described. We recently showed that mutant EGFR-L858R inhibits the expression of the p14ARF tumor-suppressor protein to promote cell survival. In this study, we defined the molecular bases by which EGFR controls Arf expression. Using various lung tumor models, we showed that EGF stimulation inhibits Arf transcription by a mechanism involving the nuclear transport and recruitment of EGFR to the Arf promoter. We unraveled the vesicular trafficking protein Vps34 as a mediator of EGFR nuclear trafficking and showed that its neutralization prevents the accumulation of EGFR to the Arf promoter in response to ligand activation. Finally, in lung tumor cells that carry mutant EGFR-L858R, we demonstrated that inhibition of Vps34 using small interfering RNA restrains nuclear EGFR location and restores Arf expression leading to apoptosis. These findings identify the Arf tumor suppressor as a new transcriptional target of nuclear EGFR and highlight Vps34 as an important regulator of the nuclear EGFR/Arf survival pathway. As a whole, they provide a mechanistic explanation to the inverse correlation between nuclear expression of EGFR and overall survival in NSCLC patients. PMID:26686095

  13. Dietary influences over proliferating cell nuclear antigen expression in the locust midgut

    OpenAIRE

    Zudaire, E. (Enrique); Simpson, S J; Illa, I.; Montuenga, L M

    2004-01-01

    We have studied the influence of variations in dietary protein (P) and digestible carbohydrate (C), the quantity of food eaten, and insect age during the fifth instar on the expression of the proliferating cell nuclear antigen (PCNA) in the epithelial cells of the midgut (with special reference to the midgut caeca) in the African migratory locust, Locusta migratoria. Densitometric analysis of PCNA-immunostained cells was used as an indirect measure of the levels of expression of PCNA, and a P...

  14. Search for Conditions to Detect Epigenetic Marks and Nuclear Proteins in Immunostaining of the Testis and Cartilage

    Directory of Open Access Journals (Sweden)

    Hisashi Ideno

    2014-01-01

    Full Text Available The localization of nuclear proteins and modified histone tails changes during cell differentiation at the tissue as well as at the cellular level. Immunostaining in paraffin sections is the most powerful approach available to evaluate protein localization. Since nuclear proteins are sensitive to fixation, immunohistochemical conditions should be optimized in light of the particular antibodies and tissues employed. In this study, we searched for optimal conditions to detect histone modification at histone H3 lysine 9 (H3K9 and H3K9 methyltransferase G9a in the testis and cartilage in paraffin sections. In the testis, antigen retrieval (AR was indispensable for detecting H3K9me1 and me3, G9a, and nuclear protein proliferating cell nuclear antigen (PCNA. With AR, shorter fixation times yielded better results for the detection of G9a and PCNA. Without AR, H3K9me2 and H3K9ac could be detected at shorter fixation times in primary spermatocytes of the testis. In contrast to the testis, all antibodies tested could detect their epitopes irrespective of AR application in the growth plate cartilage. Thus, conditions for the detection of epigenetic marks and nuclear proteins should be optimized in consideration of fixation time and AR application in different tissues and antibodies.

  15. Communication Between the Cell Membrane and the Nucleus: Role of Protein Compartmentalization

    Energy Technology Data Exchange (ETDEWEB)

    Lelievre, Sophie A; Bissell, Mina J

    1998-10-21

    Understanding how the information is conveyed from outside to inside the cell is a critical challenge for all biologists involved in signal transduction. The flow of information initiated by cell-cell and cell-extracellular matrix contacts is mediated by the formation of adhesion complexes involving multiple proteins. Inside adhesion complexes, connective membrane skeleton (CMS) proteins are signal transducers that bind to adhesion molecules, organize the cytoskeleton, and initiate biochemical cascades. Adhesion complex-mediated signal transduction ultimately directs the formation of supramolecular structures in the cell nucleus, as illustrated by the establishment of multi complexes of DNA-bound transcription factors, and the redistribution of nuclear structural proteins to form nuclear subdomains. Recently, several CMS proteins have been observed to travel to the cell nucleus, suggesting a distinctive role for these proteins in signal transduction. This review focuses on the nuclear translocation of structural signal transducers of the membrane skeleton and also extends our analysis to possible translocation of resident nuclear proteins to the membrane skeleton. This leads us to envision the communication between spatially distant cellular compartments (i.e., membrane skeleton and cell nucleus) as a bidirectional flow of information (a dynamic reciprocity) based on subtle multilevel structural and biochemical equilibria. At one level, it is mediated by the interaction between structural signal transducers and their binding partners, at another level it may be mediated by the balance and integration of signal transducers in different cellular compartments.

  16. Structural components of the nuclear body in nuclei of Allium cepa cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Nuclear bodies have long been noted in interphase nuclei of plant cells,but their structural component,origin and function are still unclear by now.The present work showed in onion cells the nuclear bodies appeared as a spherical structure about 0.3 to 0.8 μm in diameter.They possibly were formed in nucleolus and subsequently released,and entered into nucleoplasm.Observation through cytochemical staining method at the ultrastructural level confirmed that nuclear bodies consisted of ribonucleoproteins (RNPs) and silver-stainable proteins.Immunocytochemical results revealed that nuclear bodies contained no DNA and ribosomal gene transcription factor (UBF).Based on these data,we suggested that nuclear bodies are not related to the ribosome or other gene transcription activities,instead they may act as subnuclear structures for RNPs transport from nucleolus to cytoplasm,and may also be involved in splicing of pre-mRNAs.

  17. Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

    Science.gov (United States)

    O'Day, Danton H; Poloz, Yekaterina; Myre, Michael A

    2009-02-01

    The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia. PMID:19000924

  18. Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

    Science.gov (United States)

    Smidt, M P; Russchen, B; Snippe, L; Wijnholds, J; Ab, G

    1995-07-11

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. PMID:7630716

  19. LaRbp38: A Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

    International Nuclear Information System (INIS)

    Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38 kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function

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

    Science.gov (United States)

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

    2013-02-01

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. PMID:23174505

  1. In silico synchronization reveals regulators of nuclear ruptures in lamin A/C deficient model cells

    Science.gov (United States)

    Robijns, J.; Molenberghs, F.; Sieprath, T.; Corne, T. D. J.; Verschuuren, M.; de Vos, W. H.

    2016-07-01

    The nuclear lamina is a critical regulator of nuclear structure and function. Nuclei from laminopathy patient cells experience repetitive disruptions of the nuclear envelope, causing transient intermingling of nuclear and cytoplasmic components. The exact causes and consequences of these events are not fully understood, but their stochastic occurrence complicates in-depth analyses. To resolve this, we have established a method that enables quantitative investigation of spontaneous nuclear ruptures, based on co-expression of a firmly bound nuclear reference marker and a fluorescent protein that shuttles between the nucleus and cytoplasm during ruptures. Minimally invasive imaging of both reporters, combined with automated tracking and in silico synchronization of individual rupture events, allowed extracting information on rupture frequency and recovery kinetics. Using this approach, we found that rupture frequency correlates inversely with lamin A/C levels, and can be reduced in genome-edited LMNA knockout cells by blocking actomyosin contractility or inhibiting the acetyl-transferase protein NAT10. Nuclear signal recovery followed a kinetic that is co-determined by the severity of the rupture event, and could be prolonged by knockdown of the ESCRT-III complex component CHMP4B. In conclusion, our approach reveals regulators of nuclear rupture induction and repair, which may have critical roles in disease development.

  2. Nuclear area measurement on viable cells, using confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Townsend, K.M.S.; Marsden, S.J. (Medical Research Council, Harwell (United Kingdom). Radiobiological Research Unit)

    1992-04-01

    The authors describe a rapid procedure for the accurate measurement of nuclear areas on unperturbed living cells as used in radiobiological experiments, using the confocal laser scanning microscope. The microdosimetric interpretation of radiobiological data requires precise information on the nuclear area of cells as irradiated with high-LET radiation. (author).

  3. Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

    Institute of Scientific and Technical Information of China (English)

    Lan LI; Wei SHEN; Lingjiang MIN; Qingyu PAN; Yujiang SUN; Jixian DENG; Qingjie PAN

    2008-01-01

    Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.

  4. Steatosis-induced proteins adducts with lipid peroxidation products and nuclear electrophilic stress in hepatocytes

    Directory of Open Access Journals (Sweden)

    Sarit Anavi

    2015-04-01

    Full Text Available Accumulating evidence suggests that fatty livers are particularly more susceptible to several pathological conditions, including hepatic inflammation, cirrhosis and liver cancer. However the exact mechanism of such susceptibility is still largely obscure. The current study aimed to elucidate the effect of hepatocytes lipid accumulation on the nuclear electrophilic stress. Accumulation of intracellular lipids was significantly increased in HepG2 cells incubated with fatty acid (FA complex (1 mM, 2:1 oleic and palmitic acids. In FA-treated cells, lipid droplets were localized around the nucleus and seemed to induce mechanical force, leading to the disruption of the nucleus morphology. Level of reactive oxygen species (ROS was significantly increased in FA-loaded cells and was further augmented by treatment with moderate stressor (CoCl2. Increased ROS resulted in formation of reactive carbonyls (aldehydes and ketones, derived from lipid peroxidation with a strong perinuclear accumulation. Mass-spectroscopy analysis indicated that lipid accumulation per-se can results in modification of nuclear protein by reactive lipid peroxidation products (oxoLPP. 235 Modified proteins involved in transcription regulation, splicing, protein synthesis and degradation, DNA repair and lipid metabolism were identified uniquely in FA-treated cells. These findings suggest that steatosis can affect nuclear redox state, and induce modifications of nuclear proteins by reactive oxoLPP accumulated in the perinuclear space upon FA-treatment.

  5. Agrobacterium rhizogenes GALLS protein contains domains for ATP binding, nuclear localization, and type IV secretion.

    Science.gov (United States)

    Hodges, Larry D; Vergunst, Annette C; Neal-McKinney, Jason; den Dulk-Ras, Amke; Moyer, Deborah M; Hooykaas, Paul J J; Ream, Walt

    2006-12-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2. PMID:17012398

  6. Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase.

    Science.gov (United States)

    Wang, Xiao-Hu; Hong, Xin; Zhu, Lei; Wang, Yun-Tao; Bao, Jun-Ping; Liu, Lei; Wang, Feng; Wu, Xiao-Tao

    2015-04-01

    Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.

  7. MitoNuc: a database of nuclear genes coding for mitochondrial proteins. Update 2002

    Science.gov (United States)

    Attimonelli, Marcella; Catalano, Domenico; Gissi, Carmela; Grillo, Giorgio; Licciulli, Flavio; Liuni, Sabino; Santamaria, Monica; Pesole, Graziano; Saccone, Cecilia

    2002-01-01

    Mitochondria, besides their central role in energy metabolism, have recently been found to be involved in a number of basic processes of cell life and to contribute to the pathogenesis of many degenerative diseases. All functions of mitochondria depend on the interaction of nuclear and organelle genomes. Mitochondrial genomes have been extensively sequenced and analysed and data have been collected in several specialised databases. In order to collect information on nuclear coded mitochondrial proteins we developed MitoNuc, a database containing detailed information on sequenced nuclear genes coding for mitochondrial proteins in Metazoa. The MitoNuc database can be retrieved through SRS and is available via the web site http://bighost.area.ba.cnr.it/mitochondriome where other mitochondrial databases developed by our group, the complete list of the sequenced mitochondrial genomes, links to other mitochondrial sites and related information, are available. The MitoAln database, related to MitoNuc in the previous release, reporting the multiple alignments of the relevant homologous protein coding regions, is no longer supported in the present release. In order to keep the links among entries in MitoNuc from homologous proteins, a new field in the database has been defined: the cluster identifier, an alpha numeric code used to identify each cluster of homologous proteins. A comment field derived from the corresponding SWISS-PROT entry has been introduced; this reports clinical data related to dysfunction of the protein. The logic scheme of MitoNuc database has been implemented in the ORACLE DBMS. This will allow the end-users to retrieve data through a friendly interface that will be soon implemented. PMID:11752284

  8. Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals.

    Science.gov (United States)

    Yamasaki, L; Kanda, P; Lanford, R E

    1989-07-01

    The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus E1A, and Saccharomyces cerevisiae MAT alpha 2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, p100, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unlabeled peptides demonstrated that heterologous signals were able to bind the same protein and suggested that diverse signals use a common transport pathway. The subcellular distribution of the four nuclear transport signal-binding proteins suggested that nuclear transport involves both cytoplasmic and nuclear receptors. The four proteins were not bound by wheat germ agglutinin and were not associated tightly with the nuclear pore complex.

  9. Cell-to-cell propagation of infectious cytosolic protein aggregates

    Science.gov (United States)

    Hofmann, Julia P.; Denner, Philip; Nussbaum-Krammer, Carmen; Kuhn, Peer-Hendrik; Suhre, Michael H.; Scheibel, Thomas; Lichtenthaler, Stefan F.; Schätzl, Hermann M.; Bano, Daniele; Vorberg, Ina M.

    2013-01-01

    Prions are self-templating protein conformers that replicate by recruitment and conversion of homotypic proteins into growing protein aggregates. Originally identified as causative agents of transmissible spongiform encephalopathies, increasing evidence now suggests that prion-like phenomena are more common in nature than previously anticipated. In contrast to fungal prions that replicate in the cytoplasm, propagation of mammalian prions derived from the precursor protein PrP is confined to the cell membrane or endocytic vesicles. Here we demonstrate that cytosolic protein aggregates can also behave as infectious entities in mammalian cells. When expressed in the mammalian cytosol, protein aggregates derived from the prion domain NM of yeast translation termination factor Sup35 persistently propagate and invade neighboring cells, thereby inducing a self-perpetuating aggregation state of NM. Cell contact is required for efficient infection. Aggregates can also be induced in primary astrocytes, neurons, and organotypic cultures, demonstrating that this phenomenon is not specific to immortalized cells. Our data have important implications for understanding prion-like phenomena of protein aggregates associated with human diseases and for the growing number of amyloidogenic proteins discovered in mammals. PMID:23509289

  10. Origins of Protein Functions in Cells

    Science.gov (United States)

    Seelig, Burchard; Pohorille, Andrzej

    2011-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis and in vitro evolution of very large libraries of random amino acid sequences. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions yet, important clues have been uncovered. In one example (Keefe and Szostak, 2001), novel ATP binding proteins were identified that appear to be unrelated in both sequence and structure to any known ATP binding proteins. One of these proteins was subsequently redesigned computationally to bind GTP through introducing several mutations that introduce targeted structural changes to the protein, improve its binding to guanine and prevent water from accessing the active center. This study facilitates further investigations of individual evolutionary steps that lead to a change of function in primordial proteins. In a second study (Seelig and Szostak, 2007), novel enzymes were generated that can join two pieces of RNA in a reaction for which no natural enzymes are known

  11. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

    Energy Technology Data Exchange (ETDEWEB)

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.

  12. Regulation of cell proliferation by G proteins.

    Science.gov (United States)

    Dhanasekaran, N; Tsim, S T; Dermott, J M; Onesime, D

    1998-09-17

    G Proteins provide signal transduction mechanisms to seven transmembrane receptors. Recent studies have indicated that the alpha-subunits as well as the betagamma-subunits of these proteins regulate several critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Of the 17 alpha-subunits that have been cloned, at least ten of them have been shown to couple mitogenic signaling in fibroblast cells. Activating mutations in G alpha(s), G alpha(i)2, and G alpha12 have been correlated with different types of tumors. In addition, the ability of the betagamma-subunits to activate mitogenic pathways in different cell-types has been defined. The present review briefly summarizes the diverse and novel signaling pathways regulated by the alpha- as well as the betagamma-subunits of G proteins in regulating cell proliferation. PMID:9779986

  13. Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex.

    Science.gov (United States)

    Vovk, Andrei; Gu, Chad; Opferman, Michael G; Kapinos, Larisa E; Lim, Roderick Yh; Coalson, Rob D; Jasnow, David; Zilman, Anton

    2016-01-01

    Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function. PMID:27198189

  14. Cell cycle dependent association of EBP50 with protein phosphatase 2A in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Anita Boratkó

    Full Text Available Ezrin-radixin-moesin (ERM-binding phosphoprotein 50 (EBP50 is a phosphorylatable PDZ domain-containing adaptor protein that is abundantly expressed in epithelium but was not yet studied in the endothelium. We report unusual nuclear localization of EBP50 in bovine pulmonary artery endothelial cells (BPAEC. Immunofluorescent staining and cellular fractionation demonstrated that EBP50 is present in the nuclear and perinuclear region in interphase cells. In the prophase of mitosis EBP50 redistributes to the cytoplasmic region in a phosphorylation dependent manner and during mitosis EBP50 co-localizes with protein phosphatase 2A (PP2A. Furthermore, in vitro wound healing of BPAEC expressing phospho-mimic mutant of EBP50 was accelerated indicating that EBP50 is involved in the regulation of the cell division. Cell cycle dependent specific interactions were detected between EBP50 and the subunits of PP2A (A, C, and Bα with immunoprecipitation and pull-down experiments. The interaction of EBP50 with the Bα containing form of PP2A suggests that this holoenzyme of PP2A can be responsible for the dephosphorylation of EBP50 in cytokinesis. Moreover, the results underline the significance of EBP50 in cell division via reversible phosphorylation of the protein with cyclin dependent kinase and PP2A in normal cells.

  15. SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown

    OpenAIRE

    Turgay Y; Champion L; Balazs C; Held M; Toso A; Gerlich DW; Meraldi P; Kutay U.

    2014-01-01

    SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and imp...

  16. Data in support of DPF2 regulates OCT4 protein level and nuclear distribution

    Directory of Open Access Journals (Sweden)

    Chao Liu

    2015-12-01

    Full Text Available DPF2, also named ubi-d4/requiem (REQU, interacts with a protein complex containing OCT4. This paper provides data in support of the research article entitled “DPF2 regulates OCT4 protein level and nuclear distribution”. The highlights include: (1 Denature-immunoprecipitation assay revealed ubiquitination of OCT4 in pluripotent H9 cells, which was enhancedby MG132, a proteasome inhibitor. (2 Well colocalization of ectopic OCT4 and FLAG-Ub was found in HeLa cells, which was also increased by MG132. (3 MG132 treatment decreased DPF2 cytoplasmic expression in vivo. These data give insights into how proteasome inhibition contributes to studying ubiquitnation of OCT4.

  17. RanBP3 Regulates Melanoma Cell Proliferation via Selective Control of Nuclear Export.

    Science.gov (United States)

    Pathria, Gaurav; Garg, Bhavuk; Wagner, Christine; Garg, Kanika; Gschaider, Melanie; Jalili, Ahmad; Wagner, Stephan N

    2016-01-01

    Chromosome region maintenance 1-mediated nucleocytoplasmic transport has been shown as a potential anticancer target in various malignancies. However, the role of the most characterized chromosome region maintenance 1 cofactor ran binding protein 3 (RanBP3) in cancer cell biology has never been investigated. Utilizing a loss-of-function experimental setting in a vast collection of genetically varied melanoma cell lines, we observed the requirement of RanBP3 in melanoma cell proliferation and survival. Mechanistically, we suggest the reinstatement of transforming growth factor-β (TGF-β)-Smad2/3-p21(Cip1) tumor-suppressor axis as part of the RanBP3 silencing-associated antiproliferative program. Employing extensive nuclear export sequence analyses and immunofluorescence-based protein localization studies, we further present evidence suggesting the requirement of RanBP3 function for the nuclear exit of the weak nuclear export sequence-harboring extracellular signal-regulated kinase protein, although it is dispensable for general CRM1-mediated nuclear export of strong nuclear export sequence-harboring cargoes. Rendering mechanistic support to RanBP3 silencing-mediated apoptosis, consequent to extracellular signal-regulated kinase nuclear entrapment, we observed increased levels of cytoplasmically restricted nonphosphorylated/active proapoptotic Bcl-2-antagonist of cell death (BAD) protein. Last, we present evidence suggesting the frequently activated mitogen-activated protein kinase signaling in melanoma as a potential founding basis for a deregulated post-translational control of RanBP3 activity. Collectively, the presented data suggest RanBP3 as a potential target for therapeutic intervention in human melanoma.

  18. The kin17 Protein in Murine Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Anelise C. Ramos

    2015-11-01

    Full Text Available kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma.

  19. In vivo cooperation of two nuclear oncogenic proteins, P135gag-myb-ets and p61/63myc, leads to transformation and immortalization of chicken myelomonocytic cells.

    OpenAIRE

    Adelmant, G; Quatannens, B; Lagrou, C; Wernert, N; Torpier, G; Saule, S.; Stehelin, D.; Laudet, V

    1994-01-01

    To investigate a possible in vivo cooperation between the p61/63myc and P135gag-myb-ets proteins, we used a previously constructed retrovirus, named MHE226, which contains the fused v-myb and v-ets oncogenes of the E26 retrovirus and the v-myc oncogene of MH2. For that purpose, chicken neuroretina cells producing MHE226 and pseudotyped with the Rous associated virus-1 (RAV-1) helper virus were injected in 1-day-old chickens. In control experiments, we also injected chicken neuroretina cells p...

  20. Versatile protein tagging in cells with split fluorescent protein.

    Science.gov (United States)

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A; Ishikawa, Hiroaki; Leonetti, Manuel D; Marshall, Wallace F; Weissman, Jonathan S; Huang, Bo

    2016-03-18

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.

  1. Versatile protein tagging in cells with split fluorescent protein

    Science.gov (United States)

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A.; Ishikawa, Hiroaki; Leonetti, Manuel D.; Marshall, Wallace F.; Weissman, Jonathan S.; Huang, Bo

    2016-01-01

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications. PMID:26988139

  2. Versatile protein tagging in cells with split fluorescent protein.

    Science.gov (United States)

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A; Ishikawa, Hiroaki; Leonetti, Manuel D; Marshall, Wallace F; Weissman, Jonathan S; Huang, Bo

    2016-01-01

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications. PMID:26988139

  3. Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

    Energy Technology Data Exchange (ETDEWEB)

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki, E-mail: sueyoshi@ag.kagawa-u.ac.jp

    2014-03-28

    Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

  4. Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

    International Nuclear Information System (INIS)

    Research highlights: → Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. → hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. → hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

  5. Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Kwang Sung; Won, Ji Young [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Park, Jin-Ki [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Sorrell, Alice M. [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Heo, Soon Young; Kang, Jee Hyun [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Woo, Jae-Seok [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Choi, Bong-Hwan [Genomics and Bioinformatics Division, National Institute of Animal Science, Suwon (Korea, Republic of); Chang, Won-Kyong [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of)

    2010-10-01

    Research highlights: {yields} Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. {yields} hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. {yields} hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

  6. Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells

    Directory of Open Access Journals (Sweden)

    Ruley H Earl

    2004-10-01

    Full Text Available Abstract Background Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis- and trans-acting factors that influence the delivery of proteins into cells. Results In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. The most active Cre proteins tested contained either an N-terminal 6xHis tag and a nuclear localization sequence from SV40 large T antigen (HNC or the HIV Tat transduction sequence and a C-terminal 6xHis tag (TCH6. The NLS and 6xHis elements separately enhanced the delivery of the HNC protein into cells; moreover, transduction sequences from fibroblast growth factor 4, HIV Tat or consisting of the (KFF3K sequence were not required for efficient protein transduction and adversely affected enzyme solubility. Transduction of the HNC protein required 10 to 15 min for half-maximum uptake, was greatly decreased at 4°C and was inhibited by serum. Efficient recombination was observed in all cell types tested (a T-cell line, NIH3T3, Cos7, murine ES cells, and primary splenocytes, and did not require localization of the enzyme to the nucleus. Conclusions The effects of different sequences on the delivery and/or activity of Cre in cultured cells could not be predicted in advance. Consequently, the process of developing more active cell-permeant recombinases was largely empirical. The HNC protein, with an excellent combination of activity, solubility and yield, will enhance the use of cell-permeant Cre proteins to regulate gene structure and function in living cells.

  7. In vivo cell biology of cancer cells visualized with fluorescent proteins.

    Science.gov (United States)

    Hoffman, Robert M

    2005-01-01

    This chapter describes a new cell biology where the behavior of individual cells can be visualized in the living animal. Previously it has been demonstrated that fluorescent proteins can be used for whole-body imaging of metastatic tumor growth, bacterial infection, and gene expression. An example of the new cell biology is dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP)-expressing transgenic mice. These models show with great clarity the details of tumor-stroma interactions and especially tumor-induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts, and macrophages. Another example is the color coding of cells with RFP or GFP such that both cell types can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo. Mice in which the regulatory elements of the stem cell marker nestin drive GFP expression enable nascent vasculature to be visualized interacting with transplanted RFP-expressing cancer cells. Nestin-driven GFP expression can also be used to visualize hair follicle stem cells. Dual-color cells expressing GFP in the nucleus and RFP in the cytoplasm enable real-time visualization of nuclear-cytoplasm dynamics including cell cycle events and apoptosis. Highly elongated cancer cells in capillaries in living mice were observed within skin flaps. The migration velocities of the cancer cells in the capillaries were measured by capturing images of the dual-color fluorescent cells over time. The cells in the capillaries elongated to fit the width of these vessels. The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels.

  8. Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Cambier, Linda [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France); Pomies, Pascal, E-mail: pascal.pomies@crbm.cnrs.fr [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France)

    2011-06-17

    Highlights: {yields} The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. {yields} smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. {yields} The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. {yields} The LIM domain of smALP is essential for the nuclear accumulation of the protein. {yields} smALP might act in the nucleus to control some critical aspect of the muscle differentiation process. -- Abstract: The skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletal muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein.

  9. Fhit Nuclear Import Following EGF Stimulation Sustains Proliferation of Breast Cancer Cells.

    Science.gov (United States)

    Bianchi, Francesca; Sasso, Marianna; Turdo, Federica; Beretta, Giovanni L; Casalini, Patrizia; Ghirelli, Cristina; Sfondrini, Lucia; Ménard, Sylvie; Tagliabue, Elda; Campiglio, Manuela

    2015-11-01

    The tumor-suppressor protein fragile histidine triad (Fhit) exerts its functions in the cytoplasm, although some reports suggest that it may also act in the nucleus. We previously showed that cytosolic Fhit protein levels in cancer cell lines stimulated to proliferate were reduced by proteasomal degradation. Here, we demonstrate that Fhit is physiologically present in the nucleus of breast cancer cell lines and tissues at a low level and that proliferative stimulation increases nuclear levels. Breast cancer cells expressing the FhitY114F mutant, which do not undergo proteasomal degradation, contained mutated Fhit in the nucleus, while cells treated with a proteasome inhibitor accumulated nuclear Fhit during proliferation. Thus, Fhit nuclear shuttling and proteasome degradation phenomena occur independently. When Fhit was coupled to a nuclear localization sequence, the proliferation rate of the transfected cells increased together with levels of proliferation pathway mediators cyclin D1, phospho-MAPK, and phospho-STAT3. Fhit nuclear translocation upon mitogenic stimulation may represent a new regulatory mechanism that allows rapid restoration of Fhit cytoplasmic levels and promotes the proliferation cascade activated by mitogenic stimulation.

  10. Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID.

    Directory of Open Access Journals (Sweden)

    Quy Le

    2015-09-01

    Full Text Available AID (Activation Induced Deaminase deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.

  11. MitoNuc and MitoAln: two related databases of nuclear genes coding for mitochondrial proteins

    Science.gov (United States)

    Pesole, Graziano; Gissi, Carmela; Catalano, Domenico; Grillo, Giorgio; Licciulli, Flavio; Liuni, Sabino; Attimonelli, Marcella; Saccone, Cecilia

    2000-01-01

    Mitochondria, besides their central role in energy metabolism, have recently been found to be involved in a number of basic processes of cell life and to contribute to the pathogenesis of many degenerative diseases. All functions of mitochondria depend on the interaction of nuclear and organellar genomes. Mitochondrial genomes have been extensively sequenced and analysed and the data collected in several specialised databases. In order to collect information on nuclear coded mitochondrial proteins we developed MitoNuc and MitoAln, two related databases containing, respectively, detailed information on sequenced nuclear genes coding for mitochondrial proteins in Metazoa and yeast, and the multiple alignments of the relevant homologous protein coding regions. MitoNuc and MitoAln retrieval through SRS at http://bio-www.ba.cnr.it:8000/srs6/ can easily allow the extraction of sequence data, subsequences defined by specific features and nucleotide or amino acid multiple alignments. PMID:10592211

  12. Production and Characterization of Monoclonal Antibodies against Human Nuclear Protein FAM76B.

    Directory of Open Access Journals (Sweden)

    Xiaojing Zheng

    Full Text Available Human FAM76B (hFAM76B is a 39 kDa protein that contains homopolymeric histidine tracts, a targeting signal for nuclear speckles. FAM76B is highly conserved among different species, suggesting that it may play an important physiological role in normal cellular functions. However, a lack of appropriate tools has hampered study of this potentially important protein. To facilitate research into the biological function(s of FAM76B, murine monoclonal antibodies (MAbs against hFAM76B were generated by using purified, prokaryotically expressed hFAM76B protein. Six strains of MAbs specific for hFAM76B were obtained and characterized. The specificity of MAbs was validated by using FAM76B-/- HEK 293 cell line. Double immunofluorescence followed by laser confocal microscopy confirmed the nuclear speckle localization of hFAM76B, and the specific domains recognized by different MAbs were further elucidated by Western blot. Due to the high conservation of protein sequences between mouse and human FAM76B, MAbs against hFAM76B were shown to react with mouse FAM76B (mFAM76B specifically. Lastly, FAM76B was found to be expressed in the normal tissues of most human organs, though to different extents. The MAbs produced in this study should provide a useful tool for investigating the biological function(s of FAM76B.

  13. Identification of the proteins responsible for SAR DNA binding in nuclear matrix of ''Cucurbita pepo''

    International Nuclear Information System (INIS)

    The nuclear matrices from White bush (''Cucurbita pepo var. patisonina'') cell nuclei have been isolated using three methods: I, standard procedure involving extraction of cell nuclei with 2 M NaCl and 1% Triton X-100; II, the same with pre-treatment of cell nuclei with 0.5 mM CuSO4 (stabilisation step); and III, method with extraction by lithium diiodosalicylate (LIS), and compared the polypeptide pattern. The isolated matrices specifically bind SAR DNA derived from human β-interferon gene in the exogenous SAR binding assay and in the gel mobility shift assay. Using IgG against the 32 kDa endonuclease we have found in the DNA-protein blot assay that this protein is one of the proteins binding SAR DNA. We have identified three proteins with molecular mass of 65 kDa, 60 kDa and 32 kDa which are responsible for SAR DNA binding in the gel mobility shift assay experiments. (author). 21 refs, 3 figs

  14. Angiomotin promotes renal epithelial and carcinoma cell proliferation by retaining the nuclear YAP.

    Science.gov (United States)

    Lv, Meng; Li, Shuting; Luo, Changqin; Zhang, Xiaoman; Shen, Yanwei; Sui, Yan Xia; Wang, Fan; Wang, Xin; Yang, Jiao; Liu, Peijun; Yang, Jin

    2016-03-15

    Renal cell carcinoma (RCC) is one of the common tumors in the urinary system without effective therapies. Angiomotin (Amot) can interact with Yes-associated protein (YAP) to either stimulate or inhibit YAP activity, playing a potential role in cell proliferation. However, the role of Amot in regulating the proliferation of renal epithelial and RCC cells is unknown. Here, we show that Amot is expressed predominantly in the nucleus of RCC cells and tissues, and in the cytoplasm and nucleus of renal epithelial cells and paracancerous tissues. Furthermore, Amot silencing inhibited proliferation of HK-2 and 786-O cells while Amot upregulation promoted proliferation of ACHN cells. Interestingly, the location of Amot and YAP in RCC clinical samples and cells was similar. Amot interacted with YAP in HK-2 and 786-O cells, particularly in the nucleus. Moreover, Amot silencing mitigated the levels of nuclear YAP in HK-2 and 786-O cells and reduced YAP-related CTGF and Cyr61 expression in 786-O cells. Amot upregulation slightly increased the nuclear YAP and YAP-related gene expression in ACHN cells. Finally, enhanced YAP expression restored proliferation of Amot-silencing 786-O cells. Together, these data indicate that Amot is crucial for the maintenance of nuclear YAP to promote renal epithelial and RCC proliferation.

  15. Heterogeneous nuclear ribonucleoprotein A3 is the liver nuclear protein binding to age related increase element RNA of the factor IX gene.

    Directory of Open Access Journals (Sweden)

    Toshiyuki Hamada

    Full Text Available BACKGROUND: In the ASE/AIE-mediated genetic mechanism for age-related gene regulation, a recently identified age-related homeostasis mechanism, two genetic elements, ASE (age-related stability element and AIE (age-related increase element as a stem-loop forming RNA, play critical roles in producing specific age-related expression patterns of genes. PRINCIPAL FINDING: We successfully identified heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3 as a major mouse liver nuclear protein binding to the AIE-derived RNAs of human factor IX (hFIX as well as mouse factor IX (mFIX genes. HnRNP A3 bound to the AIE RNA was not phosphorylated at its Ser(359, while hnRNP A3 in the mouse liver nuclear extracts was a mixture of phosphorylated and unphosphorylated Ser(359. HepG2 cells engineered to express recombinant hFIX transduced with adenoviral vectors harboring an effective siRNA against hnRNP A3 resulted in a substantial reduction in hFIX expression only in the cells carrying a hFIX expression vector with AIE, but not in the cells carrying a hFIX expression vector without AIE. The nuclear hnRNP A3 protein level in the mouse liver gradually increased with age, while its mRNA level stayed age-stable. CONCLUSIONS: We identified hnRNP A3 as a major liver nuclear protein binding to FIX-AIE RNA. This protein plays a critical role in age-related gene expression, likely through an as yet unidentified epigenetic mechanism. The present study assigned a novel functional role to hnRNP A3 in age-related regulation of gene expression, opening up a new avenue for studying age-related homeostasis and underlying molecular mechanisms.

  16. Yes-Associated Protein (YAP) Promotes the Nuclear Import of p73

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Heng; Wu Shengnan, E-mail: wushn@scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China)

    2011-01-01

    p73 has been identified as a structural and functional homolog of the tumor suppressor p53. However, mechanisms that regulate the localization of p73 have not been fully clarified. The Yes-associated protein (YAP) is a transcriptional coactivator. As a transcriptional coactivator, YAP needs to bind transcription factors to stimulate gene expression. p73 is a reported YAP target transcription factors and YAP has been shown to positively regulate p73 in promoting apoptosis. Previous studies show that p73 interacts with YAP through its PPPY motif, and increases p73 transactivation of apoptotic genes. In this study, we focused on YAP's regulation of the localization of p73. After transient transfection into Rat pheochromocytoma (PC12) cells and Human embryonic kidney 293T cells with GFP-YAP and/or YFP-p73, and incubated for 24 hours expression. p73 was fused to YFP to allow the examination of its subcellular localization. When expressed alone, YFP-p73 was distributed throughout the cell. When coexpressed with YAP, nuclear accumulation of YFP-p73 became evident. We quantitated the effect of YAP on the redistribution of YFP-p73 by counting cells with nuclear-only YFP signal. We found that YAP can influence the subcellular distribution of p73. Altogether, coexpression with YAP affected the subcellular distribution of the p73 protein. Our studies attribute a central role to YAP in regulating p73 accumulation and YAP, at least in part, might promote the nuclear import of p73.

  17. Yes-Associated Protein (YAP) Promotes the Nuclear Import of p73

    Science.gov (United States)

    Zhang, Heng; Wu, Shengnan

    2011-01-01

    p73 has been identified as a structural and functional homolog of the tumor suppressor p53. However, mechanisms that regulate the localization of p73 have not been fully clarified. The Yes-associated protein (YAP) is a transcriptional coactivator. As a transcriptional coactivator, YAP needs to bind transcription factors to stimulate gene expression. p73 is a reported YAP target transcription factors and YAP has been shown to positively regulate p73 in promoting apoptosis. Previous studies show that p73 interacts with YAP through its PPPY motif, and increases p73 transactivation of apoptotic genes. In this study, we focused on YAP's regulation of the localization of p73. After transient transfection into Rat pheochromocytoma (PC12) cells and Human embryonic kidney 293T cells with GFP-YAP and/or YFP-p73, and incubated for 24 hours expression. p73 was fused to YFP to allow the examination of its subcellular localization. When expressed alone, YFP-p73 was distributed throughout the cell. When coexpressed with YAP, nuclear accumulation of YFP-p73 became evident. We quantitated the effect of YAP on the redistribution of YFP-p73 by counting cells with nuclear-only YFP signal. We found that YAP can influence the subcellular distribution of p73. Altogether, coexpression with YAP affected the subcellular distribution of the p73 protein. Our studies attribute a central role to YAP in regulating p73 accumulation and YAP, at least in part, might promote the nuclear import of p73.

  18. Cell cycle-dependent alteration in NAC1 nuclear body dynamics and morphology

    Science.gov (United States)

    Wu, Pei-Hsun; Hung, Shen-Hsiu; Ren, Tina; Shih, Ie-Ming; Tseng, Yiider

    2011-02-01

    NAC1, a BTB/POZ family member, has been suggested to participate in maintaining the stemness of embryonic stem cells and has been implicated in the pathogenesis of human cancer. In ovarian cancer, NAC1 upregulation is associated with disease aggressiveness and with the development of chemoresistance. Like other BTB/POZ proteins, NAC1 forms discrete nuclear bodies in non-dividing cells. To investigate the biological role of NAC1 nuclear bodies, we characterized the expression dynamics of NAC1 nuclear bodies during different phases of the cell cycle. Fluorescence recovery after photobleaching assays revealed that NAC1 was rapidly exchanged between the nucleoplasm and NAC1 nuclear bodies in interphase cells. The number of NAC1 bodies significantly increased and their size decreased in the S phase as compared to the G0/G1 and G2 phases. NAC1 nuclear bodies disappeared and NAC1 became diffuse during mitosis. NAC1 nuclear bodies reappeared immediately after completion of mitosis. These results indicate that a cell cycle-dependent regulatory mechanism controls NAC1 body formation in the nucleus and suggest that NAC1 body dynamics are associated with mitosis or cytokinesis.

  19. The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Morgane eBatzenschlager

    2013-11-01

    Full Text Available During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs are nucleated from γ-Tubulin Complexes (γ-TuCs located at the surface of the nucleus. The molecular mechanisms of γ-TuC association to the nuclear envelope are currently unknown. The γ-TuC Protein 3 (GCP3-Interacting Protein 1 (GIP1 is the smallest γ-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active γ-TuCs at interphasic and mitotic MT nucleation sites. Arabidopsis gip1gip2 mutants are impaired in establishing a fully functional mitotic spindle and exhibit severe developmental defects.In this study, gip1gip2 knock down mutants were further characterized at the cellular level. In addition to defects in both the localization of γ-TuC core proteins and MT fibre robustness, gip1gip2 mutants exhibited a severe alteration of the nuclear shape associated with an abnormal distribution of the nuclear pore complexes. Simultaneously, they showed a misorganization of the inner nuclear membrane protein AtSUN1. Furthermore, AtGIP1 was identified as an interacting partner of AtTSA1 which was detected, like the AtGIP proteins, at the nuclear envelope.These results provide the first evidence for the involvement of a γ-TuC component in both nuclear shaping and nuclear envelope organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum.

  20. Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum

    Directory of Open Access Journals (Sweden)

    Tian Shengli

    2009-08-01

    Full Text Available Abstract Background Serine/arginine (SR protein-specific kinases (SRPKs are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379 from Physarum polycephalum previously, and identified a NLS of PSRPK in this study. Results We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS. Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328 was localized in the alkaline Ω-loop of a helix-loop-helix motif (HLHM of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus. Conclusion This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Ω-loop structure could play a crucial role in the NLS function of PSRPK.

  1. HIV-1 uncoating: connection to nuclear entry and regulation by host proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ambrose, Zandrea, E-mail: zaa4@pitt.edu [Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 (United States); Aiken, Christopher [Department of Pathology, Microbiology and Immunology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

    2014-04-15

    The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed by a capsid shell that dissociates within the cell in a multistep process known as uncoating, which influences completion of reverse transcription of the viral genome. Double-stranded viral DNA is imported into the nucleus for integration into the host genome, a hallmark of retroviral infection. Reverse transcription, nuclear entry, and integration are coordinated by a capsid uncoating process that is regulated by cellular proteins. Although uncoating is not well understood, recent studies have revealed insights into the process, particularly with respect to nuclear import pathways and protection of the viral genome from DNA sensors. Understanding uncoating will be valuable toward developing novel antiretroviral therapies for HIV-infected individuals.

  2. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model*

    OpenAIRE

    Tong, Guo-qing; Heng, Boon-chin; Ng, Soon-chye

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 µmol/L strontium chloride for 5 h and sub...

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

    OpenAIRE

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

    2008-01-01

    Functionalization of monodisperse superparamagnetic magnetite (Fe3O4) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe3O4 nanoparticles functionalized with protein and nuclear locali...

  4. Identification and Ultrastructural Characterization of a Novel Nuclear Degradation Complex in Differentiating Lens Fiber Cells.

    Science.gov (United States)

    Costello, M Joseph; Brennan, Lisa A; Mohamed, Ashik; Gilliland, Kurt O; Johnsen, Sönke; Kantorow, Marc

    2016-01-01

    An unresolved issue in structural biology is how the encapsulated lens removes membranous organelles to carry out its role as a transparent optical element. In this ultrastructural study, we establish a mechanism for nuclear elimination in the developing chick lens during the formation of the organelle-free zone. Day 12-15 chick embryo lenses were examined by high-resolution confocal light microscopy and thin section transmission electron microscopy (TEM) following fixation in 10% formalin and 4% paraformaldehyde, and then processing for confocal or TEM as described previously. Examination of developing fiber cells revealed normal nuclei with dispersed chromatin and clear nucleoli typical of cells in active ribosome production to support protein synthesis. Early signs of nuclear degradation were observed about 300 μm from the lens capsule in Day 15 lenses where the nuclei display irregular nuclear stain and prominent indentations that sometimes contained a previously undescribed macromolecular aggregate attached to the nuclear envelope. We have termed this novel structure the nuclear excisosome. This complex by confocal is closely adherent to the nuclear envelope and by TEM appears to degrade the outer leaflet of the nuclear envelope, then the inner leaflet up to 500 μm depth. The images suggest that the nuclear excisosome separates nuclear membrane proteins from lipids, which then form multilamellar assemblies that stain intensely in confocal and in TEM have 5 nm spacing consistent with pure lipid bilayers. The denuded nucleoplasm then degrades by condensation and loss of structure in the range 600 to 700 μm depth producing pyknotic nuclear remnants. None of these stages display any classic autophagic vesicles or lysosomes associated with nuclei. Uniquely, the origin of the nuclear excisosome is from filopodial-like projections of adjacent lens fiber cells that initially contact, and then appear to fuse with the outer nuclear membrane. These filopodial

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

    Science.gov (United States)

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

    2015-12-01

    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.

  6. Involvement of SRSF11 in cell cycle-specific recruitment of telomerase to telomeres at nuclear speckles

    OpenAIRE

    Lee, Ji Hoon; Jeong, Sun Ah; Khadka, Prabhat; Hong, Juyeong; Chung, In Kwon

    2015-01-01

    Telomerase, a unique ribonucleoprotein complex that contains the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC) and the TERC-binding protein dyskerin, is required for continued cell proliferation in stem cells and cancer cells. Here we identify SRSF11 as a novel TERC-binding protein that localizes to nuclear speckles, subnuclear structures that are enriched in pre-messenger RNA splicing factors. SRSF11 associates with active telomerase enzyme through an interacti...

  7. Lysophosphatidic acid induced nuclear translocation of nuclear factor-κB in Panc-1 cells by mobilizing cytosolic free calcium

    Institute of Scientific and Technical Information of China (English)

    Yoshiyuki Arita; Tetsuhide Ito; Takamasa Pond; Ken Kawabe; Terumasa Hisano; Ryoichi Takayanagi

    2008-01-01

    AIM: To clarify whether Lysophosphatidic acid (LPA) activates the nuclear translocation of nuclear factor-κB (NF-κB) in pancreatic cancer.METHODS: Panc-1, a human pancreatic cancer cell line, was used throughout the study. The expression of LPA receptors was confirmed by reverse-transcript polymerase chain reaction (RT-PCR). Cytosolic free calcium was measured by fluorescent calcium indicator fura-2, and the localization of NF-κB was visualized by immunofluorescent method with or without various agents, which effect cell signaling.RESULTS: Panc-1 expressed LPA receptors, LPAA1,LPA2 and LPA3. LPA caused the elevation of cytosolic free calcium dose-dependently. LPA also caused the nuclear translocation of NF-κB. Cytosolic free calcium was attenuated by pertussis toxin (PTX) and U73122,an inhibitor of phospholipase C. The translocation of NF-κB was similarly attenuated by PTX and U73122,but phorbol ester, an activator of protein kinase C,alone did not translocate NF-κB. Furthermore, the transtocation of NF-κB was completely blocked by Ca2+ chelator BAPTA-AM. Thapsigargin, an endoplasmic-reticulum Ca2+-ATPase pump inhibitor, also promoted the translocation of NF-κB. Staurosporine, a protein kinase C inhibitor, attenuated translocation of NF-κB induced by LPA.CONCLUSlON: These findings suggest that protein kinase C is activated endogenously in Panc-1, and protein kinase C is essential for activating NF-κB with cytosolic calcium and that LPA induces the nuclear translocation of NF-κB in Panc-1 by mobilizing cytosolic free calcium.

  8. Nuclear envelope proteins and chromatin arrangement: a pathogenic mechanism for laminopathies

    Directory of Open Access Journals (Sweden)

    NM Maraldi

    2009-06-01

    Full Text Available The involvement of the nuclear envelope in the modulation of chromatin organization is strongly suggested by the increasing number of human diseases due to mutations of nuclear envelope proteins. A common feature of these diseases, named laminopathies, is the occurrence of major chromatin defects. Laminopathies share in some instances their clinical features, but each of them is characterized by a phenotype that involves one or multiple tissues.We previously reported that cells from laminopathic patients show an altered nuclear profile, and loss or detachment of heterochromatin from the nuclear envelope. Recent evidence indicates that processing of the lamin A precursor is altered in laminopathies featuring pre-mature aging and/or lipodystrophy phenotype. In these cases, pre-lamin A is accumulated in the nucleus and heterochromatin is severely disorganized. Moreover, altered distribution and solubility properties of heterochromatin-associated proteins such as HP1 are observed. These findings indicate that defects of chromatin remodeling are involved in the cascade of epigenetic events leading to the laminopathic phenotypes. Here we report evidence indicating that pre-lamin A is mis-localized in the nuclei of Emery-Dreifuss muscular dystrophy fibroblasts, either bearing lamin A/C or emerin mutations. Abnornal pre-lamin A-containing structures are formed following treatment with a farnesyl-transferase inhibitor, a drug that causes accumulation of non-farnesylated pre-lamin A. Pre-lamin A-labeled structures co-localize with heterochromatin clumps. These data indicate that in almost all laminopathies the expression of the mutant lamin A precursor disrupts the organization of heterochromatin domains so that affected cells are unable to maintain the silenced chromatin state capable to allow/preserve terminal differentiation. Our results further show that the absence of emerin expression alters the distribution of pre-lamin A and of heterochromatin

  9. Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

    OpenAIRE

    Miyamoto, C.; Smith, G. E.; Farrell-Towt, J; Chizzonite, R.; Summers, M D; Ju, G.

    1985-01-01

    A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived pr...

  10. Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells

    Directory of Open Access Journals (Sweden)

    Kuang Lin-Yun

    2008-10-01

    Full Text Available Abstract Background The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed. Results We have constructed a series of gene expression vectors, based upon the pSAT series of vectors, to facilitate the practice of multi-color BiFC. The bait protein is tagged with the C-terminal portion of CFP (cCFP, and prey proteins are tagged with the N-terminal portions of either Venus (nVenus or Cerulean (nCerulean. Interaction of cCFP-tagged proteins with nVenus-tagged proteins generates yellow fluorescence, whereas interaction of cCFP-tagged proteins with nCerulean-tagged proteins generates blue fluorescence. Additional expression of mCherry indicates transfected cells and sub-cellular structures. Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin α-1 in the cytoplasm, whereas interaction of VirE2 with a different importin α isoform, importin α-4, occurs predominantly in the nucleus. Conclusion Multi

  11. Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Li-juan LI; Hua-jun ZHANG; Cong ZHANG; Zheng-li SHI

    2009-01-01

    The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS 1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLSI were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.

  12. Role of a nuclear localization signal on the minor capsid Proteins VP2 and VP3 in BKPyV nuclear entry

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, Shauna M. [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Zhao, Linbo [Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Bosard, Catherine [Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Imperiale, Michael J., E-mail: imperial@umich.edu [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States)

    2015-01-01

    BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin β1, we found that the importin α/β pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell. - Highlights: • Polyomaviruses must deliver their genome to the nucleus to replicate. • The minor capsid proteins have a well-conserved nuclear localization signal. • Mutation of this NLS diminishes, but does not completely inhibit, infection.

  13. Nuclear export and import of human hepatitis B virus capsid protein and particles.

    Directory of Open Access Journals (Sweden)

    Hung-Cheng Li

    Full Text Available It remains unclear what determines the subcellular localization of hepatitis B virus (HBV core protein (HBc and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS, while ARD-II and ARD-IV behave like two independent nuclear export signals (NES. This conclusion is based on five independent lines of experimental evidence: i Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT. iii By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1, which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel

  14. Resolution Improvement in Multidimensional Nuclear Magnetic Resonance Spectroscopy of Proteins

    International Nuclear Information System (INIS)

    The work presented in this thesis is concerned with both liquid-state and solid-state nuclear magnetic resonance (NMR) spectroscopy. Most of this work is devoted to the investigation by solid-state NMR of C13-enriched compounds with the principal aim of presenting techniques devised for further improving the spectral resolution in multidimensional NMR of microcrystalline proteins. In fully C13-labelled compounds, the J-coupling induces a broadening of the carbon lineshapes. We show that spin-state-selective technique called IPAP can be successfully combined with standard polarisation transfer schemes in order to remove the J-broadening in multidimensional solid-state NMR correlation experiments of fully C13-enriched proteins. We present subsequently two techniques tailored for liquid-state NMR spectroscopy. The carbon directly detected techniques provide chemical shift information for all backbone hetero-nuclei. They are very attracting for the study of large bio-molecular systems or for the investigation of paramagnetic proteins. In the last part of this thesis, we study the spin-echo J-modulation for homonuclear two-spin 1/2 systems. Under magic-angle spinning, the theory of J-induced spin-echo modulation allows to derive a set of modulation regimes which give a spin-echo modulation exactly equal to the J-coupling. We show that the chemical-shift anisotropy and the dipolar interaction tend to stabilize the spin-echo J-modulation. The theoretical conclusions are supported by numerical simulations and experimental results obtained for three representative samples containing C13 spin pairs. (author)

  15. A novel bipartite nuclear localization signal guides BPM1 protein to nucleolus suggesting its Cullin3 independent function.

    Directory of Open Access Journals (Sweden)

    Dunja Leljak Levanić

    Full Text Available BPM1 belongs to the MATH-BTB family of proteins, which act as substrate-binding adaptors for the Cullin3-based E3 ubiquitin ligase. MATH-BTB proteins associate with Cullin3 via the BTB domain and with the substrate protein via the MATH domain. Few BPM1-interacting proteins with different functions are recognized, however, specific roles of BPM1, depending on its cellular localization have not been studied so far. Here, we found a novel bipartite nuclear localization signal at the C-terminus of the BPM1 protein, responsible for its nuclear and nucleolar localization and sufficient to drive the green fluorescent protein and cytoplasmic BPM4 protein into the nucleus. Co-localization analysis in live Nicotiana tabacum BY2 cells indicates a Cullin3 independent function since BPM1 localization is predominantly nucleolar and thus devoid of Cullin3. Treatment of BY2 cells with the proteasome inhibitor MG132 blocks BPM1 and Cullin3 degradation, suggesting turnover of both proteins through the ubiquitin-proteasome pathway. Possible roles of BPM1 in relation to its in vivo localization are discussed.

  16. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.

    Science.gov (United States)

    Natsume, Toyoaki; Kiyomitsu, Tomomi; Saga, Yumiko; Kanemaki, Masato T

    2016-04-01

    Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function.

  17. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors

    Directory of Open Access Journals (Sweden)

    Toyoaki Natsume

    2016-04-01

    Full Text Available Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function.

  18. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.

    Science.gov (United States)

    Natsume, Toyoaki; Kiyomitsu, Tomomi; Saga, Yumiko; Kanemaki, Masato T

    2016-04-01

    Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function. PMID:27052166

  19. Nuclear Envelope Protein SUN2 Promotes Cyclophilin-A-Dependent Steps of HIV Replication

    Directory of Open Access Journals (Sweden)

    Xavier Lahaye

    2016-04-01

    Full Text Available During the early phase of replication, HIV reverse transcribes its RNA and crosses the nuclear envelope while escaping host antiviral defenses. The host factor Cyclophilin A (CypA is essential for these steps and binds the HIV capsid; however, the mechanism underlying this effect remains elusive. Here, we identify related capsid mutants in HIV-1, HIV-2, and SIVmac that are restricted by CypA. This antiviral restriction of mutated viruses is conserved across species and prevents nuclear import of the viral cDNA. Importantly, the inner nuclear envelope protein SUN2 is required for the antiviral activity of CypA. We show that wild-type HIV exploits SUN2 in primary CD4+ T cells as an essential host factor that is required for the positive effects of CypA on reverse transcription and infection. Altogether, these results establish essential CypA-dependent functions of SUN2 in HIV infection at the nuclear envelope.

  20. Nuclear import and dimerization of tomato ASR1, a water stress-inducible protein exclusive to plants.

    Directory of Open Access Journals (Sweden)

    Martiniano M Ricardi

    Full Text Available The ASR (for ABA/water stress/ripening protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS in the primary sequence. However, here we prove that such an "NLS" of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE.

  1. Long Unfolded Linkers Facilitate Membrane Protein Import Through the Nuclear Pore Complex

    NARCIS (Netherlands)

    Meinema, Anne C.; Laba, Justyna K.; Hapsari, Rizqiya A.; Otten, Renee; Mulder, Frans A. A.; Kralt, Annemarie; van den Bogaart, Geert; Lusk, C. Patrick; Poolman, Bert; Veenhoff, Liesbeth M.

    2011-01-01

    Active nuclear import of soluble cargo involves transport factors that shuttle cargo through the nuclear pore complex (NPC) by binding to phenylalanine-glycine (FG) domains. How nuclear membrane proteins cross through the NPC to reach the inner membrane is presently unclear. We found that at least a

  2. The transport of integral membrane proteins across the nuclear pore complex

    NARCIS (Netherlands)

    Meinema, Anne C.; Poolman, Bert; Veenhoff, Liesbeth M.

    2012-01-01

    The nuclear envelope protects and organizes the genome. The nuclear pore complexes embedded in the nuclear envelope allow selective transport of macromolecules between the cytosol and nucleoplasm, and as such help to control the flow of information from DNA to RNA to proteins. A growing list of inte

  3. Quantitative Analysis of Membrane Protein Transport Across the Nuclear Pore Complex

    NARCIS (Netherlands)

    Meinema, Anne C.; Poolman, Bert; Veenhoff, Liesbeth M.

    2013-01-01

    Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on

  4. Ubiquitin-regulated nuclear-cytoplasmic trafficking of the Nipah virus matrix protein is important for viral budding.

    Directory of Open Access Journals (Sweden)

    Yao E Wang

    Full Text Available Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4 pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS and the leucine-rich nuclear export signal (NES found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC(50 of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an "off-the-shelf" therapeutic against acute NiV infection.

  5. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China); Zhao, Bo; Kieff, Elliott [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States); Peng, Chih-Wen, E-mail: pengcw@mail.tcu.edu.tw [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)

    2013-01-18

    Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

  6. SU-E-J-61: Electrodynamics and Nano-Scale Fluid Dynamics in Protein Localization of Nuclear Pore Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Cunningham, J; Gatenby, R [Moffitt Cancer Research Institute, Tampa, FL (United States)

    2014-06-01

    Purpose: To develop a simulation to catalyze a reevaluation of common assumptions about 3 dimensional diffusive processes and help cell biologists gain a more nuanced, intuitive understanding of the true physical hurdles of protein signaling cascades. Furthermore, to discuss the possibility of intracellular electrodynamics as a critical, unrecognized component of cellular biology and protein dynamics that is necessary for optimal information flow from the cell membrane to the nucleus. Methods: The Unity 3D gaming physics engine was used to build an accurate virtual scale model of the cytoplasm within a few hundred nanometers of the nuclear membrane. A cloud of simulated pERK proteins is controlled by the physics simulation, where diffusion is based on experimentally measured values and the electrodynamics are based on theoretical nano-fluid dynamics. The trajectories of pERK within the cytoplasm and through the 1250 nuclear pores on the nuclear surface is recorded and analyzed. Results: The simulation quickly demonstrates that pERKs moving solely by diffusion will rarely locate and come within capture distance of a nuclear pore. The addition of intracellular electrodynamics between charges on the nuclear pore complexes and on pERKs increases the number of successful translocations by allowing the electro-physical attractive effects to draw in pERKs from the cytoplasm. The effects of changes in intracellular shielding ion concentrations allowed for estimation of the “capture radius” under varying conditions. Conclusion: The simulation allows a shift in perspective that is paramount in attempting to communicate the scale and dynamics of intracellular protein cascade mechanics. This work has allowed researchers to more fully understand the parameters involved in intracellular electrodynamics, such as shielding anion concentration and protein charge. As these effects are still far below the spatial resolution of currently available measurement technology this

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

    Energy Technology Data Exchange (ETDEWEB)

    Sakamoto, Hikaru [Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri-shi, Hokkaido 093-2422 (Japan); Sakata, Keiko; Kusumi, Kensuke [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Kojima, Mikiko; Sakakibara, Hitoshi [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan); Iba, Koh, E-mail: koibascb@kyushu-u.org [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)

    2012-06-29

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

  8. Mapping of the nuclear localization signals in open reading frame 2 protein from porcine circovirus type 1

    Institute of Scientific and Technical Information of China (English)

    Jiangbing Shuai; Wei Wei; Lingli Jiang; Xiaoliang Li; Ning Chen; Weihuan Fang

    2008-01-01

    Porcine circovirus type 1 (PCV1) contains two major open reading frames encoding the replication-associated proteins and the major structural capsid (Cap) protein.PCV1 Cap has an N-terminus carrying several potential monopartite or bipartite nuclear localization signals (NLS).The contribution of these partially overlapping motifs to nuclear importing was identified by expression of mutated PCV1 Cap versions fused to enhanced green fluorescent protein (EGFP).The Cterminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells similar to the wild-type PCV1 Cap-EGFP,whereas truncation of the N-terminus rendered the fusion protein distributed into cytoplasm,indicating that the nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region.Substitutions of basic residues in stretches 9RRRR12 or the right part of 25RRPYLAHPAFRNRYRWRRK43 resulted in a diffused distribution of the fusion protein in both nuclei and cytoplasm,indicating that the two NLSs were responsible for restricted nuclear targeting of PCV1 Cap.

  9. Calmodulin Involvement in Stress-Activated Nuclear Localization of Albumin in JB6 Epithelial Cells.

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Thomas J.; Negash, Sewite; Smallwood, Heather S.; Ramos, Kenneth S.; Thrall, Brian D.; Squier, Thomas C.

    2004-06-15

    We report that in response to oxidative stress, albumin is translocated to the nucleus where it binds in concert with known transcription factors to an antioxidant response element (ARE), which controls the expression of glutathione-S-transferase and other antioxidant enzymes, functioning to mediate adaptive cellular responses. To investigate the mechanisms underlying this adaptive cell response, we have identified linkages between calcium signaling and the nuclear translocation of albumin in JB6 epithelial cells. Under resting conditions, albumin and the calcium regulatory protein, calmodulin (CaM), co-immunoprecipitate using antibodies against either protein, indicating a tight association. Calcium activation of CaM disrupts the association between CaM and albumin, suggesting that transient increases in cytosolic calcium levels function to mobilize intracellular albumin to facilitate its translocation into the nucleus. Likewise, nuclear translocation of albumin is induced by exposure of cells to hydrogen peroxide or a phorbol ester, indicating a functional linkage between reactive oxygen species, calcium, and PKC-signaling pathways. Inclusion of an antioxidant enzyme (i.e., superoxide dismutase) blocks nuclear translocation, suggesting that the oxidation of sensitive proteins functions to coordinate the adaptive cellular response. These results suggest that elevated calcium transients, and associated increases in reactive oxygen species, contribute to adaptive cellular responses through the mobilization and nuclear translocation of cellular albumin to mediate the transcriptional regulation of antioxidant responsive elements.

  10. Unconventional Protein Secretion in Animal Cells.

    Science.gov (United States)

    Ng, Fanny; Tang, Bor Luen

    2016-01-01

    All eukaryotic cells secrete a range of proteins in a constitutive or regulated manner through the conventional or canonical exocytic/secretory pathway characterized by vesicular traffic from the endoplasmic reticulum, through the Golgi apparatus, and towards the plasma membrane. However, a number of proteins are secreted in an unconventional manner, which are insensitive to inhibitors of conventional exocytosis and use a route that bypasses the Golgi apparatus. These include cytosolic proteins such as fibroblast growth factor 2 (FGF2) and interleukin-1β (IL-1β), and membrane proteins that are known to also traverse to the plasma membrane by a conventional process of exocytosis, such as α integrin and the cystic fibrosis transmembrane conductor (CFTR). Mechanisms underlying unconventional protein secretion (UPS) are actively being analyzed and deciphered, and these range from an unusual form of plasma membrane translocation to vesicular processes involving the generation of exosomes and other extracellular microvesicles. In this chapter, we provide an overview on what is currently known about UPS in animal cells. PMID:27665549

  11. Mapping functional group free energy patterns at protein occluded sites: nuclear receptors and G-protein coupled receptors.

    Science.gov (United States)

    Lakkaraju, Sirish Kaushik; Yu, Wenbo; Raman, E Prabhu; Hershfeld, Alena V; Fang, Lei; Deshpande, Deepak A; MacKerell, Alexander D

    2015-03-23

    Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-γ (PPARγ) NRs and the metabotropic glutamate (mGluR) and β2-adreneric (β2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the β2AR LBP were used in virtual screening to identify high efficacy agonists targeting β2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents.

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

    Directory of Open Access Journals (Sweden)

    Aviad Levin

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

  13. Role of Heat Shock Proteins in Stem Cell Behavior

    OpenAIRE

    Fan, Guo-Chang

    2012-01-01

    Stress response is well appreciated to induce the expression of heat shock proteins (Hsps) in the cell. Numerous studies have demonstrated that Hsps function as molecular chaperones in the stabilization of intracellular proteins, repairing damaged proteins, and assisting in protein translocation. Various kinds of stem cells (embryonic stem cells, adult stem cells, or induced pluripotent stem cells) have to maintain their stemness and, under certain circumstances, undergo stress. Therefore, Hs...

  14. Biological Evaluation of Single Cell Protein

    International Nuclear Information System (INIS)

    In this study, the nutritional value of single cell protein (SCP) was evaluated as a non conventional protein source produced by fermenting fungal local strains of Trichoderma longibrachiatum, Aspergillus niger, Aspergillus terreus and Penicillium funiculosum with alkali treated sugar cane bagasse. Amino acid analysis revealed that the produced SCP contains essential and non essential amino acids. Male mice were fed on normal (basal) diet which contains 18% conventional protein and served as control group. In the second (T1) and the third (T2) group, the animals were fed on a diet in which 15% and 30% of conventional protein source were replaced by SCP, respectively. At intervals of 15, 30, 45 and 60 days, mice were sacrificed and the blood samples were collected for the biochemical evaluation. The daily averages of body weight were significantly higher with group T2 than group T1. Where as, the kidney weights in groups (T1) and (T2) were significantly increased as compared with control. A non significant difference between the tested groups in the enzyme activities of AST, ALT and GSH content of liver tissue were recorded. While, cholesterol and triglycerides contents showed a significant decrease in both (T1) and (T2) groups as compared with control. The recorded values of the serum hormone (T4), ALP activities, albumin and A/G ratio did not changed by the previous treatments. Serum levels of total protein, urea, creatinine and uric acid were higher for groups (T1) and (T2) than the control group. In conclusion, partial substitution of soy bean protein in mice diet with single cell protein (15%) improved the mice growth without any adverse effects on some of the physiological functions tested

  15. Gestational protein restriction alters cell proliferation in rat placenta.

    Science.gov (United States)

    Rebelato, Hércules Jonas; Esquisatto, Marcelo Augusto Marreto; de Sousa Righi, Eloá Fernanda; Catisti, Rosana

    2016-04-01

    We recently showed that gestational protein restriction (GPR) alters the structure of the rat placenta on day 19 of gestation (dG). The aim of the study was to investigate the spatial and temporal immunolocalization of proliferating cell antigen Ki67 in normal and GPR placental development. Pregnant Wistar rats were divided into two groups: normal (NP, 17 % casein) or low-protein diet (LP, 6 % casein). Placentas and fetus were collected and weighed at 15, 17, 19 and 21 dG. Morphological, morphometric and ultrastructural analyses were performed. Immunoperoxidase was used to identify nuclear antigen Ki67 in placental sections. We observed a significant reduction in the number of trophoblast giant cells and glycogen cells in the LP group. Placental weight was significantly reduced only at 17 dG in the LP group, in parallel to a decrease in glycogen cells. From 15 to 21 dG, the thickness of the junctional zone (JZ) decreased in NP and LP animals, while that of the labyrinth zone (LZ) increased in parallel to a reduction in the number of proliferating cells in this LZ zone. GPR significantly inhibits cell proliferation in the JZ, especially at 15 and 17 dG. The ultrastructural appearance of the cytoplasm of giant and cytotrophoblastic cells indicates degeneration from 15 to 21 dG and this effect is enhanced in LP animals suggesting early aging. Offspring of NP dams were significantly heavier than offspring of LP dams at 21 dG. GPR causes modifications in specific regions of the placenta, cell proliferation inhibition and fetal growth restriction.

  16. 20-Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis-related protein in the silkworm, Bombyx mori.

    Science.gov (United States)

    Ji, M-M; Liu, A-Q; Sima, Y-H; Xu, S-Q

    2016-10-01

    The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx. PMID:27329527

  17. 70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY*

    Science.gov (United States)

    Kaur, Gurpreet; Lieu, Kim G.; Jans, David A.

    2013-01-01

    We recently showed that the developmentally important family of SOX (SRY (sex determining region on the Y chromosome)-related high mobility group (HMG) box) proteins require the calcium-binding protein calmodulin (CaM) for optimal nuclear accumulation, with clinical mutations in SRY that specifically impair nuclear accumulation via this pathway resulting in XY sex reversal. However, the mechanism by which CaM facilitates nuclear accumulation is unknown. Here, we show, for the first time, that the 70-kDa heat shock cognate protein hsc70 plays a key role in CaM-dependent nuclear import of SRY. Using a reconstituted nuclear import assay, we show that antibodies to hsc70 significantly reduce nuclear accumulation of wild type SRY and mutant derivatives thereof that retain CaM-dependent nuclear import, with an increased rate of nuclear accumulation upon addition of both CaM and hsc70, in contrast to an SRY mutant derivative with impaired CaM binding. siRNA knockdown of hsc70 in intact cells showed similar results, indicating clear dependence upon hsc70 for CaM-dependent nuclear import. Analysis using the technique of fluorescence recovery after photobleaching indicated that hsc70 is required for the maximal rate of SRY nuclear import in living cells but has no impact upon SRY nuclear retention/nuclear dynamics. Finally, we demonstrate direct binding of hsc70 to the SRY·CaM complex, with immunoprecipitation experiments from cell extracts showing association of hsc70 with wild type SRY, but not with a mutant derivative with impaired CaM binding, dependent on Ca2+. Our novel findings strongly implicate hsc70 in CaM-dependent nuclear import of SRY. PMID:23235156

  18. A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation.

    Science.gov (United States)

    Edens, Lisa J; Levy, Daniel L

    2016-01-01

    A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies. PMID:27584618

  19. Cell wall proteins: a new insight through proteomics

    OpenAIRE

    Jamet, Elisabeth; Canut, Hervé; Boudart, Georges; Pont-Lezica, Rafael F

    2006-01-01

    Cell wall proteins are essential constituents of plant cell walls; they are involved in modifications of cell wall components, wall structure, signaling and interactions with plasma membrane proteins at the cell surface. The application of proteomic approaches to the cell wall compartment raises important questions: are there technical problems specific to cell wall proteomics? What kinds of proteins can be found in Arabidopsis walls? Are some of them unexpected? What sort of post-translation...

  20. Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Kara G Lassen

    2006-07-01

    Full Text Available HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART. We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

  1. Generation of bovine transgenics using somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Stice Steven L

    2003-11-01

    Full Text Available Abstract The ability to produce transgenic animals through the introduction of exogenous DNA has existed for many years. However, past methods available to generate transgenic animals, such as pronuclear microinjection or the use of embryonic stem cells, have either been inefficient or not available in all animals, bovine included. More recently somatic cell nuclear transfer has provided a method to create transgenic animals that overcomes many deficiencies present in other methods. This review summarizes the benefits of using somatic cell nuclear transfer to create bovine transgenics as well as the possible opportunities this method creates for the future.

  2. Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: Implications for cell proliferation control

    Energy Technology Data Exchange (ETDEWEB)

    Radulescu, Razvan T., E-mail: ratura@gmx.net [Molecular Concepts Research (MCR), Muenster (Germany); Duckworth, William C. [Department of Medicine, Phoenix VA Health Care System, Phoenix, AZ (United States); Levy, Jennifer L. [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States); Fawcett, Janet, E-mail: janet.fawcett@va.gov [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States)

    2010-04-30

    Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110 kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.

  3. Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells.

    Science.gov (United States)

    Thomas, T; Shah, N; Klinge, C M; Faaland, C A; Adihkarakunnathu, S; Gallo, M A; Thomas, T J

    1999-04-01

    We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells. PMID:10194516

  4. A novel single cell method to identify the genetic composition at a single nuclear body

    Science.gov (United States)

    Anchel, David; Ching, Reagan W.; Cotton, Rachel; Li, Ren; Bazett-Jones, David P.

    2016-01-01

    Gene loci make specific associations with compartments of the nucleus (e.g. the nuclear envelope, nucleolus, and transcription factories) and this association may determine or reflect a mechanism of genetic control. With current methods, it is not possible to identify sets of genes that converge to form a “gene hub” as there is a reliance on loci-specific probes, or immunoprecipitation of a particular protein from bulk cells. We introduce a method that will allow for the identification of loci contained within the vicinity of a single nuclear body in a single cell. For the first time, we demonstrate that the DNA sequences originating from a single sub-nuclear structure in a single cell targeted by two-photon irradiation can be determined, and mapped to a particular locus. Its application to single PML nuclear bodies reveals ontologically related loci that frequently associate with each other and with PML bodies in a population of cells, and a possible nuclear body targeting role for specific transcription factor binding sites. PMID:27389808

  5. Nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(A)-binding protein are distinct processes mediated by two Epstein Barr virus proteins.

    Science.gov (United States)

    Park, Richard; El-Guindy, Ayman; Heston, Lee; Lin, Su-Fang; Yu, Kuan-Ping; Nagy, Mate; Borah, Sumit; Delecluse, Henri-Jacques; Steitz, Joan; Miller, George

    2014-01-01

    Many viruses target cytoplasmic polyA binding protein (PABPC) to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs). During lytic replication of Epstein Barr Virus (EBV) we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E), was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors. PMID:24705134

  6. Nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(A-binding protein are distinct processes mediated by two Epstein Barr virus proteins.

    Directory of Open Access Journals (Sweden)

    Richard Park

    Full Text Available Many viruses target cytoplasmic polyA binding protein (PABPC to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs. During lytic replication of Epstein Barr Virus (EBV we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E, was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors.

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

    International Nuclear Information System (INIS)

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

  8. Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix- associated PML bodies

    OpenAIRE

    1995-01-01

    The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes ...

  9. Nuclear vlimata and aneuploidy in embryonic cells is caused by meiosis. Behaviour and properties of meiotic cells

    OpenAIRE

    Logothetou-Rella, H.

    1995-01-01

    This study demonstrates that human embryonic cells divide by meiosis. The use of trophoblastic tissue cells (early embryo) and amniotic cells (late embryo) exhibited the following characteristic events of meiosis: nuclear (NVs) and nucleolar (NuVs) vlimata formation; NV invasion in host cells; extrusion of chromosomes; nuclear fusion; metaphase fusion; hybrid cell formation; nuclear, nucleolar and cytoplasmic bridges, chromosomal transfer, variablesized nuc...

  10. Sumoylation regulates nuclear localization of lipin-1alpha in neuronal cells.

    Directory of Open Access Journals (Sweden)

    Guang-Hui Liu

    Full Text Available Lipin-1 is a protein that has dual functions as a phosphatidic acid phosphohydrolase (PAP and a nuclear transcriptional coactivator. It remains unknown how the nuclear localization and coactivator functions of lipin-1 are regulated. Here, we show that lipin-1 (including both the alpha and beta isoforms is modified by sumoylation at two consensus sumoylation sites. We are unable to detect sumoylation of the related proteins lipin-2 and lipin-3. Lipin-1 is sumoylated at relatively high levels in brain, where lipin-1alpha is the predominant form. In cultured embryonic cortical neurons and SH-SY5Y neuronal cells, ectopically expressed lipin-1alpha is localized in both the nucleus and the cytoplasm, and the nuclear localization is abrogated by mutating the consensus sumyolation motifs. The sumoylation site mutant of lipin-1alpha loses the capacity to coactivate the transcriptional (co- activators PGC-1alpha and MEF2, consistent with its nuclear exclusion. Thus, these results show that sumoylation facilitates the nuclear localization and transcriptional coactivator behavior of lipin-1alpha that we observe in cultured neuronal cells, and suggest that lipin-1alpha may act as a sumoylation-regulated transcriptional coactivator in brain.

  11. Nuclear motility in glioma cells reveals a cell-line dependent role of various cytoskeletal components.

    Directory of Open Access Journals (Sweden)

    Alexa Kiss

    Full Text Available Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns--thereby forced into a bipolar morphology--displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved.

  12. The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1–AMPK complex

    International Nuclear Information System (INIS)

    Highlights: ► The nuclear protein Artemis physically interacts with AMPKα2. ► Artemis co-localizes with AMPKα2 in the nucleus. ► Artemis promotes phosphorylation and activation of AMPK. ► The interaction between AMPKα2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic α subunit and regulatory β and γ subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the α-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPKα2-binding protein. Artemis was found to co-immunoprecipitate with AMPKα2, and the co-localization of Artemis with AMPKα2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPKα2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPKα2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1–AMPK complex.

  13. The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1-AMPK complex

    Energy Technology Data Exchange (ETDEWEB)

    Nakagawa, Koji, E-mail: k_nakagawa@pharm.hokudai.ac.jp [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Asaka, Masahiro [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Takeda, Hiroshi [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Kobayashi, Masanobu [Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); School of Nursing and Social Services, Health Sciences University of Hokkaido, Ishikari-Toubetsu, Hokkaido 061-0293 (Japan)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer The nuclear protein Artemis physically interacts with AMPK{alpha}2. Black-Right-Pointing-Pointer Artemis co-localizes with AMPK{alpha}2 in the nucleus. Black-Right-Pointing-Pointer Artemis promotes phosphorylation and activation of AMPK. Black-Right-Pointing-Pointer The interaction between AMPK{alpha}2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic {alpha} subunit and regulatory {beta} and {gamma} subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the {alpha}-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPK{alpha}2-binding protein. Artemis was found to co-immunoprecipitate with AMPK{alpha}2, and the co-localization of Artemis with AMPK{alpha}2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPK{alpha}2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPK{alpha}2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex.

  14. Nuclear lipid microdomain as resting place of dexamethasone to impair cell proliferation.

    Science.gov (United States)

    Cataldi, Samuela; Codini, Michela; Cascianelli, Giacomo; Tringali, Sabina; Tringali, Anna Rita; Lazzarini, Andrea; Floridi, Alessandro; Bartoccini, Elisa; Garcia-Gil, Mercedes; Lazzarini, Remo; Ambesi-Impiombato, Francesco Saverio; Curcio, Francesco; Beccari, Tommaso; Albi, Elisabetta

    2014-01-01

    The action of dexamethasone is initiated by, and strictly dependent upon, the interaction of the drug with its receptor followed by its translocation into the nucleus where modulates gene expression. Where the drug localizes at the intranuclear level is not yet known. We aimed to study the localization of the drug in nuclear lipid microdomains rich in sphingomyelin content that anchor active chromatin and act as platform for transcription modulation. The study was performed in non-Hodgkin's T cell human lymphoblastic lymphoma (SUP-T1 cell line). We found that when dexamethasone enters into the nucleus it localizes in nuclear lipid microdomains where influences sphingomyelin metabolism. This is followed after 24 h by a cell cycle block accompanied by the up-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin-dependent kinase inhibitor 1B (CDKN1B), growth arrest and DNA-damage 45A (GADD45A), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes and by the reduction of signal transducer and activator of transcription 3 (STAT3) and phospho signal transducer and activator of transcription 3 (phoshoSTAT3) proteins. After 48 h some cells show morphological changes characteristic of apoptosis while the number of the cells that undergo cell division and express B-cell lymphoma-2 (Bcl-2) is very low. We suggest that the integrity of nuclear lipid microdomains is important for the response to glucocorticoids of cancer cells.

  15. Nuclear Lipid Microdomain as Resting Place of Dexamethasone to Impair Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Samuela Cataldi

    2014-10-01

    Full Text Available The action of dexamethasone is initiated by, and strictly dependent upon, the interaction of the drug with its receptor followed by its translocation into the nucleus where modulates gene expression. Where the drug localizes at the intranuclear level is not yet known. We aimed to study the localization of the drug in nuclear lipid microdomains rich in sphingomyelin content that anchor active chromatin and act as platform for transcription modulation. The study was performed in non-Hodgkin’s T cell human lymphoblastic lymphoma (SUP-T1 cell line. We found that when dexamethasone enters into the nucleus it localizes in nuclear lipid microdomains where influences sphingomyelin metabolism. This is followed after 24 h by a cell cycle block accompanied by the up-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A, cyclin-dependent kinase inhibitor 1B (CDKN1B, growth arrest and DNA-damage 45A (GADD45A, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH genes and by the reduction of signal transducer and activator of transcription 3 (STAT3 and phospho signal transducer and activator of transcription 3 (phoshoSTAT3 proteins. After 48 h some cells show morphological changes characteristic of apoptosis while the number of the cells that undergo cell division and express B-cell lymphoma-2 (Bcl-2 is very low. We suggest that the integrity of nuclear lipid microdomains is important for the response to glucocorticoids of cancer cells.

  16. Nuclear microscopy of sperm cell elemental structure

    International Nuclear Information System (INIS)

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility. (orig.)

  17. Recent Progress of Somatic Cell Nuclear Transfer in Pigs

    Institute of Scientific and Technical Information of China (English)

    Xu Xiaoming; Dou Zhongying

    2005-01-01

    Research in the field of somatic cell nuclear transfer (SCNT) and transgenic cloning in pigs has become a global hotspot, because porcine organs probably can be the first source of donor organs for human xenotransplantation. In recent years, though great progress has been made in porcine SCNT, the efficiency of nuclear transfer remains very low (<1% ). Thus, it is necessary to improve the procedure of nuclear transfer and to investigate some basic problems further. Recent progress and the related problems of SCNT in pigs are reviewed and analyzed so as to offer some beneficial illumination to researchers.

  18. Identification of nuclear phosphatidylinositol 4,5-bisphosphate-interacting proteins by neomycin extraction.

    Science.gov (United States)

    Lewis, Aurélia E; Sommer, Lilly; Arntzen, Magnus Ø; Strahm, Yvan; Morrice, Nicholas A; Divecha, Nullin; D'Santos, Clive S

    2011-02-01

    Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(X(n= 3-7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase IIα. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions.

  19. Synthetic protein interactions reveal a functional map of the cell

    Science.gov (United States)

    Berry, Lisa K; Ólafsson, Guðjón; Ledesma-Fernández, Elena; Thorpe, Peter H

    2016-01-01

    To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.13053.001 PMID:27098839

  20. Karyopherin α 3 and karyopherin α 4 proteins mediate the nuclear import of methyl-CpG binding protein 2.

    Science.gov (United States)

    Baker, Steven Andrew; Lombardi, Laura Marie; Zoghbi, Huda Yahya

    2015-09-11

    Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein with important roles in regulating chromatin structure and gene expression, and mutations in MECP2 cause Rett syndrome (RTT). Within the MeCP2 protein sequence, the nuclear localization signal (NLS) is reported to reside between amino acids 255-271, and certain RTT-causing mutations overlap with the MeCP2 NLS, suggesting that they may alter nuclear localization. One such mutation, R270X, is predicted to interfere with the localization of MeCP2, but recent in vivo studies have demonstrated that this mutant remains entirely nuclear. To clarify the mechanism of MeCP2 nuclear import, we isolated proteins that interact with the NLS and identified karyopherin α 3 (KPNA3 or Kap-α3) and karyopherin α 4 (KPNA4 or Kap-α4) as key binding partners of MeCP2. MeCP2-R270X did not interact with KPNA4, consistent with a requirement for an intact NLS in this interaction. However, this mutant retains binding to KPNA3, accounting for the normal localization of MeCP2-R270X to the nucleus. These data provide a mechanism for MeCP2 nuclear import and have implications for the design of therapeutics aimed at modulating the function of MeCP2 in RTT patients.

  1. Epstein - Barr virus latent membrane protein 1 suppresses reporter activity through modulation of promyelocytic leukemia protein-nuclear bodies

    Directory of Open Access Journals (Sweden)

    Flemington Erik K

    2011-10-01

    Full Text Available Abstract The Epstein-Barr virus (EBV encoded Latent Membrane Protein 1 (LMP1 has been shown to increase the expression of promyelocytic leukemia protein (PML and the immunofluorescent intensity of promyelocytic leukemia nuclear bodies (PML NBs. PML NBs have been implicated in the modulation of transcription and the association of reporter plasmids with PML NBs has been implicated in repression of reporter activity. Additionally, repression of various reporters in the presence of LMP1 has been noted. This study demonstrates that LMP1 suppresses expression of reporter activity in a dose responsive manner and corresponds with the LMP1 induced increase in PML NB intensity. Disruption of PML NBs with arsenic trioxide or a PML siRNA restores reporter activity. These data offer an explanation for previously conflicting data on LMP1 signaling and calls attention to the possibility of false-positives and false-negatives when using reporter assays as a research tool in cells expressing LMP1.

  2. Hydrogen peroxide activates activator protein-1 and mitogen-activated protein kinases in pancreatic stellate cells.

    Science.gov (United States)

    Kikuta, Kazuhiro; Masamune, Atsushi; Satoh, Masahiro; Suzuki, Noriaki; Satoh, Kennichi; Shimosegawa, Tooru

    2006-10-01

    Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis, where oxidative stress is thought to play a key role. Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) may act as a second messenger to mediate the actions of growth factors and cytokines. But the role of reactive oxygen species in the activation and regulation of cell functions in PSCs remains largely unknown. We here examined the effects of H(2)O(2) on the activation of signal transduction pathways and cell functions in PSCs. PSCs were isolated from the pancreas of male Wistar rats, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay. Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. The effects of H(2)O(2) on proliferation, alpha(1)(I)procollagen gene expression, and monocyte chemoattractant protein-1 production were evaluated. The effect of H(2)O(2) on the transformation of freshly isolated PSCs in culture was also assessed. H(2)O(2) at non-cytotoxic concentrations (up to 100 microM) induced oxidative stress in PSCs. H(2)O(2) activated activator protein-1, but not nuclear factor kappaB. In addition, H(2)O(2) activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. H(2)O(2) induced alpha(1)(I)procollagen gene expression but did not induce proliferation or monocyte chemoattractant protein-1 production. H(2)O(2) did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype. Specific activation of these signal transduction pathways and collagen gene expression by H(2)O(2) may play a role in the pathogenesis of pancreatic fibrosis.

  3. Hot-cell for dismantling of nuclear gauges

    CERN Document Server

    Reis, L C A

    2000-01-01

    This work objectives the design of a hot-cell that will be used for dismantling of nuclear gauges. In the hot-cell, nuclear gauges received as radioactive waste at the Centro de Desenvolvimento da Tecnologia Nuclear - CDTN will be dismantled, in order to decrease the volume of radioactive waste to be stored at the Center. Sources originally conditioned as special form radioactive material will be tested and in case do not present leakage, the respective gauges will be disposable for reusing by radioisotope users. The remaining sources will be taken off the original shielding and conditioned in special packages adequate for storage and disposal. All steps of work, the hot-cell design and methodology for conditioning are also described.

  4. Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

    Institute of Scientific and Technical Information of China (English)

    GONG Guochun; LI Rong; LI Ning; DAI Yunping; FAN Baoliang; ZHU Huabing; WANG Haiping; WANG Lili; FANG Changge; WAN Rong; LIU Ying

    2004-01-01

    The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 μg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.

  5. Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells.

    Science.gov (United States)

    Ikari, Akira; Watanabe, Ryo; Sato, Tomonari; Taga, Saeko; Shimobaba, Shun; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Endo, Satoshi; Matsunaga, Toshiyuki; Sugatani, Junko

    2014-09-01

    Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.

  6. Glucose enhances collectrin protein expression in insulin-producing MIN6 {beta} cells

    Energy Technology Data Exchange (ETDEWEB)

    Saisho, Kenji; Fukuhara, Atsunori [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan); Yasuda, Tomoko [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan); Sato, Yoshifumi; Fukui, Kenji; Iwahashi, Hiromi; Imagawa, Akihisa [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan); Hatta, Mitsutoki [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan); Shimomura, Iichiro [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan); Yamagata, Kazuya, E-mail: k-yamaga@kumamoto-u.ac.jp [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan)

    2009-11-06

    Collectrin is a novel target gene of hepatocyte nuclear factor-1{alpha} in pancreatic {beta}-cells and controls insulin exocytosis. Although glucose is known to stimulate the expression of genes of the insulin secretory pathway, there is no information on how glucose regulates collectrin expression. We investigated the effects of glucose on the expression of collectrin in MIN6 {beta}-cell line. Glucose, in a dose-dependent manner, increased collectrin protein levels without changing collectrin mRNA levels and protein stability, indicating that glucose stimulation of collectrin protein expression is primarily mediated at a translational level. Although mannose and pyruvate also increased collectrin protein expression level, neither 2-deoxyglucose, mitochondrial fuels leucine and glutamate, sulphonylurea nor Ca{sup 2+} channel blockers, mimicked the effects of glucose. These data indicate the involvement of mitochondrial TCA cycle intermediates, distal to pyruvate, in the regulation of collectrin protein expression in {beta}-cells.

  7. Intracellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Absence of Nucleolar Accumulation during Infection and after Expression as a Recombinant Protein in Vero Cells

    OpenAIRE

    Rowland, Raymond R. R.; Chauhan, Vinita; Fang, Ying; Pekosz, Andrew; Kerrigan, Maureen; Burton, Miriam D.

    2005-01-01

    The nucleocapsid (N) protein of several members within the order Nidovirales localizes to the nucleolus during infection and after transfection of cells with N genes. However, confocal microscopy of N protein localization in Vero cells infected with the severe acute respiratory syndrome coronavirus (SARS-CoV) or transfected with the SARS-CoV N gene failed to show the presence of N in the nucleoplasm or nucleolus. Amino acids 369 to 389, which contain putative nuclear localization signal (NLS)...

  8. Nuclear-localized Calcineurin Homologous Protein CHP1 Interacts with Upstream Binding Factor and Inhibits Ribosomal RNA Synthesis*

    OpenAIRE

    Jiménez-Vidal, Maite; Srivastava, Jyoti; Putney, Luanna K; Barber, Diane L.

    2010-01-01

    Calcineurin homologous protein 1 (CHP1) is a widely expressed, 22-kDa myristoylated EF-hand Ca2+-binding protein that shares a high degree of similarity with the regulatory B subunit of calcineurin (65%) and with calmodulin (59%). CHP1 localizes to the plasma membrane, the Golgi apparatus, and the nucleus and functions to regulate trafficking of early secretory vesicles, activation of T cells, and expression and transport of the Na-H exchanger NHE1. Although CHP1 contains nuclear export signa...

  9. Analyzing Cell Death by Nuclear Staining with Hoechst 33342.

    Science.gov (United States)

    Crowley, Lisa C; Marfell, Brooke J; Waterhouse, Nigel J

    2016-01-01

    The nuclei of healthy cells are generally spherical, and the DNA is evenly distributed. During apoptosis the DNA becomes condensed, but this process does not occur during necrosis. Nuclear condensation can therefore be used to distinguish apoptotic cells from healthy cells or necrotic cells. Dyes that bind to DNA, such as Hoechst 33342 or 4',6-diamidino-2-phenylindole (DAPI), can be used to observe nuclear condensation. These dyes fluoresce at 461 nm when excited by ultraviolet light and can therefore be visualized using conventional fluorescent microscopes equipped with light sources that emit light at ∼350 nm and filter sets that permit the transmission of light at ∼460 nm. This protocol describes staining and visualization of cells stained with Hoechst 33342, but it can be adapted for staining with DAPI or other dyes. PMID:27587774

  10. KIFC1-like motor protein associates with the cephalopod manchette and participates in sperm nuclear morphogenesis in Octopus tankahkeei.

    Directory of Open Access Journals (Sweden)

    Wei Wang

    Full Text Available BACKGROUND: Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery. METHODOLOGY/PRINCIPAL FINDINGS: We detected the presence of a KIFC1-like protein in the testis, muscle, and liver of O. tankahkeei by Western Blot. Then we tracked its dynamic localization in spermatic cells at various stages using Immunofluorescence and Immunogold Electron Microscopy. The KIFC1-like protein was not expressed at early stages of spermatogenesis when no significant morphological changes occur, began to be present in early spermatid, localized around and in the nucleus of intermediate and late spermatids where the nucleus was dramatically elongated and compressed, and concentrated at one end of final spermatid. Furthermore, distribution of the motor protein during nuclear elongation and condensation overlapped with that of the cephalopod counterpart of manchette at a significant level. CONCLUSIONS/SIGNIFICANCE: The results support the assumption that the protein is actively involved in sperm nuclear morphogenesis in O. tankahkeei possibly through bridging the manchette-like perinuclear microtubules to the nucleus and assisting in the nucleocytoplasmic trafficking of specific cargoes. This study represents the first description of the role of a motor protein in sperm nuclear shaping in cephalopod.

  11. Single-cell protein from waste cellulose

    Science.gov (United States)

    Dunlap, C. E.; Callihan, C. D.

    1973-01-01

    The recycle, reuse, or reclamation of single cell protein from liquid and solid agricultural waste fibers by a fermentation process is reported. It is shown that cellulose comprises the bulk of the fibers at 50% to 55% of the dry weight of the refuse and that its biodegradability is of prime importance in the choice of a substrate. The application of sodium hydroxide followed by heat and pressure serves to de-polymerize and disrupt lignin structure while swelling the cellulose to increase water uptake and pore volume. Some of the lignin, hemi-celluloses, ash, and cellulose of the material is hydrolized and solubilized. Introduction of microorganisms to the substrate fibers mixed with nutrients produces continuous fermentation of cellulose for further protein extraction and purification.

  12. Biosynthesis and release of proteins by isolated pulmonary Clara cells

    International Nuclear Information System (INIS)

    The major proteins synthesized and released by Clara cells were identified and compared with those synthesized and released by mixed lung cells. Highly purified Clara cells (85.9 +/- 2.4%) and mixed lung cells (Clara cells 4%, Type II cells 33%, granulocytes 18%, macrophages 2.7%, ciliated cells 1.2%) were isolated from rabbit lungs, incubated with Ham's F12 medium in collagen/fibronectin-coated plastic culture dishes in the presence of 35S-methionine for periods of 4 and 18 hrs. Radiolabelled proteins were isolated from the cells and from the culture medium, electrophoresed on polyacrylamide gels in the presence of SDS under reducing conditions, and then autoradiographed. After 4 and 18 hr of incubation of the Clara cells the major radiolabelled cell-associated proteins were those with molecular weights of 6, 48, and 180 Kd. The major radiolabelled proteins released by Clara cells into the medium after 4 hrs of incubation had molecular weights of 6, 48, and 180 Kd, accounting for 42, 16, and 10%, respectively, of the total extracellular protein-associated radioactivity. After 18 hr of incubation the 6 and 48 Kd proteins represented 30 and 18% of the total released radioactivity, and the relative amount of the 180 Kd protein had decreased to 3%. With the mixed lung cells, the major proteins released into the medium had molecular weights of 6 and 48 Kd. Under nonreducing conditions the 6 Kd protein released by Clara cells had an apparent molecular weight of 12 Kd. Labelling isolated Clara cells with a mixture of 14C-amino acids also identified this low molecular weight protein as the major secretory product of the Clara cell. The 6 Kd protein did not label when the cells were incubated with 14C-glucosamine indicating that it was not a glycoprotein. Data demonstrate the release of several proteins from isolated Clara cells but the major protein had a M.W. of 6 Kd

  13. Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

    NARCIS (Netherlands)

    Kralt, Annemarie; Jagalur, Noorjahan B.; van den Boom, Vincent; Lokareddy, Ravi K.; Steen, Anton; Cingolani, Gino; Fornerod, Maarten; Veenhoff, Liesbeth M.

    2015-01-01

    Endoplasmic reticulum-synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a

  14. Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA

    Science.gov (United States)

    Orzalli, Megan H.; Conwell, Sara E.; Berrios, Christian; DeCaprio, James A.; Knipe, David M.

    2013-01-01

    Mammalian cells have evolved mechanisms to silence foreign DNA introduced by viruses or by transfection. Upon herpesviral infection of cells, the viral genome is chromatinized in an attempt by the host cell to restrict expression of the viral genome. HSV ICP0 acts to counter host-intrinsic and innate responses to viral infection. We have found that nuclear interferon (IFN)-inducible protein 16 (IFI16) acts as a restriction factor against ICP0-null herpes simplex virus 1 (HSV-1) to limit viral replication and immediate–early gene expression. IFI16 promoted the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin. IFI16 also restricted the expression of plasmid DNAs introduced by transfection but did not restrict SV40 DNA introduced into the cellular nucleus in the form of nucleosomal chromatin by viral infection. These results argue that IFI16 restricts unchromatinized DNA when it enters the cell nucleus by promoting the loading of nucleosomes and the addition of heterochromatin marks. Furthermore, these results indicate that IFI16 provides a broad surveillance role against viral and transfected DNA by promoting restriction of gene expression from the exogenous DNA and inducing innate immune responses. PMID:24198334

  15. Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA.

    Science.gov (United States)

    Orzalli, Megan H; Conwell, Sara E; Berrios, Christian; DeCaprio, James A; Knipe, David M

    2013-11-19

    Mammalian cells have evolved mechanisms to silence foreign DNA introduced by viruses or by transfection. Upon herpesviral infection of cells, the viral genome is chromatinized in an attempt by the host cell to restrict expression of the viral genome. HSV ICP0 acts to counter host-intrinsic and innate responses to viral infection. We have found that nuclear interferon (IFN)-inducible protein 16 (IFI16) acts as a restriction factor against ICP0-null herpes simplex virus 1 (HSV-1) to limit viral replication and immediate-early gene expression. IFI16 promoted the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin. IFI16 also restricted the expression of plasmid DNAs introduced by transfection but did not restrict SV40 DNA introduced into the cellular nucleus in the form of nucleosomal chromatin by viral infection. These results argue that IFI16 restricts unchromatinized DNA when it enters the cell nucleus by promoting the loading of nucleosomes and the addition of heterochromatin marks. Furthermore, these results indicate that IFI16 provides a broad surveillance role against viral and transfected DNA by promoting restriction of gene expression from the exogenous DNA and inducing innate immune responses. PMID:24198334

  16. Ribosome Protein L4 is essential for Epstein-Barr Virus Nuclear Antigen 1 function.

    Science.gov (United States)

    Shen, Chih-Lung; Liu, Cheng-Der; You, Ren-In; Ching, Yung-Hao; Liang, Jun; Ke, Liangru; Chen, Ya-Lin; Chen, Hong-Chi; Hsu, Hao-Jen; Liou, Je-Wen; Kieff, Elliott; Peng, Chih-Wen

    2016-02-23

    Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. We have now found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP-mediated episome binding and maintenance, whereas the NCL C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection. PMID:26858444

  17. A Cell-Based Protein-Protein Interaction Method Using a Permuted Luciferase Reporter

    OpenAIRE

    Eishingdrelo, Haifeng; Cai, Jidong; Weissensee, Paul; Sharma, Praveen; Tocci, Michael J; Wright, Paul S

    2011-01-01

    We have developed a novel cell-based protein-protein interaction assay method. The method relies on conversion of an inactive permuted luciferase containing a Tobacco Etch Virus protease (TEV) cleavage sequence fused onto protein (A) to an active luciferase upon interaction and cleavage by another protein (B) fused with the TEV protease. We demonstrate assay applicability for ligand-induced protein-protein interactions including G-protein coupled receptors, receptor tyrosine kinases and nucle...

  18. Sde2: A novel nuclear protein essential for telomeric silencing and genomic stability in Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Sugioka-Sugiyama, Rie [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Initiative for the Promotion of Young Scientists' Independent Research, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Sugiyama, Tomoyasu, E-mail: sugiyamt@biol.tsukuba.ac.jp [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Initiative for the Promotion of Young Scientists' Independent Research, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012 (Japan)

    2011-03-18

    Research highlights: {yields} Sde2 is essential for telomere silencing. {yields} Sde2 is involved in the maintenance of genomic stability. {yields} Sde2 promotes the recruitment of SHREC, a histone deacetylase complex, to telomeres. -- Abstract: Telomeres, specialized domains assembled at the ends of linear chromosomes, are essential for genomic stability in eukaryotes. The formation and maintenance of telomeres are governed by numerous factors such as telomeric repeats, telomere-binding proteins, heterochromatin proteins, and telomerase. Here, we report Sde2, a novel nuclear protein essential for telomeric silencing and genomic stability in the fission yeast Schizosaccharomyces pombe. A deficiency in sde2 results in the derepression of the ura4{sup +} gene inserted near telomeric repeats, and the noncoding transcripts from telomeric regions accumulate in sde2{Delta} cells. The loss of Sde2 function compromises transcriptional silencing at telomeres, and this silencing defect is accompanied by increased levels of acetylated histone H3K14 and RNA polymerase II occupancy at telomeres as well as reduced recruitment of the SNF2 ATPase/histone deacetylase-containing complex SHREC to telomeres. Deletion of sde2 also leads to a higher frequency of mitotic minichromosome loss, and sde2{Delta} cells often form asci that contain spores in abnormal numbers, shapes, or both. In addition, sde2{Delta} cells are highly sensitive to several stresses, including high/low temperatures, bleomycin, which induces DNA damage, and thiabendazole, a microtubule-destabilizing agent. Furthermore, Sde2 genetically interacts with the telomere regulators Taz1, Pof3, and Ccq1. These findings demonstrate that Sde2 cooperates with other telomere regulators to maintain functional telomeres, thereby preventing genomic instability.

  19. Research of BH3 domain protein inducing cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    FENG Wan-yu; LIU Yang; ZHANG Zhi-cheng

    2008-01-01

    Objective BH3 domain protein plays an important role in control mechanism of cell apoptosis. The article mainly discusses its mechanism of promoting cell apoptosis and control. Methods The article analyzed and evaluated the mechanism of BH3 domain protein promoting cell apoptosis by internal and overseas literature. Results Activation of BH3 domain protein could promote the increase of mitochondrial membrane permeability, then it would start mitoehondrial apoptosis pathway, and at the last the cell apoptosis. Conclusions BH3 domain protein is the necessary condition of starting cell apoptosis. Its activation can cause cell apoptosis.

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

    Directory of Open Access Journals (Sweden)

    Alain Y Dury

    2013-10-01

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

  1. The Effect of the LysoPC-induced Endothelial Cell Conditioned Medium on Proliferating Cell Nuclear Antigen Expression of the Calf Thoracic Aorta Smooth Muscle Cells

    Institute of Scientific and Technical Information of China (English)

    周洪莲; 姚济华; 余枢

    2002-01-01

    In order to study the effect of and mechanism of lysophosphatidylcholine (LysoPC) on proliferation of the calf thoracic aorta smooth muscle cells (ASMCs), the ASMCs were used to observe the effects of LysoPC-induced endothelial cell conditioned medium on the DNA content and proliferating cell nuclear antigen (PCNA) expression in the calf thoracic ASMCs by flow cytometry and Western Blot technique. It was found that LysoPC-induced endothelial cell conditioned medium could significantly promote PCNA expression of the calf ASMCs, induce the converting of ASMCs from G0/G1 phase to S phase of DNA synthesis, and increase the tyrosine phosphorylation protein expression. Tyrosine protein kinase inhibitor (TPKi) RG50864 could obviously inhibit proliferation of LysoPC-induced ASMCs in a dose-dependence manner. The results indicated that the effect of LysoPC promoting the proliferation of ASMCs is partly evoked by endothelial cell derived growth factors such as PDGF and so on.

  2. Cell tracking using a photoconvertible fluorescent protein.

    Science.gov (United States)

    Hatta, Kohei; Tsujii, Hitomi; Omura, Tomomi

    2006-01-01

    The tracking of cell fate, shape and migration is an essential component in the study of the development of multicellular organisms. Here we report a protocol that uses the protein Kaede, which is fluorescent green after synthesis but can be photoconverted red by violet or UV light. We have used Kaede along with confocal laser scanning microscopy to track labeled cells in a pattern of interest in zebrafish embryos. This technique allows the visualization of cell movements and the tracing of neuronal shapes. We provide illustrative examples of expression by mRNA injection, mosaic expression by DNA injection, and the creation of permanent transgenic fish with the UAS-Gal4 system to visualize morphogenetic processes such as neurulation, placode formation and navigation of early commissural axons in the hindbrain. The procedure can be adapted to other photoconvertible and reversible fluorescent molecules, including KikGR and Dronpa; these molecules can be used in combination with two-photon confocal microscopy to specifically highlight cells buried in tissues. The total time needed to carry out the protocol involving transient expression of Kaede by injection of mRNA or DNA, photoconversion and imaging is 2-8 d.

  3. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

  4. Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix?

    Science.gov (United States)

    Johansen, Kristen M; Forer, Arthur; Yao, Changfu; Girton, Jack; Johansen, Jørgen

    2011-04-01

    The idea of a spindle matrix has long been proposed in order to account for poorly understood features of mitosis. However, its molecular nature and structural composition have remained elusive. Here, we propose that the spindle matrix may be constituted by mainly nuclear-derived proteins that reorganize during the cell cycle to form an elastic gel-like matrix. We discuss this hypothesis in the context of recent observations from phylogenetically diverse organisms that nuclear envelope and intranuclear proteins form a highly dynamic and malleable structure that contributes to mitotic spindle function. We suggest that the viscoelastic properties of such a matrix may constrain spindle length while at the same time facilitating microtubule growth and dynamics as well as chromosome movement. A corollary to this hypothesis is that a key determinant of spindle size may be the amount of nuclear proteins available to form the spindle matrix. Such a matrix could also serve as a spatial regulator of spindle assembly checkpoint proteins during open and semi-open mitosis. PMID:21274615

  5. KAR5 Encodes a Novel Pheromone-inducible Protein Required for Homotypic Nuclear Fusion

    OpenAIRE

    Beh, Christopher T.; Brizzio, Valeria; Rose, Mark D.

    1997-01-01

    KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is i...

  6. Nuclear entry of hyperbranched polylysine nanoparticles into cochlear cells

    Directory of Open Access Journals (Sweden)

    Weikai Zhang

    2011-03-01

    Full Text Available Weikai Zhang1, Ya Zhang1, Marian Löbler2, Klaus-Peter Schmitz2, Aqeel Ahmad3, Ilmari Pyykkö1, Jing Zou11Department of Otolaryngology, University of Tampere, Medical School, Tampere, Finland; 2University of Rostock, Institute for Biomedical Engineering, Rostock, Germany; 3Department of Biomedical Engineering and Computational Science, Aalto University, Espoo, FinlandBackground: Gene therapy is a potentially effective therapeutic modality for treating sensorineural hearing loss. Nonviral gene delivery vectors are expected to become extremely safe and convenient, and nanoparticles are the most promising types of vectors. However, infrequent nuclear localization in the cochlear cells limits their application for gene therapy. This study aimed to investigate the potential nuclear entry of hyperbranched polylysine nanoparticles (HPNPs for gene delivery to cochlear targets.Methods: Rat primary cochlear cells and cochlear explants generated from newborn rats were treated with different concentrations of HPNPs. For the in vivo study, HPNPs were administered to the rats' round window membranes. Subcellular distribution of HPNPs in different cell populations was observed with confocal microscope 24 hours after administration.Results: Nuclear entry was observed in various cochlear cell types in vitro and in vivo. In the primary cochlear cell culture, concentration-dependent internalization was observed. In the cochlear organotypic culture, abundant HPNPs were found in the modiolus, including the spiral ganglion, organ of Corti, and lateral wall tissues. In the in vivo study, a gradient distribution of HPNPs through different layers of the round window membrane was observed. HPNPs were also distributed in the cells of the middle ear tissue. Additionally, efficient internalization of HPNPs was observed in the organ of Corti and spiral ganglion cells. In primary cochlear cells, HPNPs induced higher transfection efficiency than did Lipofectamine

  7. Calreticulin: Roles in Cell-Surface Protein Expression

    Directory of Open Access Journals (Sweden)

    Yue Jiang

    2014-09-01

    Full Text Available In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins.

  8. Putting things in place for fertilization: discovering roles for importin proteins in cell fate and spermatogenesis

    Directory of Open Access Journals (Sweden)

    Kate L. Loveland

    2015-01-01

    Full Text Available Importin proteins were originally characterized for their central role in protein transport through the nuclear pores, the only intracellular entry to the nucleus. This vital function must be tightly regulated to control access by transcription factors and other nuclear proteins to genomic DNA, to achieve appropriate modulation of cellular behaviors affecting cell fate. Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets. Knowledge of importin function has expanded substantially in regard to three key developmental systems: embryonic stem cells, muscle cells and the germ line. In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate. This review presents key findings from studies of mammalian spermatogenesis that reveal potential new pathways by which male fertility and infertility arise. These studies of germline genesis illuminate new ways in which importin proteins govern cellular differentiation, including via directing proteins to distinct intracellular compartments and by determining cellular stress responses.

  9. Somatic cell nuclear transfer: pros and cons.

    Science.gov (United States)

    Sumer, Huseyin; Liu, Jun; Tat, Pollyanna; Heffernan, Corey; Jones, Karen L; Verma, Paul J

    2009-01-01

    Even though the technique of mammalian SCNT is just over a decade old it has already resulted in numerous significant advances. Despite the recent advances in the reprogramming field, SCNT remains the bench-mark for the generation of both genetically unmodified autologous pluripotent stem cells for transplantation and for the production of cloned animals. In this review we will discuss the pros and cons of SCNT, drawing comparisons with other reprogramming methods. PMID:20232594

  10. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import.

    LENUS (Irish Health Repository)

    Gu, Lili

    2011-03-14

    Abstract Background The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  11. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import

    Directory of Open Access Journals (Sweden)

    Sheehy Noreen

    2011-03-01

    Full Text Available Abstract Background The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  12. The complete amino acid sequence of the basic nuclear protein of bull spermatozoa

    NARCIS (Netherlands)

    Coelingh, J.P.; Monfoort, Cornelis H.; Rozijn, Thomas H.; Gevers Leuven, Jan A.; Schiphof, R.; Steyn-Parvé, Elizabeth P.; Braunitzer, Gerhard; Schrank, Barbara; Ruhfus, Annette

    1972-01-01

    The complete amino acid sequence of the basic nuclear protein of bull spermatozoa has been established. The sequence was partially deduced by characterization of peptides isolated from thermolysine and chymotryptic digests of the reduced and S-aminoethylated protein. The complete sequence of the fir

  13. Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

    Directory of Open Access Journals (Sweden)

    St-Laurent Georges

    2006-11-01

    Full Text Available Abstract Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

  14. PROTEIN L-ISOASPARTYL METHYLTRANSFERASE2 is differentially expressed in chickpea and enhances seed vigor and longevity by reducing abnormal isoaspartyl accumulation predominantly in seed nuclear proteins.

    Science.gov (United States)

    Verma, Pooja; Kaur, Harmeet; Petla, Bhanu Prakash; Rao, Venkateswara; Saxena, Saurabh C; Majee, Manoj

    2013-03-01

    PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a widely distributed protein-repairing enzyme that catalyzes the conversion of abnormal l-isoaspartyl residues in spontaneously damaged proteins to normal aspartyl residues. This enzyme is encoded by two divergent genes (PIMT1 and PIMT2) in plants, unlike many other organisms. While the biological role of PIMT1 has been elucidated, the role and significance of the PIMT2 gene in plants is not well defined. Here, we isolated the PIMT2 gene (CaPIMT2) from chickpea (Cicer arietinum), which exhibits a significant increase in isoaspartyl residues in seed proteins coupled with reduced germination vigor under artificial aging conditions. The CaPIMT2 gene is found to be highly divergent and encodes two possible isoforms (CaPIMT2 and CaPIMT2') differing by two amino acids in the region I catalytic domain through alternative splicing. Unlike CaPIMT1, both isoforms possess a unique 56-amino acid amino terminus and exhibit similar yet distinct enzymatic properties. Expression analysis revealed that CaPIMT2 is differentially regulated by stresses and abscisic acid. Confocal visualization of stably expressed green fluorescent protein-fused PIMT proteins and cell fractionation-immunoblot analysis revealed that apart from the plasma membrane, both CaPIMT2 isoforms localize predominantly in the nucleus, while CaPIMT1 localizes in the cytosol. Remarkably, CaPIMT2 enhances seed vigor and longevity by repairing abnormal isoaspartyl residues predominantly in nuclear proteins upon seed-specific expression in Arabidopsis (Arabidopsis thaliana), while CaPIMT1 enhances seed vigor and longevity by repairing such abnormal proteins mainly in the cytosolic fraction. Together, our data suggest that CaPIMT2 has most likely evolved through gene duplication, followed by subfunctionalization to specialize in repairing the nuclear proteome.

  15. VP22 herpes simplex virus protein can transduce proteins into stem cells

    International Nuclear Information System (INIS)

    The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations

  16. VP22 herpes simplex virus protein can transduce proteins into stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gabanyi, I.; Lojudice, F.H.; Kossugue, P.M. [Centro de Terapia Celular e Molecular, Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP (Brazil); Rebelato, E. [Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil); Demasi, M.A.; Sogayar, M.C. [Centro de Terapia Celular e Molecular, Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP (Brazil)

    2013-02-01

    The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.

  17. Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Pedersen, Hanne Gervi; Jakobsen, Anne Sørig;

    2007-01-01

    nuclear transfer (SCNT) using fluorescence in situ hybridization (FISH) with an rDNA probe and subsequent visualization of the nucleolar proteins by silver staining. In the 205 IVP embryos investigated, all two-cell embryos (n = 34) were categorized as transcriptionally inactive. At the late four...

  18. Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes.

    Science.gov (United States)

    Brabec, Viktor; Kasparkova, Jana; Kostrhunova, Hana; Farrell, Nicholas P

    2016-01-01

    Nuclear DNA is the target responsible for anticancer activity of platinum anticancer drugs. Their activity is mediated by altered signals related to programmed cell death and the activation of various signaling pathways. An example is activation of nuclear factor kappaB (NF-κB). Binding of NF-κB proteins to their consensus sequences in DNA (κB sites) is the key biochemical activity responsible for the biological functions of NF-κB. Using gel-mobility-shift assays and surface plasmon resonance spectroscopy we examined the interactions of NF-κB proteins with oligodeoxyribonucleotide duplexes containing κB site damaged by DNA adducts of three platinum complexes. These complexes markedly differed in their toxic effects in tumor cells and comprised highly cytotoxic trinuclear platinum(II) complex BBR3464, less cytotoxic conventional cisplatin and ineffective transplatin. The results indicate that structurally different DNA adducts of these platinum complexes exhibit a different efficiency to affect the affinity of the platinated DNA (κB sites) to NF-κB proteins. Our results support the hypothesis that structural perturbations induced in DNA by platinum(II) complexes correlate with their higher efficiency to inhibit binding of NF-κB proteins to their κB sites and cytotoxicity as well. However, the full generalization of this hypothesis will require to evaluate a larger series of platinum(II) complexes. PMID:27574114

  19. Fluorescence lifetime imaging microscopy (FLIM) to quantify protein-protein interactions inside cells.

    Science.gov (United States)

    Duncan, R R

    2006-11-01

    Recent developments in cellular imaging spectroscopy now permit the minimally invasive study of protein dynamics inside living cells. These advances are of interest to cell biologists, as proteins rarely act in isolation, but rather in concert with others in forming cellular machinery. Until recently, all protein interactions had to be determined in vitro using biochemical approaches: this biochemical legacy has provided cell biologists with the basis to test defined protein-protein interactions not only inside cells, but now also with high spatial resolution. These techniques can detect and quantify protein behaviours down to the single-molecule level, all inside living cells. More recent developments in TCSPC (time-correlated single-photon counting) imaging are now also driving towards being able to determine protein interaction rates with similar spatial resolution, and together, these experimental advances allow investigators to perform biochemical experiments inside living cells. PMID:17052173

  20. Nuclear transport factor directs localization of protein synthesis during mitosis

    NARCIS (Netherlands)

    Bogaart, Geert van den; Meinema, Anne C.; Krasnikov, Viktor; Veenhoff, Liesbeth M.; Poolman, Bert

    2009-01-01

    Export of messenger RNA from the transcription site in the nucleus and mRNA targeting to the translation site in the cytoplasm are key regulatory processes in protein synthesis. In yeast, the mRNA-binding proteins Nab2p and Nab4p/Hrp1p accompany transcripts to their translation site, where the karyo

  1. A comparison between protein profiles of B cell subpopulations and mantle cell lymphoma cells

    Directory of Open Access Journals (Sweden)

    Lehtiö Janne

    2009-11-01

    Full Text Available Abstract Background B-cell lymphomas are thought to reflect different stages of B-cell maturation. Based on cytogenetics and molecular markers, mantle cell lymphoma (MCL is presumed to derive predominantly from naïve, pre-germinal centre (pre-GC B lymphocytes. The aim of this study was to develop a method to investigate the similarity between MCL cells and different B-cell compartments on a protein expression level. Methods Subpopulations of B cells representing the germinal centre (GC, the pre-GC mantle zone and the post-GC marginal zone were isolated from tonsils using automated magnetic cell sorting (AutoMACS of cells based on their expression of CD27 and IgD. Protein profiling of the B cell subsets, of cell lines representing different lymphomas and of primary MCL samples was performed using top-down proteomics profiling by surface-enhanced laser detection/ionization time-of-flight mass spectrometry (SELDI-TOF-MS. Results Quantitative MS data of significant protein peaks (p-value Conclusion AutoMACS sorting generates sufficient purity to enable a comparison between protein profiles of B cell subpopulations and malignant B lymphocytes applying SELDI-TOF-MS. Further validation with an increased number of patient samples and identification of differentially expressed proteins would enable a search for possible treatment targets that are expressed during the early development of MCL.

  2. Nuclear Magnetic Resonance Characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa.

    Science.gov (United States)

    Chaudhury, Sukanya; Nordhues, Bryce A; Kaur, Kawaljit; Zhang, Na; De Guzman, Roberto N

    2015-11-01

    Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS), to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologues are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residue 16 to 41 and from residue 55 to 76. The helices of PcrG are partially formed, have similar backbone dynamics, and are flexible. NMR titrations show that the entire length of PcrG residues from position 9 to 76 is involved in binding to PcrV. PcrG adds to the growing list of partially folded or unstructured proteins with important roles in type III secretion.

  3. Phosphorylated nucleolin interacts with translationally controlled tumor protein during mitosis and with Oct4 during interphase in ES cells.

    Directory of Open Access Journals (Sweden)

    Helena Johansson

    Full Text Available BACKGROUND: Reprogramming of somatic cells for derivation of either embryonic stem (ES cells, by somatic cell nuclear transfer (SCNT, or ES-like cells, by induced pluripotent stem (iPS cell procedure, provides potential routes toward non-immunogenic cell replacement therapies. Nucleolar proteins serve as markers for activation of embryonic genes, whose expression is crucial for successful reprogramming. Although Nucleolin (Ncl is one of the most abundant nucleolar proteins, its interaction partners in ES cells have remained unidentified. METHODOLOGY: Here we explored novel Ncl-interacting proteins using in situ proximity ligation assay (PLA, colocalization and immunoprecipitation (IP in ES cells. PRINCIPAL FINDINGS: We found that phosphorylated Ncl (Ncl-P interacted with translationally controlled tumor protein (Tpt1 in murine ES cells. The Ncl-P/Tpt1 complex peaked during mitosis and was reduced upon retinoic acid induced differentiation, signifying a role in cell proliferation. In addition, we showed that Ncl-P interacted with the transcription factor Oct4 during interphase in human as well as murine ES cells, indicating of a role in transcription. The Ncl-P/Oct4 complex peaked during early stages of spontaneous human ES cell differentiation and may thus be involved in the initial differentiation event(s of mammalian development. CONCLUSIONS: Here we described two novel protein-protein interactions in ES cells, which give us further insight into the complex network of interacting proteins in pluripotent cells.

  4. Serum levels of pancreatic stone protein (PSP)/reg1A as an indicator of beta-cell apoptosis suggest an increased apoptosis rate in hepatocyte nuclear factor 1 alpha (HNF1A-MODY) carriers from the third decade of life onward

    LENUS (Irish Health Repository)

    Bacon, Siobhan

    2012-07-18

    AbstractBackgroundMutations in the transcription factor hepatocyte nuclear factor-1-alpha (HNF1A) result in the commonest type of maturity onset diabetes of the young (MODY). HNF1A-MODY carriers have reduced pancreatic beta cell mass, partially due to an increased rate of apoptosis. To date, it has not been possible to determine when apoptosis is occurring in HNF1A-MODY.We have recently demonstrated that beta cell apoptosis stimulates the expression of the pancreatic stone protein\\/regenerating (PSP\\/reg) gene in surviving neighbour cells, and that PSP\\/reg1A protein is subsequently secreted from these cells. The objective of this study was to determine whether serum levels of PSP\\/reg1A are elevated during disease progression in HNF1A-MODY carriers, and whether it may provide information regarding the onset of beta-cell apoptosis.MethodsWe analysed serum PSP\\/reg1A levels and correlated with clinical and biochemical parameters in subjects with HNF1A-MODY, glucokinase (GCK-MODY), and type 1 diabetes mellitus. A control group of normoglycaemic subjects was also analysed.ResultsPSP\\/reg1A serum levels were significantly elevated in HNF1A-MODY (n = 37) subjects compared to controls (n = 60) (median = 12.50 ng\\/ml, IQR = 10.61-17.87 ng\\/ml versus median = 10.72 ng\\/ml, IQR = 8.94-12.54 ng\\/ml, p = 0.0008). PSP\\/reg1A correlated negatively with insulin levels during OGTT, (rho = −0.40, p = 0.02). Interestingly we noted a significant positive correlation of PSP\\/reg1A with age of the HNF1A-MODY carriers (rho = 0.40 p = 0.02) with an age of 25 years separating carriers with low and high PSP\\/reg1A levels. Patients with type 1 diabetes mellitus also had elevated serum levels of PSP\\/reg1A compared to controls, however this was independent of the duration of diabetes.ConclusionOur data suggest that beta cell apoptosis contributes increasingly to the pathophysiology of HNF1A-MODY in patients 25 years and

  5. A Cell-Based Assay Reveals Nuclear Translocation of Intracellular Domains Released by SPPL Proteases.

    Science.gov (United States)

    Mentrup, Torben; Häsler, Robert; Fluhrer, Regina; Saftig, Paul; Schröder, Bernd

    2015-08-01

    During regulated intramembrane proteolysis (RIP) a membrane-spanning substrate protein is cleaved by an ectodomain sheddase and an intramembrane cleaving protease. A cytoplasmic intracellular domain (ICD) is liberated, which can migrate to the nucleus thereby influencing transcriptional regulation. Signal peptide peptidase-like (SPPL) 2a and 2b have been implicated in RIP of type II transmembrane proteins. Even though SPPL2a might represent a potential pharmacological target for treatment of B-cell-mediated autoimmunity, no specific and potent inhibitors for this enzyme are currently available. We report here on the first quantitative cell-based assay for measurement of SPPL2a/b activity. Demonstrating the failure of standard Gal4/VP16 reporter assays for SPPL2a/b analysis, we have devised a novel system employing β-galactosidase (βGal) complementation. This is based on detecting nuclear translocation of the proteolytically released substrate ICDs, which results in specific restoration of βGal activity. Utilizing this potentially high-throughput compatible new setup, we demonstrate nuclear translocation of the ICDs from integral membrane protein 2B (ITM2B), tumor necrosis factor (TNF) and CD74 and identify secreted frizzled-related protein 2 (SFRP2) as potential transcriptional downstream target of the CD74 ICD. We show that the presented assay is easily adaptable to other intramembrane proteases and therefore represents a valuable tool for the functional analysis and development of new inhibitors of this class of enzymes. PMID:25824657

  6. CRISPR-Cas9 nuclear dynamics and target recognition in living cells.

    Science.gov (United States)

    Ma, Hanhui; Tu, Li-Chun; Naseri, Ardalan; Huisman, Maximiliaan; Zhang, Shaojie; Grunwald, David; Pederson, Thoru

    2016-08-29

    The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9-guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time.

  7. Detection and characterization of protein interactions in vivo by a simple live-cell imaging method.

    Directory of Open Access Journals (Sweden)

    Oriol Gallego

    Full Text Available Over the last decades there has been an explosion of new methodologies to study protein complexes. However, most of the approaches currently used are based on in vitro assays (e.g. nuclear magnetic resonance, X-ray, electron microscopy, isothermal titration calorimetry etc. The accurate measurement of parameters that define protein complexes in a physiological context has been largely limited due to technical constrains. Here, we present PICT (Protein interactions from Imaging of Complexes after Translocation, a new method that provides a simple fluorescence microscopy readout for the study of protein complexes in living cells. We take advantage of the inducible dimerization of FK506-binding protein (FKBP and FKBP-rapamycin binding (FRB domain to translocate protein assemblies to membrane associated anchoring platforms in yeast. In this assay, GFP-tagged prey proteins interacting with the FRB-tagged bait will co-translocate to the FKBP-tagged anchor sites upon addition of rapamycin. The interactions are thus encoded into localization changes and can be detected by fluorescence live-cell imaging under different physiological conditions or upon perturbations. PICT can be automated for high-throughput studies and can be used to quantify dissociation rates of protein complexes in vivo. In this work we have used PICT to analyze protein-protein interactions from three biological pathways in the yeast Saccharomyces cerevisiae: Mitogen-activated protein kinase cascade (Ste5-Ste11-Ste50, exocytosis (exocyst complex and endocytosis (Ede1-Syp1.

  8. Protein dynamics in individual human cells: experiment and theory.

    Directory of Open Access Journals (Sweden)

    Ariel Aharon Cohen

    Full Text Available A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

  9. Nuclear proteome analysis of benzo(a)pyrene-treated HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan Chunlan; Chen Zhaojun; Li Huanrong; Zhang Guanglin [The First Affiliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003 (China); Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang 310058 (China); Li Feng [The First Renmin Hospital, Houma, Shanxi 043000 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [The First Affiliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003 (China); Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang 310036 (China)

    2012-03-01

    Previously, we employed a proteomics-based 2-D gel electrophoresis assay to show that exposure to 10 {mu}M benzo(a)pyrene (BaP) during a 24 h frame can lead to changes in nuclear protein expression and alternative splicing. To further expand our knowledge about the DNA damage response (DDR) induced by BaP, we investigated the nuclear protein expression profiles in HeLa cells treated with different concentrations of BaP (0.1, 1, and 10 {mu}M) using this proteomics-based 2-D gel electrophoresis assay. We found 125 differentially expressed proteins in BaP-treated cells compared to control cells. Among them, 79 (63.2%) were down-regulated, 46 (36.8%) were up-regulated; 8 showed changes in the 1 {mu}M and 10 {mu}M BaP-treated groups, 2 in the 0.1 {mu}M and 10 {mu}M BaP-treated groups, 4 in the 0.1 {mu}M and 1 {mu}M BaP-treated groups, and only one showed changes in all three groups. Fifty protein spots were chosen for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and of these, 39 were identified, including subunits of the 26S proteasome and Annexin A1. The functions of some identified proteins were further examined and the results showed that they might be involved in BaP-induced DDR. Taken together, these data indicate that proteomics is a valuable approach in the study of environmental chemical-host interactions, and the identified proteins could provide new leads for better understanding BaP-induced mutagenesis and carcinogenesis.

  10. Neocarzinostatin-induced Rad51 nuclear focus formation is cell cycle regulated and aberrant in AT cells

    International Nuclear Information System (INIS)

    DNA double-stranded breaks are the most detrimental form of DNA damage and, if not repaired properly, may lead to an accumulation of chromosomal aberrations and eventually tumorigenesis. Proteins of the Rad51/Rad52 epitasis group are crucial for the recombinational repair of DNA double-stranded breaks, whereas the Rad50/NBS1/Mre11 nuclease complex is involved in both the recombinational and the end-joining repair of DNA double-stranded breaks. Herein, we demonstrate that the chemotherapeutic enediyne antibiotic neocarzinostatin induced Rad51, but not NBS1, nuclear focus formation in a cell- cycle-dependent manner. Furthermore, neocarzinostatin-induced Rad51 foci formation revealed a slower kinetic change in AT cells, but not in wild-type or NBS cells. In summary, our results suggest that neocarzinostatin induces Rad51 focus formation through an ATM- and cell-cycle-dependent, but NBS1-independent, pathway

  11. A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth.

    Directory of Open Access Journals (Sweden)

    Myeong Kyu Choi

    Full Text Available The nonvirion (NV protein of infectious hematopoietic necrosis virus (IHNV has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP, a nuclear localization of NV was demonstrated. Deletion analyses showed that the (32EGDL(35 residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the (32EGDL(35 was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.. While treatment with poly I∶C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I∶C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of (32EGDL(35 responsible for nuclear localization are important for the inhibitory activity of NV.

  12. A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth

    Science.gov (United States)

    Choi, M.K.; Moon, C.H.; Ko, M.S.; Lee, U.-H.; Cho, W.J.; Cha, S.J.; Do, J.W.; Heo, G.J.; Jeong, S.G.; Hahm, Y.S.; Harmache, A.; Bremont, M.; Kurath, G.; Park, J.-W.

    2011-01-01

    The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the 32EGDL35 residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the 32EGDL35 was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I:C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I:C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of 32EGDL35 responsible for nuclear localization are important for the inhibitory activity of NV.

  13. Nuclear APC.

    Science.gov (United States)

    Neufeld, Kristi L

    2009-01-01

    Mutational inactivation of the tumor suppressor gene APC (Adenomatous polyposis coli) is thought to be an initiating step in the progression of the vast majority ofcolorectal cancers. Attempts to understand APC function have revealed more than a dozen binding partners as well as several subcellular localizations including at cell-cell junctions, associated with microtubules at the leading edge of migrating cells, at the apical membrane, in the cytoplasm and in the nucleus. The present chapter focuses on APC localization and functions in the nucleus. APC contains two classical nuclear localization signals, with a third domain that can enhance nuclear import. Along with two sets of nuclear export signals, the nuclear localization signals enable the large APC protein to shuttle between the nucleus and cytoplasm. Nuclear APC can oppose beta-catenin-mediated transcription. This down-regulation of nuclear beta-catenin activity by APC most likely involves nuclear sequestration of beta-catenin from the transcription complex as well as interaction of APC with transcription corepressor CtBP. Additional nuclear binding partners for APC include transcription factor activator protein AP-2alpha, nuclear export factor Crm1, protein tyrosine phosphatase PTP-BL and perhaps DNA itself. Interaction of APC with polymerase beta and PCNA, suggests a role for APC in DNA repair. The observation that increases in the cytoplasmic distribution of APC correlate with colon cancer progression suggests that disruption of these nuclear functions of APC plays an important role in cancer progression. APC prevalence in the cytoplasm of quiescent cells points to a potential function for nuclear APC in control of cell proliferation. Clear definition of APC's nuclear function(s) will expand the possibilities for early colorectal cancer diagnostics and therapeutics targeted to APC. PMID:19928349

  14. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

    International Nuclear Information System (INIS)

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type

  15. Quantification of the spatial organization of the nuclear lamina as a tool for cell classification

    NARCIS (Netherlands)

    Righolt, C.H.; Zatreanu, D.A.; Raz, V.

    2013-01-01

    The nuclear lamina is the structural scaffold of the nuclear envelope that plays multiple regulatory roles in chromatin organization and gene expression as well as a structural role in nuclear stability. The lamina proteins, also referred to as lamins, determine nuclear lamina organization and defin

  16. Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits.

    Science.gov (United States)

    Verheyen, Toon; Görnemann, Janina; Verbinnen, Iris; Boens, Shannah; Beullens, Monique; Van Eynde, Aleyde; Bollen, Mathieu

    2015-07-13

    Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the α, β and γ isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1β emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits. PMID:25990731

  17. The insulator protein SU(HW fine-tunes nuclear lamina interactions of the Drosophila genome.

    Directory of Open Access Journals (Sweden)

    Joke G van Bemmel

    Full Text Available Specific interactions of the genome with the nuclear lamina (NL are thought to assist chromosome folding inside the nucleus and to contribute to the regulation of gene expression. High-resolution mapping has recently identified hundreds of large, sharply defined lamina-associated domains (LADs in the human genome, and suggested that the insulator protein CTCF may help to demarcate these domains. Here, we report the detailed structure of LADs in Drosophila cells, and investigate the putative roles of five insulator proteins in LAD organization. We found that the Drosophila genome is also organized in discrete LADs, which are about five times smaller than human LADs but contain on average a similar number of genes. Systematic comparison to new and published insulator binding maps shows that only SU(HW binds preferentially at LAD borders and at specific positions inside LADs, while GAF, CTCF, BEAF-32 and DWG are mostly absent from these regions. By knockdown and overexpression studies we demonstrate that SU(HW weakens genome - NL interactions through a local antagonistic effect, but we did not obtain evidence that it is essential for border formation. Our results provide insights into the evolution of LAD organization and identify SU(HW as a fine-tuner of genome - NL interactions.

  18. Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

    NARCIS (Netherlands)

    A. Kralt (Annemarie); N.B. Jagalur (Noorjahan ); V. van den Boom (Vincent); R.K. Lokareddy (Ravi K.); A.F.W. van der Steen (Anton); G. Cingolani (Gino); M.W.J. Fornerod (Maarten); L.M. Veenhoff (Liesbeth M.)

    2015-01-01

    textabstractEndoplasmic reticulum-synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these

  19. Nuclear translocation of p21{sup WAF1/CIP1} protein prior to its cytosolic degradation by UV enhances DNA repair and survival

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Young; Kim, Hee Suk; Kim, Joo Young [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Sohn, Jeongwon, E-mail: biojs@korea.ac.kr [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of)

    2009-12-25

    We previously reported that UV induced rapid proteasomal degradation of p21 protein in an ubiquitination-independent manner. Here, UV-induced p21 proteolysis was found to occur in the cytosol. Before cytosolic degradation, however, p21 protein translocated to and transiently accumulated in the nucleus. Nuclear translocation of p21 was not required for its degradation, but rather promoted DNA repair and cell survival. Overexpression of the wild type p21, but not the one with defective nuclear localization signal (NLS), reduced UV-induced DNA damage and cell death. Some of p21 protein translocated to the nucleus were associated with chromatin-bound PCNA and saved from UV-induced proteolysis. These data together show that p21 translocates to the nucleus to participate in DNA repair, while the rest is rapidly degraded in the cytosol. We propose that our findings reflect a mechanism to facilitate removal of damaged cells, enhancing DNA repair at the same time.

  20. Nuclear translocation of p21WAF1/CIP1 protein prior to its cytosolic degradation by UV enhances DNA repair and survival

    International Nuclear Information System (INIS)

    We previously reported that UV induced rapid proteasomal degradation of p21 protein in an ubiquitination-independent manner. Here, UV-induced p21 proteolysis was found to occur in the cytosol. Before cytosolic degradation, however, p21 protein translocated to and transiently accumulated in the nucleus. Nuclear translocation of p21 was not required for its degradation, but rather promoted DNA repair and cell survival. Overexpression of the wild type p21, but not the one with defective nuclear localization signal (NLS), reduced UV-induced DNA damage and cell death. Some of p21 protein translocated to the nucleus were associated with chromatin-bound PCNA and saved from UV-induced proteolysis. These data together show that p21 translocates to the nucleus to participate in DNA repair, while the rest is rapidly degraded in the cytosol. We propose that our findings reflect a mechanism to facilitate removal of damaged cells, enhancing DNA repair at the same time.

  1. The bovine papillomavirus type 1 E2 transactivator and repressor proteins use different nuclear localization signals.

    Science.gov (United States)

    Skiadopoulos, M H; McBride, A A

    1996-02-01

    The E2 gene of bovine papillomavirus type 1 encodes at least three nuclear phosphoproteins that regulate viral transcription and DNA replication. All three proteins have a common C-terminal domain that has DNA-binding and dimerization activities. A basic region in this domain forms an alpha helix which makes direct contact with the DNA target. In this study, it is shown that in addition to its role in DNA binding, this basic region functions as a nuclear localization signal both in the E2 DNA-binding domain and in a heterologous protein. Deletion of this signal sequence resulted in increased accumulation of the E2 transactivator and repressor proteins in the cytoplasm, but nuclear localization was not eliminated. In the full-length transactivator protein, another signal, present in the N-terminal transactivation domain, is used for transport to the nucleus, and the C-terminal nuclear localization signal(s) are masked. The use of different nuclear localization signals could potentially allow differential regulation of the subcellular localization of the E2 transactivator and repressor proteins at some stage in the viral life cycle. PMID:8551571

  2. A combined gas cooled nuclear reactor and fuel cell cycle

    Science.gov (United States)

    Palmer, David J.

    Rising oil costs, global warming, national security concerns, economic concerns and escalating energy demands are forcing the engineering communities to explore methods to address these concerns. It is the intention of this thesis to offer a proposal for a novel design of a combined cycle, an advanced nuclear helium reactor/solid oxide fuel cell (SOFC) plant that will help to mitigate some of the above concerns. Moreover, the adoption of this proposal may help to reinvigorate the Nuclear Power industry while providing a practical method to foster the development of a hydrogen economy. Specifically, this thesis concentrates on the importance of the U.S. Nuclear Navy adopting this novel design for its nuclear electric vessels of the future with discussion on efficiency and thermodynamic performance characteristics related to the combined cycle. Thus, the goals and objectives are to develop an innovative combined cycle that provides a solution to the stated concerns and show that it provides superior performance. In order to show performance, it is necessary to develop a rigorous thermodynamic model and computer program to analyze the SOFC in relation with the overall cycle. A large increase in efficiency over the conventional pressurized water reactor cycle is realized. Both sides of the cycle achieve higher efficiencies at partial loads which is extremely important as most naval vessels operate at partial loads as well as the fact that traditional gas turbines operating alone have poor performance at reduced speeds. Furthermore, each side of the cycle provides important benefits to the other side. The high temperature exhaust from the overall exothermic reaction of the fuel cell provides heat for the reheater allowing for an overall increase in power on the nuclear side of the cycle. Likewise, the high temperature helium exiting the nuclear reactor provides a controllable method to stabilize the fuel cell at an optimal temperature band even during transients helping

  3. Design of nuclear cells with re linking of trajectories

    International Nuclear Information System (INIS)

    Presently work the results obtained with the Ohtli-RT system obtained when implementing the combinatory optimization technique well-known as Trajectories re linking or Path Re linking in English. The problem to solve is the radial design of nuclear fuel, taking like base nuclear fuel assembles for boiling water reactors (BWR Boiling Water Reactor by its initials in English). To evaluate the objective function used in the system the code in two dimensions Heliums 1.5 was used, which calculates the cross sections of the proposed design. The parameters that were considered for the evaluation of the objective function are the Power peak factor of the bar that generates bigger power in the cell and the Infinite Multiplication Factor. To prove the system its were used assembles 10x10 with 2 water channels. The obtained radial designs of nuclear fuel fulfilled the restrictions imposed to the considered limits, with regard to the involved parameters. (Author)

  4. Functions of Heterogeneous Nuclear Ribonucleoproteins in Stem Cell Potency and Differentiation

    Directory of Open Access Journals (Sweden)

    Qishan Chen

    2013-01-01

    Full Text Available Stem cells possess huge importance in developmental biology, disease modelling, cell replacement therapy, and tissue engineering in regenerative medicine because they have the remarkable potential for self-renewal and to differentiate into almost all the cell types in the human body. Elucidation of molecular mechanisms regulating stem cell potency and differentiation is essential and critical for extensive application. Heterogeneous nuclear ribonucleoproteins (hnRNPs are modular proteins consisting of RNA-binding motifs and auxiliary domains characterized by extensive and divergent functions in nucleic acid metabolism. Multiple roles of hnRNPs in transcriptional and posttranscriptional regulation enable them to be effective gene expression regulators. More recent findings show that hnRNP proteins are crucial factors implicated in maintenance of stem cell self-renewal and pluripotency and cell differentiation. The hnRNPs interact with certain sequences in target gene promoter regions to initiate transcription. In addition, they recognize 3′UTR or 5′UTR of specific gene mRNA forming mRNP complex to regulate mRNA stability and translation. Both of these regulatory pathways lead to modulation of gene expression that is associated with stem cell proliferation, cell cycle control, pluripotency, and committed differentiation.

  5. Sub-nuclear distribution and mobility of nuclear proteins involved in histone acetylation and pre-mRNA splicing

    International Nuclear Information System (INIS)

    The mitotic relationship between levels of highly acetylated chromatin, chromatin condensation, and HAT/HDAC organization was examined. HATs and HDACs were found to dissociate from chromosomes along with a loss of highly acetylated histones in condensed chromatin in mitosis. We demonstrate that, rather than being enzymatically inactivated, HAT and HDAC activities are decreased in mitosis because the enzymes are sequestered to a non-chromatin domain. Highly acetylated histone species reappear coincident with the reassociation of HATs and HDACs in late telophase/early interphase and before reinitiation of transcription. We propose that HATs and HDACs are spatially regulated through the cell cycle and that this regulation influences which chromatin domains are available for acetylation and deacetylation. We examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF:GFP) using timelapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We found that ASF:GFP moves significantly slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of transcription inhibitors and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites. Through a careful analysis of HDAC4 expression we found that HDAC4-containing MAD bodies are not a consistent component of the interphase nucleus. By comparing MAD bodies to PML bodies we found that the assembly, maintenance and distribution of PML bodies is regulated. We investigated the involvement of chromatin condensation in establishing mitotic transcription repression, by analyzing transcriptional activity in

  6. [The cell-free protein synthesis-based protein microarray technology].

    Science.gov (United States)

    Lu, Linli; Lin, Bicheng

    2010-12-01

    The major bottle-neck in the way of constructing high density protein microarray is the availability and stability of proteins. The traditional methods of generating protein arrays require the in-vivo expression, purification and immobilization of hundreds or thousands of proteins. The cell-free protein array technology employs cell-free expression systems to produce proteins directly onto surface from co-distributed or pre-arrayed DNA or RNA, thus avoiding the laborious and often costly processes of protein preparation in the traditional approach. Here we provide an overview of recently developed novel technology in cell free based protein microarray and their applications in protein interaction analysis, in antibody specificity and vaccine screening, and in biomarker assay. PMID:21375003

  7. The evolution of human cells in terms of protein innovation.

    Science.gov (United States)

    Sardar, Adam J; Oates, Matt E; Fang, Hai; Forrest, Alistair R R; Kawaji, Hideya; Gough, Julian; Rackham, Owen J L

    2014-06-01

    Humans are composed of hundreds of cell types. As the genomic DNA of each somatic cell is identical, cell type is determined by what is expressed and when. Until recently, little has been reported about the determinants of human cell identity, particularly from the joint perspective of gene evolution and expression. Here, we chart the evolutionary past of all documented human cell types via the collective histories of proteins, the principal product of gene expression. FANTOM5 data provide cell-type-specific digital expression of human protein-coding genes and the SUPERFAMILY resource is used to provide protein domain annotation. The evolutionary epoch in which each protein was created is inferred by comparison with domain annotation of all other completely sequenced genomes. Studying the distribution across epochs of genes expressed in each cell type reveals insights into human cellular evolution in terms of protein innovation. For each cell type, its history of protein innovation is charted based on the genes it expresses. Combining the histories of all cell types enables us to create a timeline of cell evolution. This timeline identifies the possibility that our common ancestor Coelomata (cavity-forming animals) provided the innovation required for the innate immune system, whereas cells which now form the brain of human have followed a trajectory of continually accumulating novel proteins since Opisthokonta (boundary of animals and fungi). We conclude that exaptation of existing domain architectures into new contexts is the dominant source of cell-type-specific domain architectures.

  8. Interspecific variation of intracellular localization and postirradiation movement of Ku70-protein in fibroblastic cells

    International Nuclear Information System (INIS)

    Ku (Ku70 and Ku80) Proteins are known as components of DNA-dependent protein kinase (DNA-PK) and play an important role for DNA repair. We previously reported that more than 70% of Ku proteins were located in cytoplasm of rat cells, the Ku proteins moved into nuclei of normal rat cells after X-irradiation, Ku proteins also moved into nuclei after X-irradiation but were not retained in nucleus of radiosensitive LEC rat cells. While reports have been shown about mechanisms on nuclear localization of Ku proteins, how Ku proteins export from nucleus is poorly understood. Here we show that C-terminal region of Ku70 protein is important for its cytoplasmic localization. When transfected into LEC rat cells, exogenous intact Ku70 (1-609) tagged with enhanced green fluorescent protein (EGFP-Ku70) localized mainly in the cytoplasm, whereas C-terminal-deletion mutant of Ku70 (1-593) tagged with EGFP (EGFP-Ku70D) was mainly localized in the nucleus. After X-irradiation, the endogenous intact EGFP-Ku70 once moved into nucleus, but returned into the cytoplasm. On the other hand, EGFP-Ku70D was retained in nucleus for two hours after X-irradiation. These results suggest that C-terminal region of Ku70 is included in the postirradiation nuclear export. Next, we investigated the intracellular localization of Ku70 proteins and the movement after X-irradiation of fibroblastic cells prepared from some mammalian species. Ku70 proteins were localized in nucleus and the postirradiation-extranuclear transport was not observed in human and African green monkey cells. On the other hand, Ku70 proteins were mainly localized in cytoplasm and moved into nucleus in mouse, Chinese hamster, Golden hamster, cotton rat, squirrel, cat and dog cells. These results may show that alternatively Ku70 protein is localized in the cytoplasm or nucleus depends on species and translocation of cytoplasmic Ku70 into nucleus is a response against low dose irradiation in fibroblasts of rodents, cats and dogs

  9. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wen-Ta [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Li, Hui-Chun [Department of Biochemistry, Tzu Chi University, Hualien, Taiwan (China); Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Lo, Shih-Yen, E-mail: losylo@mail.tcu.edu.tw [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan (China)

    2013-05-24

    Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.

  10. The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein.

    Directory of Open Access Journals (Sweden)

    Chao Shi

    Full Text Available Since the identification of poly-alanine expanded poly(A binding protein nuclear 1 (PABPN1 as the genetic cause of oculopharyngeal muscular dystrophy (OPMD, considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear.In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90. Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs. Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP. The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells.Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD.

  11. A suspended cell line from Trichoplusia ni (Lepidoptera):Characterization and expression of recombinant proteins

    Institute of Scientific and Technical Information of China (English)

    Min-Juan Meng; Tian-Long Li; Chang-You Li; Guo-Xun Li

    2008-01-01

    A suspended cell line from Trichoplusia ni embryos was established, and its susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)infection was investigated. This cell line had characteristics distinct from the BTI-Tn5B 14 cell line (Tn5B 1-4) from T. ni in growth, and showed approximately the same responses to AcMNPV infection, production of occlusion bodies, and levels of recombinant protein expression. No clumps were observed at maximum cell density at late-log phase in shakeflask or T-flask cultures, and thus the cells represent a useful new contribution for baculovirus research. The cells consist of two major morphological types: approximately 70% spindle-shaped cells and 30% round cells. The cell line was highly susceptible to virus infection and produced around 107 AcMNPV occlusion bodies per cell, on average.Production of β-galactosidase and secreted alkaline phosphatase was high with 3.97 + 0.13×104 IU/mL and 3.48±0.40 IU/mL, respectively. This cell line may be applicable for studies of scale-up production of viruses or baculovirus-insect cell expression. We also believe the new line can be a source for cell clones with higher production of virus and recombinant proteins compared to the parent or other existing cell lines such as Tn5B 1-4.

  12. A case of anti-nuclear matrix protein 2 antibody positive myopathy associated with lung cancer.

    Science.gov (United States)

    Ohta, Shin; Unoda, Ki-Ichi; Nakajima, Hideto; Ikeda, Soichiro; Hamaguchi, Yasuhito; Kimura, Fumiharu

    2016-08-31

    Myositis-specific autoantibodies (MSAs) are associated with myositis. Anti-nuclear matrix protein 2 (NXP-2) antibody was recently identified as a major MSA and was observed mostly in juvenile dermatomyositis. We report the case of a 44-year-old man who presented with myopathy with anti-NXP-2 antibody and large cell carcinoma of the lung. He was hospitalized because of myalgia and edema of limbs. Neurological examination revealed mild proximal-dominant weakness in all four extremities, and laboratory studies showed elevated creatine kinase level (6,432 IU/l). Needle electromyography showed myogenic patterns. MRI of the lower limbs demonstrated inflammatory lesions in the thighs. Biopsied specimen from the left quadriceps femoris muscle showed mild mononuclear inflammatory infiltrate surrounding muscle fibres but no fiber necrosis. He was diagnosed with myopathy based on neurological examinations and clinical symptoms. His chest X-ray and CT showed tumor shadow on the right upper lung field, but CT didn't indicate the findings of interstitial lung disease. This was surgically removed, and a histological diagnosis of non-small cell lung cancer was suspected. He was also treated with definitive chemoradiotherapy before and after operation. His symptoms of myopathy promptly remitted with the preoperative chemotherapy. His serum analysis was positive for the anti-NXP-2. Further investigation and experience of MSAs are necessary to evaluate the therapeutic strategy against cancer-associated myopathy/myositis. PMID:27477574

  13. Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells.

    Science.gov (United States)

    Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

    2016-01-01

    Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal. PMID:27033801

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

    Energy Technology Data Exchange (ETDEWEB)

    Tannukit, Sissada [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States); Crabb, Tara L.; Hertel, Klemens J. [Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA 92697-4025 (United States); Wen, Xin [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States); Jans, David A. [Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, Monash University, Clayton, Victoria 3800 (Australia); Paine, Michael L., E-mail: paine@usc.edu [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States)

    2009-12-18

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

  15. Differential ligand-dependent protein–protein interactions between nuclear receptors and a neuronal-specific cofactor

    OpenAIRE

    Greiner, Erich F.; Kirfel, Jutta; Greschik, Holger; Huang, DongYa; Becker, Peter; Kapfhammer, Josef P.; Schüle, Roland

    2000-01-01

    Nuclear receptors are transcription factors that require multiple protein–protein interactions to regulate target gene expression. We have cloned a 27-kDa protein, termed NIX1 (neuronal interacting factor X 1), that directly binds nuclear receptors in vitro and in vivo. Protein–protein interaction between NIX1 and ligand-activated or constitutive active nuclear receptors, including retinoid-related orphan receptor β (RORβ) (NR1F2), strictly depends on the conserved receptor C-terminal activat...

  16. Effects of notoginosides on proliferation and upregulation of GR nuclear transcription factor in hematopoietic cells

    Institute of Scientific and Technical Information of China (English)

    Rui-lan GAO; Xiao-hong CHEN; Xiao-jie LIN; Xu-dai QIAN; Wei-hong XU; Beng Hock CHONC

    2007-01-01

    Aim: To investigate the effects of panax notoginosides (PNS) on the proliferation of human hematopoietic stem/progenitor cells, and to explore the signaling path-way of the nuclear transcription factor of the glucocorticoid receptor (GR-NTF) initiated by PNS related with the proliferation. Methods: The human CD34+ cells and bone marrow nuclear cells were exposed to PNS at a concentration of 0, 10, 25,50, and 100 mg/L, respectively, in semi-solid culture system to observe colony forming unite of all lineages, granulocyte, erythrocyte, and megakaryocyte (CFU-GEMM, CFU-GM, CFU-E, and CFU-MK). Three lineages of human hematopoietic cell lines, including granulocytic HL-60, erythrocytic K562, megakaryocytic CHRF-288, and Meg-01 cells were incubated with PNS at 20 mg/L for 14 d. Meanwhile,dexamethasone (Dex) was used as a positive control. The nuclear protein of the cells was analyzed by Western blotting with monoclonal antibodies against the amino or carboxyl terminus of GR-NTF. Electrophoretic mobility shift assay per-formed by using the 32p-radiolabeled GR-NTF consensus oligonucleotide. Results:PNS promoted the proliferation of CD34+ cells and significantly raised the colony numbers of CFU-GEMM by 34.7%~±16.0% over the non-PNS control (P<0.01).PNS also enhanced the proliferation of CFU-GM, CFU-E, and CFU-MK by 39.3%±5.7%, 33.3%±7.3%, and 26.2%±3.2%, respectively. GR-NTF protein levels of either the amino or carboxyl terminus in K562, CHRF-288, and Meg-01 treated by PNS increased by 2.4- 2.8 fold and 1.3- 3.9 fold over the untreated cells. GR-NTF binding activity, initiated by either PNS or Dex, was apparently elevated to form the complex of GR-NTF with DNA as higher density bands in K562 and CHRF-288 cells, and some activity appeared as a band in HL-60 cells induced by PNS.Conclusion: PNS displayed the action of hematopoietic growth factor-like or syn-ergistic efficacy to promote proliferation of human progenitor cells, may play a role in the upregulation of gene

  17. Nuclear lamins and oxidative stress in cell proliferation and longevity.

    Science.gov (United States)

    Shimi, Takeshi; Goldman, Robert D

    2014-01-01

    In mammalian cells, the nuclear lamina is composed of a complex fibrillar network associated with the inner membrane of the nuclear envelope. The lamina provides mechanical support for the nucleus and functions as the major determinant of its size and shape. At its innermost aspect it associates with peripheral components of chromatin and thereby contributes to the organization of interphase chromosomes. The A- and B-type lamins are the major structural components of the lamina, and numerous mutations in the A-type lamin gene have been shown to cause many types of human diseases collectively known as the laminopathies. These mutations have also been shown to cause a disruption in the normal interactions between the A and B lamin networks. The impact of these mutations on nuclear functions is related to the roles of lamins in regulating various essential processes including DNA synthesis and damage repair, transcription and the regulation of genes involved in the response to oxidative stress. The major cause of oxidative stress is the production of reactive oxygen species (ROS), which is critically important for cell proliferation and longevity. Moderate increases in ROS act to initiate signaling pathways involved in cell proliferation and differentiation, whereas excessive increases in ROS cause oxidative stress, which in turn induces cell death and/or senescence. In this review, we cover current findings about the role of lamins in regulating cell proliferation and longevity through oxidative stress responses and ROS signaling pathways. We also speculate on the involvement of lamins in tumor cell proliferation through the control of ROS metabolism.

  18. Overexpression of human selenoprotein H in neuronal cells enhances mitochondrial biogenesis and function through activation of protein kinase A, protein kinase B, and cyclic adenosine monophosphate response element-binding protein pathway.

    Science.gov (United States)

    Mehta, Suresh L; Mendelev, Natalia; Kumari, Santosh; Andy Li, P

    2013-03-01

    Mitochondrial biogenesis is activated by nuclear encoded transcription co-activator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which is regulated by several upstream factors including protein kinase A and Akt/protein kinase B. We have previously shown that selenoprotein H enhances the levels of nuclear regulators for mitochondrial biogenesis, increases mitochondrial mass and improves mitochondrial respiratory rate, under physiological condition. Furthermore, overexpression of selenoprotein H protects neuronal HT22 cells from ultraviolet B irradiation-induced cell damage by lowering reactive oxygen species production, and inhibiting activation of caspase-3 and -9, as well as p53. The objective of this study is to identify the cell signaling pathways by which selenoprotein H initiates mitochondrial biogenesis. We first confirmed our previous observation that selenoprotein H transfected HT22 cells increased the protein levels of nuclear-encoded mitochondrial biogenesis factors, peroxisome proliferator-activated receptor γ coactivator-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A. We then observed that total and phosphorylation of protein kinase A, Akt/protein kinase B and cyclic adenosine monophosphate response element-binding protein (CREB) were significantly increased in selenoprotein H transfected cells compared to vector transfected HT22 cells. To verify whether the observed stimulating effects on mitochondrial biogenesis pathways are caused by selenoprotein H and mediated through CREB, we knocked down selenoprotein H mRNA level using siRNA and inhibited CREB with napthol AS-E phosphate in selenoprotein H transfected cells and repeated the measurements of the aforementioned biomarkers. Our results revealed that silencing of selenoprotein H not only decreased the protein levels of PGC-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A, but also decreased the total and

  19. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    Science.gov (United States)

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed.

  20. Localization of Cellular Retinol-Binding Protein and Retinol-Binding Protein in Cells Comprising the Blood-Brain Barrier of Rat and Human

    Science.gov (United States)

    MacDonald, Paul N.; Bok, Dean; Ong, David E.

    1990-06-01

    Brain is not generally recognized as an organ that requiries vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the endothelial cells of the brain microvasculature and within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.

  1. Statistical analysis in the design of nuclear fuel cells

    International Nuclear Information System (INIS)

    This work presents the preliminary results of a statistical analysis carried out for the design of nuclear fuel cells. The analysis consists in verifying the behavior of a cell, related with the frequency of the pines used for its design. In this preliminary study was analyzed the behavior of infinite multiplication factor and the peak factor of local power. On the other hand, the mentioned analysis was carried out using a pines group of enriched uranium previously established, for which varies the pines frequency used in the design. To carry out the study, the CASMO-IV code was used. The obtained designs are for the different axial areas of a fuel assembly. A balance cycle of the unit 1 of the nuclear power plant of Laguna Verde was used like reference. To obtain the result of the present work, systems that are already had and in which have already been implemented the heuristic techniques of ant colonies, neural networks and a hybrid between the dispersed search and the trajectories re-chaining. The results show that is possible to design nuclear fuel cells with a good performance, if is considered a statistical behavior in the frequency of the used pines, in a same way. (Author)

  2. Growth inhibiting effects of antisense eukaryotic expression vector of proliferating cell nuclear antigen gene on human bladder cancer cells

    Institute of Scientific and Technical Information of China (English)

    童强松; 曾甫清; 林晨; 赵军; 鲁功成

    2003-01-01

    Objective To explore the growth inhibiting effects on human bladder cancer by antisense RNA targeting the proliferating cell nuclear antigen (PCNA) gene. Methods The eukaryotic expression vector for antisense PCNA cDNA was constructed and transferred into a bladder cancer EJ cell line. The PCNA expression in the cancer cells was detected by RT-PCR and Western blotting assays. The in vitro proliferation activities of the transferred cells were observed by growth curve, tetrazolium bromide (MTT) colorimetry, tritiated thymidine (3H-TdR)incorporation, flow cytometry and clone formation testing, while its in vivo anti-tumor effects were detected on nude mice allograft models.Results After the antisense vector, pLAPSN, was transferred, cellular PCNA expression was inhibited at both protein and mRNA levels. The growth rates of EJ cells were reduced from 27.91% to 62.07% (P<0.01), with an inhibition of DNA synthesis rate by 52.31% (P<0.01). Transferred cells were blocked at G0/G1 phases in cell-cycle assay, with the clone formation ability decreased by 50.81% (P<0.01). The in vivo carcinogenic abilities of the transferred cancer cells were decreased by 54.23% (P<0.05). Conclusions Antisense PCNA gene transfer could inhibit the growth of bladder cancer cells in vitro and in vivo, which provided an ideal strategy for gene therapy of human cancers.

  3. Using somatic-cell nuclear transfer to study aging.

    Science.gov (United States)

    Kishigami, Satoshi; Lee, Ah Reum; Wakayama, Teruhiko

    2013-01-01

    In mammals, a diploid genome following fertilization of haploid cells, an egg, and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual's inevitable demise. Since it was first reported in 1997 that Dolly the sheep had been cloned, many mammalian species have been cloned successfully using somatic-cell nuclear transfer (SCNT). The success of SCNT in mammals enables us not only to reproduce offspring without germ cells, that is, to "passage" a unique diploid genome, but also to address valuable biological questions on development, nuclear reprogramming, and epigenetic memory. Successful cloning can also support epigenetic reprogramming where the aging clock is reset or reversed. Recent work using iPS cell technology has explored the practicality and led to the recapitulation of premature aging with iPSCs from progeroid laminopathies. As a result, reprogramming tools are also expected to contribute to studying biological age. However, the efficiency of animal cloning is still low in most cases and the mechanism of reprogramming in cloned embryos is still largely unclear. Here, based on recent advances, we describe an improved, more efficient mouse cloning protocol using histone deacetylase inhibitors (HDACis) and latrunculin A, which increases the success rates of producing cloned mice or establishing ES cells fivefold. This improved method of cloning will provide a strong tool to address many issues including biological aging more easily and with lower cost. PMID:23929101

  4. Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

  5. Nuclear microprobe imaging of gallium nitrate in cancer cells

    Science.gov (United States)

    Ortega, Richard; Suda, Asami; Devès, Guillaume

    2003-09-01

    Gallium nitrate is used in clinical oncology as treatment for hypercalcemia and for cancer that has spread to the bone. Its mechanism of antitumor action has not been fully elucidated yet. The knowledge of the intracellular distribution of anticancer drugs is of particular interest in oncology to better understand their cellular pharmacology. In addition, most metal-based anticancer compounds interact with endogenous trace elements in cells, altering their metabolism. The purpose of this experiment was to examine, by use of nuclear microprobe analysis, the cellular distribution of gallium and endogenous trace elements within cancer cells exposed to gallium nitrate. In a majority of cellular analyses, gallium was found homogeneously distributed in cells following the distribution of carbon. In a smaller number of cells, however, gallium appeared concentrated together with P, Ca and Fe within round structures of about 2-5 μm diameter located in the perinuclear region. These intracellular structures are typical of lysosomial material.

  6. Nuclear microprobe imaging of gallium nitrate in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, Richard E-mail: ortega@cenbg.in2p3.fr; Suda, Asami; Deves, Guillaume

    2003-09-01

    Gallium nitrate is used in clinical oncology as treatment for hypercalcemia and for cancer that has spread to the bone. Its mechanism of antitumor action has not been fully elucidated yet. The knowledge of the intracellular distribution of anticancer drugs is of particular interest in oncology to better understand their cellular pharmacology. In addition, most metal-based anticancer compounds interact with endogenous trace elements in cells, altering their metabolism. The purpose of this experiment was to examine, by use of nuclear microprobe analysis, the cellular distribution of gallium and endogenous trace elements within cancer cells exposed to gallium nitrate. In a majority of cellular analyses, gallium was found homogeneously distributed in cells following the distribution of carbon. In a smaller number of cells, however, gallium appeared concentrated together with P, Ca and Fe within round structures of about 2-5 {mu}m diameter located in the perinuclear region. These intracellular structures are typical of lysosomial material.

  7. Gangliosides in cell recognition and membrane protein regulation

    OpenAIRE

    Lopez, Pablo H. H.; Schnaar, Ronald L.

    2009-01-01

    Gangliosides, sialic acid-bearing glycosphingolipids, are expressed on all vertebrate cells, and are the major glycans on nerve cells. They are anchored to the plasma membrane through their ceramide lipids with their varied glycans extending into the extracellular space. Through sugar-specific interactions with glycan binding proteins on apposing cells, gangliosides function as receptors in cell-cell recognition, regulating natural killer cell cytotoxicity via Siglec-7 binding, myelin-axon in...

  8. Charged MVB protein 5 is involved in T-cell receptor signaling.

    Science.gov (United States)

    Wi, Sae Mi; Min, Yoon; Lee, Ki-Young

    2016-01-01

    Charged multivesicular body protein 5 (CHMP5) has a key role in multivesicular body biogenesis and a critical role in the downregulation of signaling pathways through receptor degradation. However, the role of CHMP5 in T-cell receptor (TCR)-mediated signaling has not been previously investigated. In this study, we utilized a short hairpin RNA-based RNA interference approach to investigate the functional role of CHMP5. Upon TCR stimulation, CHMP5-knockdown (CHMP5(KD)) Jurkat T cells exhibited activation of TCR downstream signaling molecules, such as PKCθ and IKKαβ, and resulted in the activation of nuclear factor-κB and the marked upregulation of TCR-induced gene expression. Moreover, we found that activator protein-1 and nuclear factor of activated T-cells transcriptional factors were markedly activated in CHMP5(KD) Jurkat cells in response to TCR stimulation, which led to a significant increase in interleukin-2 secretion. Biochemical studies revealed that CHMP5 endogenously forms high-molecular-weight complexes, including TCR molecules, and specifically interacts with TCRβ. Interestingly, flow cytometry analysis also revealed that CHMP5(KD) Jurkat T cells exhibit upregulation of TCR expression on the cell surface compared with control Jurkat T cells. Taken together, these findings demonstrated that CHMP5 might be involved in the homeostatic regulation of TCR on the cell surface, presumably through TCR recycling or degradation. Thus CHMP5 is implicated in TCR-mediated signaling. PMID:26821576

  9. Charged MVB protein 5 is involved in T-cell receptor signaling.

    Science.gov (United States)

    Wi, Sae Mi; Min, Yoon; Lee, Ki-Young

    2016-01-29

    Charged multivesicular body protein 5 (CHMP5) has a key role in multivesicular body biogenesis and a critical role in the downregulation of signaling pathways through receptor degradation. However, the role of CHMP5 in T-cell receptor (TCR)-mediated signaling has not been previously investigated. In this study, we utilized a short hairpin RNA-based RNA interference approach to investigate the functional role of CHMP5. Upon TCR stimulation, CHMP5-knockdown (CHMP5(KD)) Jurkat T cells exhibited activation of TCR downstream signaling molecules, such as PKCθ and IKKαβ, and resulted in the activation of nuclear factor-κB and the marked upregulation of TCR-induced gene expression. Moreover, we found that activator protein-1 and nuclear factor of activated T-cells transcriptional factors were markedly activated in CHMP5(KD) Jurkat cells in response to TCR stimulation, which led to a significant increase in interleukin-2 secretion. Biochemical studies revealed that CHMP5 endogenously forms high-molecular-weight complexes, including TCR molecules, and specifically interacts with TCRβ. Interestingly, flow cytometry analysis also revealed that CHMP5(KD) Jurkat T cells exhibit upregulation of TCR expression on the cell surface compared with control Jurkat T cells. Taken together, these findings demonstrated that CHMP5 might be involved in the homeostatic regulation of TCR on the cell surface, presumably through TCR recycling or degradation. Thus CHMP5 is implicated in TCR-mediated signaling.

  10. Cloned calves produced by nuclear transfer from cultured cumulus cells

    Institute of Scientific and Technical Information of China (English)

    AN; Xiaorong(安晓荣); GOU; Kemian(苟克勉); ZHU; Shien(朱士恩); GUAN; Hong(关宏); HOU; Jian(侯健); LIN; Aixing(林爱星); ZENG; Shenming(曾申明); TIAN; Jianhui(田见辉); CHEN; Yongfu(陈永福)

    2002-01-01

    Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2-3 h, was significantly lower than that of the 3-6 h groups (31.0%), while not significantly different among 3-4 h (P < 0.05), 4-5 h, and 5-6 h groups (P ≥ 0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.

  11. A mass spectrometric-derived cell surface protein atlas.

    Directory of Open Access Journals (Sweden)

    Damaris Bausch-Fluck

    Full Text Available Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa. The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.

  12. A mass spectrometric-derived cell surface protein atlas.

    Science.gov (United States)

    Bausch-Fluck, Damaris; Hofmann, Andreas; Bock, Thomas; Frei, Andreas P; Cerciello, Ferdinando; Jacobs, Andrea; Moest, Hansjoerg; Omasits, Ulrich; Gundry, Rebekah L; Yoon, Charles; Schiess, Ralph; Schmidt, Alexander; Mirkowska, Paulina; Härtlová, Anetta; Van Eyk, Jennifer E; Bourquin, Jean-Pierre; Aebersold, Ruedi; Boheler, Kenneth R; Zandstra, Peter; Wollscheid, Bernd

    2015-01-01

    Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments. PMID:25894527

  13. Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells.

    Science.gov (United States)

    Cui, Yong; Gao, Caiji; Zhao, Qiong; Jiang, Liwen

    2016-01-01

    Studies of protein subcellular localization and dynamics are helpful in understanding the cellular functions of proteins in an organism. In the past decade, the use of green fluorescent protein (GFP) as a fusion tag has dramatically extended our knowledge in this field. Transient expression and stable transformation of GFP-tagged proteins have been wildly used to study protein localization in vivo in different systems. Although GFP-based tags provide a fast and convenient way to characterize protein properties in living cells, several reports have demonstrated that GFP fusions might not accurately reflect the localization of the native protein as GFP tags may alter the protein properties. To facilitate proper usage of GFP tags in plant cell biology study, we describe detailed protocols to identify possible inhibitory effects of fluorescent tags on protein subcellular localization and to determine if a fluorescently tagged protein is localized to the correct subcellular compartment. Using Arabidopsis Endomembrane protein 12 (EMP12) as an example, we first show the possible inhibitory effect of GFP tags on proper protein localization and then describe the immunofluorescence labeling method to verify the correct localization of GFP fusion proteins. Next, a method is presented using the ImageJ program with the Pearson-Spearman correlation (PSC) colocalization plug-in for statistical quantification of colocalization ratios of two fluorophores. Finally we provide a detailed method for protein dynamics studies using spinning disk confocal microscopy in Arabidopsis cells. PMID:27515077

  14. Human NTH1 physically interacts with p53 and proliferating cell nuclear antigen

    International Nuclear Information System (INIS)

    Thymine glycol (Tg) is one of predominant oxidative DNA lesions caused by ionizing radiation and other oxidative stresses. Human NTH1 is a bifunctional enzyme with DNA glycosylase and AP lyase activities and removes Tg as the first step of base excision repair (BER). We have searched for the factors interacting with NTH1 by using a pull-down assay and found that GST-NTH1 fusion protein precipitates proliferating cell nuclear antigen (PCNA) and p53 as well as XPG from human cell-free extracts. GST-NTH1 also bound to recombinant FLAG-tagged XPG, PCNA, and (His)6-tagged p53 proteins, indicating direct protein-protein interaction between those proteins. Furthermore, His-p53 and FLAG-XPG, but not PCNA, stimulated the Tg DNA glycosylase/AP lyase activity of GST-NTH1 or NTH1. These results provide an insight into the positive regulation of BER reaction and also suggest a possible linkage between BER of Tg and other cellular mechanisms

  15. Epigallocatechin-3-gallate regulates cell growth, cell cycle and phosphorylated nuclear factor-KB in human dermal fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Dong-Wook HAN; Mi Hee LEE; Hak Hee KIM; Suong-Hyu HYON; Jong-Chul PARK

    2011-01-01

    Aim: To investigate the effects of (-)epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, on cell growth, cell cycle and phosphorylated nuclear factor-kB (pNF-KB) expression in neonatal human dermal fibroblasts (nHDFs).Methods: The proliferation and cell-cycle of nHDFs were determined using WST-8 cell growth assay and flow cytometry, respectively. The apoptosis was examined using DNA ladder and Annexin V-FITC assays. The expression levels of pNF-kB and cell cycle-related genes and proteins in nHDFs were measured using cDNA microarray analyses and Western blot. The cellular uptake of EGCG was examined using fluorescence (FITC)-Iabeled EGCG (FITC-EGCG) in combination with confocal microscopy.Results: The effect of EGCG on the growth of nHDFs depended on the concentration tested. At a low concentration (200 μmol/L), EGCG resulted in a slight decrease in the proportion of ceils in the S and G/M phases of cell cycle with a concomitant increase in the proportion of cells in G/G phase. At the higher doses (400 and 800 pmol/L), apoptosis was induced. The regulation of EGCG on the expression of pNF-kB was also concentration-dependent, whereas it did not affect the unphosphorylated NF-kB expression, cDNA microarray analysis showed that cell cycle-related genes were down-regulated by EGCG (200 μmol/L). The expression of cyclins A/B and cyclin-dependent kinase 1 was reversibly regulated by EGCG (200 μmol/L). FITC-EGCG was found to be internalized into the cyto-plasm and translocated into the nucleus of nHDFs.Conclusion: EGCG, through uptake into cytoplasm, reversibly regulated the cell growth and expression of cell cycle-related proteins and genes in normal fibroblasts.

  16. Control of Oxidative Stress and Generation of Induced Pluripotent Stem Cell-like Cells by Jun Dimerization Protein 2

    International Nuclear Information System (INIS)

    We report here that the Jun dimerization protein 2 (JDP2) plays a critical role as a cofactor for the transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and MafK in the regulation of the antioxidants and production of reactive oxygen species (ROS). JDP2 associates with Nrf2 and MafK (Nrf2-MafK) to increase the transcription of antioxidant response element-dependent genes. Oxidative-stress-inducing reagent led to an increase in the intracellular accumulation of ROS and cell proliferation in Jdp2 knock-out mouse embryonic fibroblasts. In Jdp2-Cre mice mated with reporter mice, the expression of JDP2 was restricted to granule cells in the brain cerebellum. The induced pluripotent stem cells (iPSC)-like cells were generated from DAOY medulloblastoma cell by introduction of JDP2, and the defined factor OCT4. iPSC-like cells expressed stem cell-like characteristics including alkaline phosphatase activity and some stem cell markers. However, such iPSC-like cells also proliferated rapidly, became neoplastic, and potentiated cell malignancy at a later stage in SCID mice. This study suggests that medulloblastoma cells can be reprogrammed successfully by JDP2 and OCT4 to become iPSC-like cells. These cells will be helpful for studying the generation of cancer stem cells and ROS homeostasis

  17. Control of Oxidative Stress and Generation of Induced Pluripotent Stem Cell-like Cells by Jun Dimerization Protein 2

    Directory of Open Access Journals (Sweden)

    Naoto Yamaguchi

    2013-07-01

    Full Text Available We report here that the Jun dimerization protein 2 (JDP2 plays a critical role as a cofactor for the transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2 and MafK in the regulation of the antioxidants and production of reactive oxygen species (ROS. JDP2 associates with Nrf2 and MafK (Nrf2-MafK to increase the transcription of antioxidant response element-dependent genes. Oxidative-stress-inducing reagent led to an increase in the intracellular accumulation of ROS and cell proliferation in Jdp2 knock-out mouse embryonic fibroblasts. In Jdp2-Cre mice mated with reporter mice, the expression of JDP2 was restricted to granule cells in the brain cerebellum. The induced pluripotent stem cells (iPSC-like cells were generated from DAOY medulloblastoma cell by introduction of JDP2, and the defined factor OCT4. iPSC-like cells expressed stem cell-like characteristics including alkaline phosphatase activity and some stem cell markers. However, such iPSC-like cells also proliferated rapidly, became neoplastic, and potentiated cell malignancy at a later stage in SCID mice. This study suggests that medulloblastoma cells can be reprogrammed successfully by JDP2 and OCT4 to become iPSC-like cells. These cells will be helpful for studying the generation of cancer stem cells and ROS homeostasis.

  18. Control of Oxidative Stress and Generation of Induced Pluripotent Stem Cell-like Cells by Jun Dimerization Protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Chiou, Shyh-Shin, E-mail: chiouss@kmu.edu.tw [Division of Hematology-Oncology, Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan (China); Department of Pediatrics, Faculty of Medicine, School of Medicine, Kaohsiung Medical University, 807 Kaohsiung 807, Taiwan (China); Wang, Sophie Sheng-Wen; Wu, Deng-Chyang [Department of Gastroenterology, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan (China); Lin, Ying-Chu [School of Dentistry, College of Dentistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Kao, Li-Pin [Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, 807 Kaohsiung 807, Taiwan (China)

    2013-07-26

    We report here that the Jun dimerization protein 2 (JDP2) plays a critical role as a cofactor for the transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and MafK in the regulation of the antioxidants and production of reactive oxygen species (ROS). JDP2 associates with Nrf2 and MafK (Nrf2-MafK) to increase the transcription of antioxidant response element-dependent genes. Oxidative-stress-inducing reagent led to an increase in the intracellular accumulation of ROS and cell proliferation in Jdp2 knock-out mouse embryonic fibroblasts. In Jdp2-Cre mice mated with reporter mice, the expression of JDP2 was restricted to granule cells in the brain cerebellum. The induced pluripotent stem cells (iPSC)-like cells were generated from DAOY medulloblastoma cell by introduction of JDP2, and the defined factor OCT4. iPSC-like cells expressed stem cell-like characteristics including alkaline phosphatase activity and some stem cell markers. However, such iPSC-like cells also proliferated rapidly, became neoplastic, and potentiated cell malignancy at a later stage in SCID mice. This study suggests that medulloblastoma cells can be reprogrammed successfully by JDP2 and OCT4 to become iPSC-like cells. These cells will be helpful for studying the generation of cancer stem cells and ROS homeostasis.

  19. Tetramethylpyrazine protected photoreceptor cells of rats by modulating nuclear translocation of NF-κB

    Institute of Scientific and Technical Information of China (English)

    Jin-nan YANG; Jin-mao CHEN; Lin LUO; Shao-chun LIN; Dai LI; Shi-xing HU

    2005-01-01

    Aim: To evaluate the effect of tetramethylpyrazine (TMP) injection on retinal damage induced by N-methyl-N-nitrosourea (MNU) in rats and on nuclear factorkappa B (NF-κB) family members.Methods: Female Sprague-Dawley (SD) rats were randomly divided into groups: (i), control group; (ii), model group; and (iii),TMP-injection groups, in which the rats were subdivided into 40 mg/kg, 80 mg/kg and 160 mg/kg groups.Drugs were injected ip into 47-day-old SD rats once a day.physiological saline.All rats were killed at different times after MNU or physiological saline treatment.The apoptotic index of photoreceptor cells was calculated by TUNEL labeling; retinal damage was evaluated based on retinal thickness and the expression of NF-κB family members was detected by Western blot.Results: TMP injections, in a dose-dependent manner, suppressed photoreceptor cell apoptosis and decreased its loss in the peripheral retina.As compared with the MNU-treated group, TMP injection at a dose of 160 mg/kg also timedependently upregulated the NF-κB/p65 protein level in the nucleus and downregulated the Iκ Bα protein level in the cytoplasm.However, no protective effect of TMP injection on MNU-induced central retinal damage was found.Conclusion: TMP injection partially protects against MNU-induced retiral damage by upregulating the nuclear translocation of p65 to inhibit photoreceptor cells apoptosis.

  20. Analysis of nucleolar morphology and protein localization as an indicator of nuclear reprogramming

    DEFF Research Database (Denmark)

    Østrup, Olga; Pedersen, Hanne Skovsgaard; Holm, Hanne M.;

    2015-01-01

    to cloning by somatic cell nuclear transfer. However, when cells are reprogrammed by less fundamental means, as for example treatment by Xenopus extract or expression of pluripotency genes, more subtle nucleolar modulations can also be noted. The monitoring and understanding of the reprogramming...

  1. SIRT1 interacts with and protects glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from nuclear translocation: Implications for cell survival after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Joo, Hyun-Yoo [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Woo, Seon Rang; Shen, Yan-Nan; Yun, Mi Yong; Shin, Hyun-Jin; Park, Eun-Ran; Kim, Su-Hyeon; Park, Jeong-Eun; Ju, Yeun-Jin; Hong, Sung Hee; Hwang, Sang-Gu [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Cho, Myung-Haing [Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742 (Korea, Republic of); Kim, Joon, E-mail: joonkim@korea.ac.kr [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Lee, Kee-Ho, E-mail: khlee@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer SIRT1 serves to retain GAPDH in the cytosol, preventing GAPDH nuclear translocation. Black-Right-Pointing-Pointer When SIRT1 is depleted, GAPDH translocation occurs even in the absence of stress. Black-Right-Pointing-Pointer Upon irradiation, SIRT1 interacts with GAPDH. Black-Right-Pointing-Pointer SIRT1 prevents irradiation-induced nuclear translocation of GAPDH. Black-Right-Pointing-Pointer SIRT1 presence rather than activity is essential for inhibiting GAPDH translocation. -- Abstract: Upon apoptotic stimulation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytosolic enzyme normally active in glycolysis, translocates into the nucleus and activates an apoptotic cascade therein. In the present work, we show that SIRT1 prevents nuclear translocation of GAPDH via interaction with GAPDH. SIRT1 depletion triggered nuclear translocation of cytosolic GAPDH even in the absence of apoptotic stress. Such translocation was not, however, observed when SIRT1 enzymatic activity was inhibited, indicating that SIRT1 protein per se, rather than the deacetylase activity of the protein, is required to inhibit GAPDH translocation. Upon irradiation, SIRT1 prevented irradiation-induced nuclear translocation of GAPDH, accompanied by interaction of SIRT1 and GAPDH. Thus, SIRT1 functions to retain GAPDH in the cytosol, protecting the enzyme from nuclear translocation via interaction with these two proteins. This serves as a mechanism whereby SIRT1 regulates cell survival upon induction of apoptotic stress by means that include irradiation.

  2. Taperin (c9orf75, a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1α docking protein

    Directory of Open Access Journals (Sweden)

    Tony Ferrar

    2012-02-01

    The promiscuous activity of protein phosphatase one (PP1 is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75, encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic ‘RVxF’ motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and IIα. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway.

  3. Association of Bovine Papillomavirus E2 Protein with Nuclear Structures In Vivo

    OpenAIRE

    Kurg, Reet; Sild, Kristiina; Ilves, Aigi; Sepp, Mari; Ustav, Mart

    2005-01-01

    Papillomaviruses are small DNA viruses which have the capacity to establish a persistent infection in mammalian epithelial cells. The papillomavirus E2 protein is a central coordinator of viral gene expression, genome replication, and maintenance. We have investigated the distribution of bovine papillomavirus E2 protein in nuclei of proliferating cells and found that E2 is associated with cellular chromatin. This distribution does not change during the entire cell cycle. The N-terminal transa...

  4. The hepatitis E virus ORF3 protein regulates the expression of liver-specific genes by modulating localization of hepatocyte nuclear factor 4.

    Directory of Open Access Journals (Sweden)

    Vivek Chandra

    Full Text Available The hepatitis E virus (HEV is a small RNA virus and the cause of acute viral hepatitis E. The open reading frame 3 protein (pORF3 of HEV appears to be a pleiotropic regulatory protein that helps in the establishment, propagation and progression of viral infection. However, the global cellular effects of this protein remain to be explored. In the absence of traditional in vitro viral infection systems or efficient replicon systems, we made an adenovirus based ORF3 protein expression system to study its effects on host cell gene expression. We infected Huh7 hepatoma cells with recombinant adenoviruses expressing pORF3 and performed microarray-based gene expression analyses. Several genes down regulated in pORF3-expressing cells were found to be under regulation of the liver-enriched hepatocyte nuclear factor 4 (HNF4, which regulates hepatocyte-specific gene expression. While HNF4 localizes to the nucleus, its phosphorylation results in impaired nuclear localization of HNF4. Here we report that pORF3 increases HNF4 phosphorylation through the ERK and Akt kinases, which results in impaired nuclear translocation of HNF4 and subsequently the down modulation of HNF4-responsive genes in pORF3-expressing cells. We propose that modulation of several hepatocyte specific genes by pORF3 will create an environment favorable for viral replication and pathogenesis.

  5. Monodisperse magnetite nanoparticles coupled with nuclear localization signal peptide for cell-nucleus targeting.

    Science.gov (United States)

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

    2008-03-01

    Functionalization of monodisperse superparamagnetic magnetite (Fe(3)O(4)) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe(3)O(4) nanoparticles functionalized with protein and nuclear localization signal (NLS) peptide. These NLS-coated nanoparticles were introduced into the HeLa cell cytoplasm and nucleus, where the particles were monodispersed and non-aggregated. The success of labeling was examined and identified by fluorescence microscopy and MRI. The work demonstrates that monodisperse magnetic nanoparticles can be readily functionalized and stabilized for potential diagnostic and therapeutic applications. PMID:18080259

  6. Ephemeral protein binding to DNA shapes stable nuclear bodies and chromatin domains

    CERN Document Server

    Brackley, C A; Michieletto, D; Mouvet, F; Cook, P R; Marenduzzo, D

    2016-01-01

    Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear "bodies" exchange rapidly with the soluble pool whilst the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins; these proteins switch between two states -- active (binding) and inactive (non-binding). This system provides a model for any DNA-binding protein that can be modified post-translationally to change its affinity for DNA (e.g., like the phosphorylation of a transcription factor). Due to this out-of-equilibrium process, proteins spontaneously assemble into clusters of self-limiting size, as individual proteins in a cluster exchange with the soluble pool with kinetics like those seen in photo-bleaching experiments. This behavior contrasts sharply with that exhibited by "equilibrium", or non-switching, proteins that exis...

  7. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins

    DEFF Research Database (Denmark)

    Weber, Daniela; Davies, Michael J.; Grune, Tilman

    2015-01-01

    in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented...... different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used...

  8. Protein expression of G-protein inwardly rectifying potassium channels (GIRK in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Plummer Howard K

    2006-08-01

    Full Text Available Abstract Background Previous data from our laboratory has indicated that a functional link exists between the G-protein-coupled inwardly rectifying potassium (GIRK channel and the beta-adrenergic receptor pathway in breast cancer cell lines, and these pathways were involved in growth regulation of these cells. Alcohol is an established ri