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Sample records for latent nuclear antigen

  1. High expression of the Epstein-Barr virus latent protein EB nuclear antigen-2 on pyothorax-associated lymphomas.

    PubMed Central

    Sasajima, Y.; Yamabe, H.; Kobashi, Y.; Hirai, K.; Mori, S.

    1993-01-01

    Pyothorax-associated lymphoma (PAL) is a rare tumor associated with long-standing tuberculous pyothorax. Most of these lymphomas are B-cell lymphomas of high-grade malignancy. Over 50 cases have been reported in Japan, but no cases have been described in Western countries. Its pathogenesis remains unknown. We studied immunohistologically the expression of Epstein-Barr virus- (EBV) encoded latent gene products, EB nuclear antigen-2 and LMP-1, in four cases of PAL. Fifty B-cell lymphomas unrelated to pyothorax, and five EBV-bearing lymphoblastic tumors produced in severe combined immune deficient mice (severe combined immune deficient-EBV+ tumors) were also studied as controls. Marked expression of EB nuclear antigen-2 was demonstrated on all four PALs. LMP-1 was also present in all cases, but both the staining intensity and the number of stained cells remained less than on severe combined immune deficient-EBV+ tumors. Neither EB nuclear antigen-2 nor LMP-1 was observed in the 50 control B-cell lymphomas. Additional molecular genetic analysis revealed that EBVs are incorporated into each PAL clonally. These results confirm the definite association of EBV with PALs, although the significance of weak expression of LMP-1 awaits further study. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8238246

  2. Tissue Specificity of the Kaposi's Sarcoma-Associated Herpesvirus Latent Nuclear Antigen (LANA/orf73) Promoter in Transgenic Mice

    PubMed Central

    Jeong, Joseph H.; Hines-Boykin, Rebecca; Ash, John D.; Dittmer, Dirk P.

    2002-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is a human-oncogenic herpesvirus. Cells from KSHV-associated tumors, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), are of endothelial and B-cell origin, respectively. KSHV persists indefinitely in these cell lineages during latent infection. Indeed, cellular latency is a hallmark of all herpesviruses that is intimately linked to their pathogenesis. We previously characterized the promoter for the KSHV latency-associated nuclear antigen LANA/orf73. LANA is required for latent episome maintenance and has also been implicated in oncogenesis. Hence, regulation of LANA expression is critical to KSHV persistence. We find that a region extending to bp −1299 upstream of the LANA transcription start site is able to drive lacZ-reporter gene expression in several lines of transgenic mice. In agreement with KSHV's natural tropism, we detected reporter gene expression in CD19-positive B cells but not in CD3-positive T cells. We also detected expression in the kidney and, at a lower level, in the liver. In contrast to KS tumors, transgene expression was localized to kidney tubular epithelium rather than vascular endothelial cells. This suggests that our promoter fragment contains all cis-regulatory elements sufficient for B-cell specificity but not those required for endothelial specificity. Alternatively, while the trans-acting factors required for LANA expression in B cells are evolutionarily conserved, those that regulate endothelial cell-specific expression are unique to humans. Our in vivo studies address a conundrum in KSHV biology: in culture, KSHV is able to infect a variety of cell types indiscriminately, while in healthy latent carriers KSHV is found in B lymphocytes. The transgenic-mouse experiments reported here suggest that tissue-restricted LANA gene expression could explain B-cell-specific viral persistence. PMID:12368345

  3. DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication

    SciTech Connect

    Cha, Seho; Lim, Chunghun; Lee, Jae Young; Song, Yoon-Jae; Park, Junsoo; Choe, Joonho; Seo, Taegun

    2010-04-16

    During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

  4. Immunohistochemical detection of the latent nuclear antigen-1 of the human herpesvirus type 8 to differentiate cutaneous epidemic Kaposi sarcoma and its histological simulators.

    PubMed

    Pereira, Patricia Fonseca; Cuzzi, Tullia; Galhardo, Maria Clara Gutierrez

    2013-01-01

    Kaposi's sarcoma is the most common neoplasia diagnosed in AIDS patients and the expression of the human herpesvirus-8 (HHV-8) latent nuclear antigen-1 has been useful for its histological diagnosis. The aim of this study is to confirm that immunohistochemistry is a valuable tool for differentiating KS from its simulators in skin biopsies of HIV patients. Immunohistochemical and histological analyses were performed in 49 Kaposi's sarcoma skin biopsies and 60 of its histological simulators. Positivity was present in the 49 Kaposi's sarcoma skin biopsies and no staining was observed in the 60 simulators analyzed, resulting in sensibility and specificity of 100%. HHV-8 immunohistochemical detection is an effective tool for diagnosing Kaposi's sarcoma, especially in early lesions in which neoplastic features are not evident. It also contributes to its histological differential diagnosis.

  5. Follicular dendritic cells in multicentric Castleman disease present human herpes virus type 8 (HHV8)-latent nuclear antigen 1 (LANA1) in a proportion of cases and is associated with an enhanced T-cell response.

    PubMed

    El-Daly, Hesham; Bower, Mark; Naresh, Kikkeri N

    2010-02-01

    Multicentric Castleman disease (MCD) is a rare human herpes virus type 8 (HHV8)-associated lymphoproliferative disorder that occurs more frequently in patients infected with human immunodeficiency virus (HIV). In tissue samples, HHV8-infected plasmablasts localise to the mantle zones of the lymphoid follicles. We revisited the immunohistological features in 25 lymph node (LN) and three spleen samples of MCD. In five (20%) LN and one (33%) spleen sample, HHV8 latent nuclear antigen 1 (LANA1) staining was also noted on the follicular dendritic cells (FDC). The HHV8-positive FDC subgroup of patients had significantly higher numbers of CD3-positive T cells infiltrating the follicles when compared to the HHV8-negative FDC subgroup (P = 0.047). Furthermore, the numbers of HHV8-positive plasmablasts and serum HHV8 viral copy numbers were lower among the HHV8-positive FDC subgroup when compared to the HHV8-negative FDC subgroup (not statistically significant). Our findings show, for the first time, possible 'presentation' of an HHV8 antigen by FDCs in MCD.

  6. Identification of Major Phosphorylation Sites of Epstein-Barr Virus Nuclear Antigen Leader Protein (EBNA-LP): Ability of EBNA-LP To Induce Latent Membrane Protein 1 Cooperatively with EBNA-2 Is Regulated by Phosphorylation†

    PubMed Central

    Yokoyama, Akihiko; Tanaka, Michiko; Matsuda, Go; Kato, Kentaro; Kanamori, Mikiko; Kawasaki, Hiroshi; Hirano, Hisashi; Kitabayashi, Issay; Ohki, Misao; Hirai, Kanji; Kawaguchi, Yasushi

    2001-01-01

    Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a phosphoprotein suggested to play important roles in EBV-induced immortalization of B cells. One of the potential functions of EBNA-LP is a cooperative induction with EBNA-2 of viral and cellular gene expression, including that of the genes for viral latent membrane protein 1 (LMP-1) and cellular cyclin D2. We report here that the phosphorylation of EBNA-LP by cellular kinase(s) is critical to its ability to cooperate with EBNA-2 in up-regulating the expression of LMP-1 in a B-lymphoma cell line. Our conclusion is based on the following observations. (i) Mass-spectrometric analysis of purified EBNA-LP and mutational analyses of EBNA-LP revealed that the serine residue at position 35 in the W2 repeat domain is the major phosphorylation site of EBNA-LP in vivo. (ii) Substitutions of this site in each W2 repeat domain with alanine markedly reduced the ability of the protein to induce LMP-1 expression in combination with EBNA-2 in Akata cells. (iii) Replacement at the major phosphorylation sites with glutamic acids restored the wild-type phenotype. It is well established that this substitution mimics constitutive phosphorylation. These results indicated that the coactivator function of EBNA-LP is regulated by phosphorylation. PMID:11333893

  7. [Characteristic of nuclear antigen 1 gene and latent membrane protein 1 gene of Epstein-Barr virus in primary EBV infection in children in Beijing area in 2005-2010].

    PubMed

    Ai, Jun-Hong; Xie, Zheng-De; Liu, Chun-Yan; Gao, Li-Wei; Yan, Jing

    2012-10-01

    To analyze the characteristic of nuclear antigen 1 gene and latent membrane protein 1 gene of Epstein-Barr virus in primary EBV infection in children in Beijing area in 2005-2012. Polymerase chain reaction (PCR) was used to amplify the EBNA-3C, EBNA1 and LMP1 genes. The amplified products were sequenced directly and the sequences were analyzed by BioEdit 7. 0. 9 and MEGA 4. 0. 2. Type A EBV was detected in 98% samples. Nucleotide sequence analysis of the carboxy-terminal region of EBNA1 showed that Vvvl was deteted in 98% samples. DNA sequence analysis of LMP1 C-terminus indicated that China 1 was 90% in this study. There were no significant differences in the frequency of Vvv1 and China 1 between the IM and HLH samples (P = 1.00). Linkage analysis of EBV types, EBNA1 and LMP1 variants indicated that 90% of EBV type A was associated with EBNA1-Vvv1 variant and LMP1-China 1 variant in 40 cases. Full length of LMP1 gene was successfully amplified in 35 cases. Four Chinese groups (CG1-4) were identified. The percentage of CG1-CG4 were 85%, 6%, 6% and 3%, respectively. EBV type A is predominant in primary EBV infection in children in Beijing Area. EBNA1-Vvv1 and LMP1-China 1 variants were predominant genotypes in this area. There is a high linkage between EBNA1-Vvv1 variant and LMP1-China 1 variant. Four Chinese groups (CG1-4) were identified according to the full length of LMP1 gene and CG1 was the most prevalent.

  8. [The clinical significance of serum Epstein-Barr virus-determined nuclear antigen 1 (EBNA1)/latent membrane protein 1 (LMP1) assay in patients with nasal type extranodal NK/T-cell lymphoma].

    PubMed

    Yao, Na; Cui, Xueying; Wang, Jingwen

    2015-02-01

    To explore the clinical significance of the serum Epstein-Barr virus-determined nuclear antigen 1 (EBNA1)/latent membrane protein 1 (LMP1) in patients with extranodal NK/T-cell lymphoma, nasal type (ENKL). The serum EBNA1 and LMP1 were detected by real-time PCR in 36 ENKL patients hospitalized in Beijing Tongren Hospital from August 2010 to August 2013. Twenty healthy volunteers were recruited as controls. The median serum EBNA1 was 1.9×10(4) (ranged from 0 to 11.0×10(4)) copies/µl in ENKL patients and 8.0 (ranged from 0 to 43.8) copies/µl in healthy volunteers. The median serum LMP1 was 3.9×10(3) (ranged from 118.3 to 24.0×10(3)) copies/µl in ENKL patients and 3.3 (ranged from 0 to 33.3) copies/µl in healthy volunteers. Both EBNA1 and LMP1 were higher in ENKL patients than healthy volunteers (all P < 0.01). The median EBNA1 and LMP1 in ENKL patients posttreatment were 1.0×10(3) (ranged from 0 to 2.0 × 10(3)) copies/µl and 300.8 (ranged from 0 to 825.7) copies/µl respectively, which were both significantly decreased than pretreatment (all P < 0.05). The EBNA1 and LMP1 were decreased in effective treatment group versus ineffective treatment group (P < 0.05). The serum EBNA1 and LMP1 were positively correlated with lactic dehydrogenase (LDH) level (r = 0.364,0.546; P = 0.040,0.012). (1) The measurement of EBNA1/LMP1 may be useful in evaluating the therapeutic effect. (2) The serum EBNA1/LMP1 may reflect the tumor load in ENKL patients.

  9. T Helper Cell Tolerance to Ubiquitous Nuclear Antigens

    PubMed Central

    NAKKEN, BRITT; DAVIS, KAREN E.; PAN, ZIJIAN; BACHMANN, MICHAEL; FARRIS, A. DARISE

    2007-01-01

    Systemic autoimmune diseases are characterized by the development of anti-nuclear autoantibodies. In order to understand the immunologic events leading to the development of such antibodies, knowledge of mechanisms of immune tolerance to nuclear antigens is required. By utilizing adoptive T cell transfer strategies with transgenic mouse models expressing nuclear neo-self antigens, T cell tolerance to the lupus-related nuclear antigens human La and nRNP A has been demonstrated. These findings also indicate the existence in normal animals of autoreactive B cells continuously presenting nuclear antigen, suggesting that nuclear antigens are not sequestered from the immune system. Investigations of CD4+ T cell tolerance to non-nuclear antigens have revealed a number of mechanisms that protect the host from autoreactivity, including autoreactive T cell deletion, regulatory T cell development and anergy induction. Recent studies using T cell receptor and neo-self nuclear antigen transgenic mice are revealing the importance of such mechanisms in maintaining tolerance to nuclear antigens. Mechanisms of tolerogenic antigen presentation, identification of tolerogenic antigen source(s), and the pathways leading to loss of tolerance to nuclear antigens in systemic autoimmune disease states are currently being sought. PMID:14629620

  10. Structure and Function of Latency-Associated Nuclear Antigen

    PubMed Central

    Verma, S. C.; Lan, K.

    2011-01-01

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

  11. A Mycobacterium tuberculosis Dormancy Antigen Differentiates Latently Infected Bacillus Calmette–Guérin-vaccinated Individuals

    PubMed Central

    Peña, Delfina; Rovetta, Ana I.; Hernández Del Pino, Rodrigo E.; Amiano, Nicolás O.; Pasquinelli, Virginia; Pellegrini, Joaquín M.; Tateosian, Nancy L.; Rolandelli, Agustín; Gutierrez, Marisa; Musella, Rosa M.; Palmero, Domingo J.; Gherardi, María M.; Iovanna, Juan; Chuluyan, H. Eduardo; García, Verónica E.

    2015-01-01

    IFN-γ release assays (IGRAs) are better indicators of Mycobacterium tuberculosis infection than the tuberculin skin test (TST) in Bacillus Calmette–Guérin (BCG)-vaccinated populations. However, IGRAs do not discriminate active and latent infections (LTBI) and no gold standard for LTBI diagnosis is available. Thus, since improved tests to diagnose M. tuberculosis infection are required, we assessed the efficacy of several M. tuberculosis latency antigens. BCG-vaccinated healthy donors (HD) and tuberculosis (TB) patients were recruited. QuantiFERON-TB Gold In-Tube, TST and clinical data were used to differentiate LTBI. IFN-γ production against CFP-10, ESAT-6, Rv2624c, Rv2626c and Rv2628 antigens was tested in peripheral blood mononuclear cells. LTBI subjects secreted significantly higher IFN-γ levels against Rv2626c than HD. Additionally, Rv2626c peptide pools to which only LTBI responded were identified, and their cumulative IFN-γ response improved LTBI discrimination. Interestingly, whole blood stimulation with Rv2626c allowed the discrimination between active and latent infections, since TB patients did not secrete IFN-γ against Rv2626c, in contrast to CFP-10 + ESAT-6 stimulation that induced IFN-γ response from both LTBI and TB patients. ROC analysis confirmed that Rv2626c discriminated LTBI from HD and TB patients. Therefore, since only LTBI recognizes specific epitopes from Rv2626c, this antigen could improve LTBI diagnosis, even in BCG-vaccinated people. PMID:26425695

  12. Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23

    SciTech Connect

    Wang, F.; Gregory, C.D.; Rowe, M.; Rickinson, A.B.; Wang, D.; Birkenbach, M.; Kikutani, H.; Kishimoto, T.; Kieff, E.

    1987-05-01

    Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells includes some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation.

  13. The EBV Latent Antigen 3C Inhibits Apoptosis through Targeted Regulation of Interferon Regulatory Factors 4 and 8

    PubMed Central

    Banerjee, Shuvomoy; Lu, Jie; Cai, Qiliang; Saha, Abhik; Jha, Hem Chandra; Dzeng, Richard Kuo; Robertson, Erle S.

    2013-01-01

    Epstein-Barr virus (EBV) is linked to a broad spectrum of B-cell malignancies. EBV nuclear antigen 3C (EBNA3C) is an encoded latent antigen required for growth transformation of primary human B-lymphocytes. Interferon regulatory factor 4 (IRF4) and 8 (IRF8) are transcription factors of the IRF family that regulate diverse functions in B cell development. IRF4 is an oncoprotein with anti-apoptotic properties and IRF8 functions as a regulator of apoptosis and tumor suppressor in many hematopoietic malignancies. We now demonstrate that EBNA3C can contribute to B-cell transformation by modulating the molecular interplay between cellular IRF4 and IRF8. We show that EBNA3C physically interacts with IRF4 and IRF8 with its N-terminal domain in vitro and forms a molecular complex in cells. We identified the Spi-1/B motif of IRF4 as critical for EBNA3C interaction. We also demonstrated that EBNA3C can stabilize IRF4, which leads to downregulation of IRF8 by enhancing its proteasome-mediated degradation. Further, si-RNA mediated knock-down of endogenous IRF4 results in a substantial reduction in proliferation of EBV-transformed lymphoblastoid cell lines (LCLs), as well as augmentation of DNA damage-induced apoptosis. IRF4 knockdown also showed reduced expression of its targeted downstream signalling proteins which include CDK6, Cyclin B1 and c-Myc all critical for cell proliferation. These studies provide novel insights into the contribution of EBNA3C to EBV-mediated B-cell transformation through regulation of IRF4 and IRF8 and add another molecular link to the mechanisms by which EBV dysregulates cellular activities, increasing the potential for therapeutic intervention against EBV-associated cancers. PMID:23658517

  14. Formation of nanometer-size wires using infiltration into latent nuclear tracks

    DOEpatents

    Musket, Ronald G.; Felter, Thomas E.

    2002-01-01

    Nanometer-size wires having a cross-sectional dimension of less than 8 nm with controllable lengths and diameters are produced by infiltrating latent nuclear or ion tracks formed in trackable materials with atomic species. The trackable materials and atomic species are essentially insoluble in each other, thus the wires are formed by thermally driven, self-assembly of the atomic species during annealing, or re-crystallization, of the damage in the latent tracks. Unlike conventional ion track lithography, the inventive method does not require etching of the latent tracks.

  15. Prediction of nuclear proteins using nuclear translocation signals proposed by probabilistic latent semantic indexing

    PubMed Central

    2012-01-01

    Background Identification of subcellular localization in proteins is crucial to elucidate cellular processes and molecular functions in a cell. However, given a tremendous amount of sequence data generated in the post-genomic era, determining protein localization based on biological experiments can be expensive and time-consuming. Therefore, developing prediction systems to analyze uncharacterised proteins efficiently has played an important role in high-throughput protein analyses. In a eukaryotic cell, many essential biological processes take place in the nucleus. Nuclear proteins shuttle between nucleus and cytoplasm based on recognition of nuclear translocation signals, including nuclear localization signals (NLSs) and nuclear export signals (NESs). Currently, only a few approaches have been developed specifically to predict nuclear localization using sequence features, such as putative NLSs. However, it has been shown that prediction coverage based on the NLSs is very low. In addition, most existing approaches only attained prediction accuracy and Matthew's correlation coefficient (MCC) around 54%~70% and 0.250~0.380 on independent test set, respectively. Moreover, no predictor can generate sequence motifs to characterize features of potential NESs, in which biological properties are not well understood from existing experimental studies. Results In this study, first we propose PSLNuc (Protein Subcellular Localization prediction for Nucleus) for predicting nuclear localization in proteins. First, for feature representation, a protein is represented by gapped-dipeptides and the feature values are weighted by homology information from a smoothed position-specific scoring matrix. After that, we incorporate probabilistic latent semantic indexing (PLSI) for feature reduction. Finally, the reduced features are used as input for a support vector machine (SVM) classifier. In addition to PSLNuc, we further identify gapped-dipeptide signatures for putative NLSs and NESs

  16. Epstein–Barr virus latent antigens EBNA3C and EBNA1 modulate epithelial to mesenchymal transition of cancer cells associated with tumor metastasis

    PubMed Central

    Gaur, Nivedita; Gandhi, Jaya; Robertson, Erle S.; Verma, Subhash C.

    2016-01-01

    Epithelial–mesenchymal transition is an important mechanism in cancer invasiveness and metastasis. We had previously reported that cancer cells expressing Epstein–Barr virus (EBV) latent viral antigens EBV nuclear antigen EBNA3C and/ or EBNA1 showed higher motility and migration potential and had a propensity for increased metastases when tested in nude mice model. We now show that both EBNA3C and EBNA1 can modulate cellular pathways critical for epithelial to mesenchymal transition of cancer cells. Our data confirms that presence of EBNA3C or EBNA1 result in upregulation of transcriptional repressor Slug and Snail, up-regulation of intermediate filament of mesenchymal origin vimentin, upregulation of transcription factor TCF8/ZEB1, downregulation as well as disruption of tight junction zona occludens protein ZO-1, downregulation of cell adhesion molecule E-cadherin, and nuclear translocation of β-catenin. We further show that the primary tumors as well as metastasized lesions derived from EBV antigen-expressing cancer cells in nude mice model display EMT markers expression pattern suggesting their greater propensity to mesenchymal transition. PMID:25501510

  17. Epstein-Barr virus latent antigens EBNA3C and EBNA1 modulate epithelial to mesenchymal transition of cancer cells associated with tumor metastasis.

    PubMed

    Gaur, Nivedita; Gandhi, Jaya; Robertson, Erle S; Verma, Subhash C; Kaul, Rajeev

    2015-04-01

    Epithelial-mesenchymal transition is an important mechanism in cancer invasiveness and metastasis. We had previously reported that cancer cells expressing Epstein-Barr virus (EBV) latent viral antigens EBV nuclear antigen EBNA3C and/ or EBNA1 showed higher motility and migration potential and had a propensity for increased metastases when tested in nude mice model. We now show that both EBNA3C and EBNA1 can modulate cellular pathways critical for epithelial to mesenchymal transition of cancer cells. Our data confirms that presence of EBNA3C or EBNA1 result in upregulation of transcriptional repressor Slug and Snail, upregulation of intermediate filament of mesenchymal origin vimentin, upregulation of transcription factor TCF8/ZEB1, downregulation as well as disruption of tight junction zona occludens protein ZO-1, downregulation of cell adhesion molecule E-cadherin, and nuclear translocation of β-catenin. We further show that the primary tumors as well as metastasized lesions derived from EBV antigen-expressing cancer cells in nude mice model display EMT markers expression pattern suggesting their greater propensity to mesenchymal transition.

  18. Close but Distinct Regions of Human Herpesvirus 8 Latency-Associated Nuclear Antigen 1 Are Responsible for Nuclear Targeting and Binding to Human Mitotic Chromosomes

    PubMed Central

    Piolot, Tristan; Tramier, Marc; Coppey, Maité; Nicolas, Jean-Claude; Marechal, Vincent

    2001-01-01

    Human herpesvirus 8 is associated with all forms of Kaposi's sarcoma, AIDS-associated body cavity-based lymphomas, and some forms of multicentric Castleman's disease. Herpesvirus 8, like other gammaherpesviruses, can establish a latent infection in which viral genomes are stably maintained as multiple episomes. The latent nuclear antigen (LANA or LNAI) may play an essential role in the stable maintenance of latent episomes, notably by interacting concomitantly with the viral genomes and the metaphase chromosomes, thus ensuring an efficient transmission of the neoduplicated episomes to the daughter cells. To identify the regions responsible for its nuclear and subnuclear localization in interphase and mitotic cells, LNAI and various truncated forms were fused to a variant of green fluorescent protein. This enabled their localization and chromosome binding activity to be studied by low-light-level fluorescence microscopy in living HeLa cells. The results demonstrate that nuclear localization of LNAI is due to a unique signal, which maps between amino acids 24 and 30. Interestingly, this nuclear localization signal closely resembles those identified in EBNA1 from Epstein-Barr virus and herpesvirus papio. A region encompassing amino acids 5 to 22 was further proved to mediate the specific interaction of LNA1 with chromatin during interphase and the chromosomes during mitosis. The presence of putative phosphorylation sites in the chromosome binding sites of LNA1 and EBNA1 suggests that their activity may be regulated by specific cellular kinases. PMID:11264383

  19. Autoantibodies to nuclear envelope antigens in chronic fatigue syndrome.

    PubMed Central

    Konstantinov, K; von Mikecz, A; Buchwald, D; Jones, J; Gerace, L; Tan, E M

    1996-01-01

    We have identified and partially characterized the autoantibodies in sera of 60 patients with chronic fatigue syndrome. Approximately 52% of the sera were found to react with nuclear envelope antigens. The combination of nuclear rim staining observed in immunofluorescence microscopy and immunoblot analysis of highly purified nuclear envelope proteins provided initial characterization of these autoantibodies. Further characterization showed that some sera immunoprecipitated the in vitro transcription and translation product of a human cDNA clone encoding the nuclear envelope protein lamin B1. The autoantibodies were of the IgG isotype. The occurrence of autoantibodies to a conserved intracellular protein like lamin B1 provides new laboratory evidence for an autoimmune component in chronic fatigue syndrome. PMID:8878441

  20. Contributions of Epstein–Barr Nuclear Antigen 1 (EBNA1) to Cell Immortalization and Survival

    PubMed Central

    Frappier, Lori

    2012-01-01

    Epstein–Barr virus (EBV) immortalizes host cells as part of its latent mode of infection. As a result of this ability to promote cell proliferation and survival, EBV infection contributes to the development of several kinds of B-cell lymphomas and epithelial tumours. The EBV Epstein–Barr nuclear antigen 1 (EBNA1) protein is the only EBV protein expressed in all EBV-associated tumours and plays multiple important roles in EBV latency. In addition to its well-studied roles in viral DNA replication, segregation and transcriptional activation, several studies have identified roles of EBNA1 in manipulating cellular processes that result in reduced apoptosis and increased cell survival. This review discusses these cellular effects of EBNA1 and mechanisms by which they occur. PMID:23170171

  1. Contributions of Epstein-Barr nuclear antigen 1 (EBNA1) to cell immortalization and survival.

    PubMed

    Frappier, Lori

    2012-09-01

    Epstein-Barr virus (EBV) immortalizes host cells as part of its latent mode of infection. As a result of this ability to promote cell proliferation and survival, EBV infection contributes to the development of several kinds of B-cell lymphomas and epithelial tumours. The EBV Epstein-Barr nuclear antigen 1 (EBNA1) protein is the only EBV protein expressed in all EBV-associated tumours and plays multiple important roles in EBV latency. In addition to its well-studied roles in viral DNA replication, segregation and transcriptional activation, several studies have identified roles of EBNA1 in manipulating cellular processes that result in reduced apoptosis and increased cell survival. This review discusses these cellular effects of EBNA1 and mechanisms by which they occur.

  2. Anthelmintic Therapy Modifies the Systemic and Mycobacterial Antigen-Stimulated Cytokine Profile in Helminth-Latent Mycobacterium tuberculosis Coinfection.

    PubMed

    Anuradha, Rajamanickam; Munisankar, Saravanan; Bhootra, Yukthi; Dolla, Chandrakumar; Kumaran, Paul; Nutman, Thomas B; Babu, Subash

    2017-04-01

    Helminth infections are known to modulate cytokine responses in latent tuberculosis (LTB). However, very few studies have examined whether this modulation is reversible upon anthelmintic therapy. We measured the systemic and mycobacterial (TB) antigen-stimulated levels of type 1, type 2, type 17, and regulatory cytokines in individuals with LTB and with or without coexistent Strongyloides stercoralis infection before and after anthelmintic therapy. Our data reveal that individuals with LTB and coexistent S. stercoralis infection have significantly lower levels of systemic and TB antigen-stimulated type 1 (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]) and type 17 (IL-17A and/or IL-17F) cytokines and significantly higher levels of systemic but not TB antigen-stimulated type 2 (IL-4 and IL-5) and regulatory (transforming growth factor beta [TGF-β]) cytokines. Anthelmintic therapy resulted in significantly increased systemic levels of type 1 and/or type 17 cytokines and in significantly decreased systemic levels of type 2 and regulatory (IL-10 and TGF-β) cytokines. In addition, anthelmintic therapy resulted in significantly increased TB antigen-stimulated levels of type 1 cytokines only. Our data therefore confirm that the modulation of systemic and TB antigen-stimulated cytokine responses in S. stercoralis-LTB coinfection is reversible (for the most part) by anthelmintic treatment. Copyright © 2017 American Society for Microbiology.

  3. Potential cellular functions of Epstein-Barr Nuclear Antigen 1 (EBNA1) of Epstein-Barr Virus.

    PubMed

    Westhoff Smith, Danielle; Sugden, Bill

    2013-01-16

    Epstein-Barr Nuclear Antigen 1 (EBNA1) is a multifunctional protein encoded by EBV. EBNA1's role in maintaining EBV in latently proliferating cells, by mediating EBV genome synthesis and nonrandom partitioning to daughter cells, as well as regulating viral gene transcription, is well characterized. Less understood are the roles of EBNA1 in affecting the host cell to provide selective advantages to those cells that harbor EBV. In this review we will focus on the interactions between EBNA1 and the host cell that may provide EBV-infected cells selective advantages beyond the maintenance of EBV.

  4. Potential Cellular Functions of Epstein-Barr Nuclear Antigen 1 (EBNA1) of Epstein-Barr Virus

    PubMed Central

    Smith, Danielle Westhoff; Sugden, Bill

    2013-01-01

    Epstein-Barr Nuclear Antigen 1 (EBNA1) is a multifunctional protein encoded by EBV. EBNA1’s role in maintaining EBV in latently proliferating cells, by mediating EBV genome synthesis and nonrandom partitioning to daughter cells, as well as regulating viral gene transcription, is well characterized. Less understood are the roles of EBNA1 in affecting the host cell to provide selective advantages to those cells that harbor EBV. In this review we will focus on the interactions between EBNA1 and the host cell that may provide EBV-infected cells selective advantages beyond the maintenance of EBV. PMID:23325328

  5. Epstein-Barr virus latent membrane protein 2A exacerbates experimental autoimmune encephalomyelitis and enhances antigen presentation function

    PubMed Central

    Chang, Rhoda A.; Miller, Stephen D.; Longnecker, Richard

    2012-01-01

    Multiple sclerosis (MS) is an inflammatory, autoimmune disease of the central nervous system. The cause of MS is still unknown but epidemiological and immunological studies have implicated Epstein-Barr virus (EBV), which infects B cells, as a possible etiological agent involved in disease. Of particular interest is EBV latent membrane protein 2A (LMP2A) because previous studies have demonstrated that LMP2A enhances the expansion and differentiation of B cells upon antigen stimulation, revealing a potential contribution of this protein in autoimmunity. Since B cells are thought to contribute to MS, we examined the role of LMP2A in the animal model experimental autoimmune encephalomyelitis (EAE). In this model, transgenic mice in which B cells express LMP2A show increased severity and incidence of disease. This difference was not due to lymphocyte recruitment into the CNS or differences in T cell activation, rather, we show that LMP2A enhances antigen presentation function. PMID:22616025

  6. Nuclear binding of cell cycle-related proteins: cyclin A versus proliferating cell nuclear antigen (PCNA).

    PubMed

    Stivala, L A; Scovassi, A I; Bianchi, L; Prosperi, E

    1995-01-01

    We have investigated the cell cycle-dependent nuclear binding of cyclin A and of the proliferating cell nuclear antigen (PCNA) in asynchronously growing human fibroblasts. To this purpose, we have applied flow cytometry immunofluorescence, a powerful technique for elucidating the cell cycle phase during which the nuclear binding occurs. We have observed that, in striking contrast with the distribution of nuclear-bound PCNA which is restricted to S phase, the immunofluorescence signal of the nuclear-bound form of cyclin A is high in the G1 and G2 phases of the cell cycle. These results suggest the involvement of nuclear-bound cyclin A in the G1/S and G2/M phase transitions.

  7. Coexistent Malnutrition Is Associated with Perturbations in Systemic and Antigen-Specific Cytokine Responses in Latent Tuberculosis Infection.

    PubMed

    Anuradha, Rajamanickam; Munisankar, Saravanan; Bhootra, Yukthi; Kumar, Nathalla Pavan; Dolla, Chandrakumar; Kumaran, Paul; Babu, Subash

    2016-04-01

    Malnutrition, as defined by low body mass index (BMI), is a major risk factor for the development of active tuberculosis (TB), although the biological basis underlying this susceptibility remains poorly characterized. To verify whether malnutrition affects the systemic and antigen-specific cytokine levels in individuals with latent TB (LTB), we examined circulating and TB antigen-stimulated levels of cytokines in individuals with LTB and low BMI (LBMI) and compared them with those in individuals with LTB and normal BMI (NBMI). Coexistent LBMI with LTB was characterized by diminished circulating levels of type 1 (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), type 2 (interleukin-4 [IL-4]), type 17 (IL-22), and other proinflammatory (IL-1α, IL-1β, and IL-6) cytokines but elevated levels of other type 2 (IL-5 and IL-13) and regulatory (IL-10 and transforming growth factor beta [TGF-β]) cytokines. In addition, LBMI with LTB was associated with diminished TB antigen-induced IFN-γ, TNF-α, IL-6, IL-1α, and IL-1β levels. Finally, there was a significant positive correlation between BMI values and TNF-α and IL-1β levels and a significant negative correlation between BMI values and IL-2, IL-10, and TGF-β levels in individuals with LTB. Therefore, our data reveal that latent TB with a coexistent low BMI is characterized by diminished protective cytokine responses and heightened regulatory cytokine responses, providing a potential biological mechanism for the increased risk of developing active TB.

  8. Induction of the autoantigen proliferating cell nuclear antigen in T lymphocytes by a mycobacterial antigen.

    PubMed Central

    Haftel, H M; Chang, Y; Hinderer, R; Hanash, S M; Holoshitz, J

    1994-01-01

    Mycobacteria have been implicated in the pathogenesis of autoimmunity. To determine the potential effect of mycobacterial antigens on peripheral blood mononuclear cells (PBMC), we analyzed PBMC incubated with the acetone-precipitable fraction of Mycobacterium tuberculosis (APMT) for changes in cellular protein expression. Two-dimensional gel analysis showed induction of a 36-kD polypeptide identified as proliferating cell nuclear antigen (PCNA), a known autoantigen, after incubation with AP-MT. PCNA plays a role in cell proliferation and is expressed as a late growth regulated factor. However, its synthesis in response to AP-MT was induced as an early event. The early induction of PCNA was regulated at a posttranscriptional level and was restricted to T cells. Treatment of PBMC with known T cell mitogens, namely PHA, anti-CD3 antibodies, and staphylococcal superantigens failed to induce an early PCNA increase. The distinct characteristics of the AP-MT effect on PCNA expression suggest a separate mechanism of induction in response to AP-MT, compared with the late increase observed in response to mitogens. The induction of PCNA in response to mycobacterial antigens may represent a pathogenically relevant mechanism in autoimmunity. Images PMID:7929811

  9. Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins

    NASA Technical Reports Server (NTRS)

    Schatten, G.; Schatten, H.; Simerly, C.; Maul, G. G.; Chaly, N.

    1985-01-01

    Nuclear structural changes during fertilization and embryogenesis in mice and sea urchins are traced using four antibodies. The oocytes from virgin female mice, morulae and blastocytes from mated females, and gametes from the sea urchin Lytechnius variegatis are studied using mouse monoclonal antibodies to nuclear lamin A/C, monoclonal antibody to P1, human autoimmune antibodies to lamin A/C, and to lamin B. The mouse fertilization data reveal no lamins on the oocyte; however, lamins are present on the pronuclei, and chromosomes are found on the oocytes and pronuclei. It is detected that on the sea urchin sperm the lamins are reduced to acrosomal and centriolar fossae and peripheral antigens are around the sperm nucleus. The mouse sperm bind lamin antibodies regionally and do not contain antigens. Lamins and antigens are observed on both pronuclei and chromosomes during sea urchin fertilization. Mouse embryogenesis reveals that lamin A/C is not recognized at morula and blastocyst stages; however, lamin B stains are retained. In sea urchin embryogenesis lamin recognition is lost at the blastrula, gastrula, and plutei stages. It is noted that nuclear lamins lost during spermatogenesis are restored at fertilization and peripheral antigens are associated with the surface of chromosomes during meiosis and mitosis and with the periphery of the pronuclei and nuclei during interphase.

  10. Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins

    NASA Technical Reports Server (NTRS)

    Schatten, G.; Schatten, H.; Simerly, C.; Maul, G. G.; Chaly, N.

    1985-01-01

    Nuclear structural changes during fertilization and embryogenesis in mice and sea urchins are traced using four antibodies. The oocytes from virgin female mice, morulae and blastocytes from mated females, and gametes from the sea urchin Lytechnius variegatis are studied using mouse monoclonal antibodies to nuclear lamin A/C, monoclonal antibody to P1, human autoimmune antibodies to lamin A/C, and to lamin B. The mouse fertilization data reveal no lamins on the oocyte; however, lamins are present on the pronuclei, and chromosomes are found on the oocytes and pronuclei. It is detected that on the sea urchin sperm the lamins are reduced to acrosomal and centriolar fossae and peripheral antigens are around the sperm nucleus. The mouse sperm bind lamin antibodies regionally and do not contain antigens. Lamins and antigens are observed on both pronuclei and chromosomes during sea urchin fertilization. Mouse embryogenesis reveals that lamin A/C is not recognized at morula and blastocyst stages; however, lamin B stains are retained. In sea urchin embryogenesis lamin recognition is lost at the blastrula, gastrula, and plutei stages. It is noted that nuclear lamins lost during spermatogenesis are restored at fertilization and peripheral antigens are associated with the surface of chromosomes during meiosis and mitosis and with the periphery of the pronuclei and nuclei during interphase.

  11. Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins

    SciTech Connect

    Schatten, G.; Schatten, H.; Simerly, C.; Maul, G.G.; Chaly, N.

    1985-07-01

    Nuclear structural changes during fertilization and embryogenesis in mice and sea urchins are traced using four antibodies. The oocytes from virgin female mice, morulae and blastocytes from mated females, and gametes from the sea urchin Lytechnius variegatis are studied using mouse monoclonal antibodies to nuclear lamin A/C, monoclonal antibody to P1, human autoimmune antibodies to lamin A/C, and to lamin B. The mouse fertilization data reveal no lamins on the oocyte; however, lamins are present on the pronuclei, and chromosomes are found on the oocytes and pronuclei. It is detected that on the sea urchin sperm the lamins are reduced to acrosomal and centriolar fossae and peripheral antigens are around the sperm nucleus. The mouse sperm bind lamin antibodies regionally and do not contain antigens. Lamins and antigens are observed on both pronuclei and chromosomes during sea urchin fertilization. Mouse embryogenesis reveals that lamin A/C is not recognized at morula and blastocyst stages; however, lamin B stains are retained. In sea urchin embryogenesis lamin recognition is lost at the blastrula, gastrula, and plutei stages. It is noted that nuclear lamins lost during spermatogenesis are restored at fertilization and peripheral antigens are associated with the surface of chromosomes during meiosis and mitosis and with the periphery of the pronuclei and nuclei during interphase. 32 references.

  12. Effect of isoniazid on antigen-specific interferon-γ secretion in latent tuberculosis

    PubMed Central

    Torres, Martha; Cruz-Hervert, Pablo; Guio, Heinner; Carranza, Claudia; Ferreyra-Reyes, Leticia; Canizales, Sergio; Molina, Susana; Ferreira-Guerrero, Elizabeth; Téllez, Norma; Montero-Campos, Rogelio; Delgado-Sánchez, Guadalupe; Mongua-Rodriguez, Norma; Sifuentes-Osornio, Jose; Ponce-de Leon, Alfredo; Sada, Eduardo; Young, Douglas B.; Wilkinson, Robert J.

    2015-01-01

    Treatment of persons with latent tuberculosis (TB) infection at greatest risk of reactivation is an important component of TB control and elimination strategies. Biomarkers evaluating the effectiveness of treatment of latent TB infection have not yet been identified. This information would enhance control efforts and assist the evaluation of new treatment regimes. We designed a two-group, two-arm, randomised clinical study of tuberculin skin test-positive participants: 26 with documented contact with TB patients and 34 with non-documented contact. Participants in each group were randomly assigned to the immediate- or deferred-isoniazid treatment arms. Assays of in vitro interferon (IFN)-γ secretion in response to recombinant Rv1737 and overlapping synthetic peptide pools from various groups of immunodominant proteins were performed. During isoniazid therapy, a significant increase from baseline in the proportion of IFN-γ responders to the 10-kDa culture filtrate protein, Rv2031, Rv0849, Rv1986, Rv2659c, Rv2693c and the recombinant Rv1737 protein was observed (p⩽0.05). The peptide pool of Rv0849 and Rv1737 recombinant proteins induced the highest percentage of IFN-γ responders after isoniazid therapy. The in vitro IFN-γ responses to these proteins might represent useful markers to evaluate changes associated with treatment of latent TB infection. PMID:25359354

  13. Nuclear Sm antigens in the sperm of different organisms.

    PubMed

    Delgado, F; Brito, M; Concha, I I; Schroeder, R; Burzio, L O

    1994-08-01

    Immunoblot analysis of sperm protein from several species revealed the presence of polypeptides recognised by anti-Sm sera obtained from patients with systemic lupus erythematosus. Immunoreactive polypeptides in human, bull, mouse and rat sperm were identified as protein B', B and D as compared with the Sm polypeptides of HeLa cells. In the sperm of rooster, the teleost fish Cyprinus carpio and the mussel Choromytilus chorus, the immunoreactive polypeptide profile was more complex. To ascertain the sperm origin of the Sm antigens, immunolocalisation with anti-Sm serum was carried out. The results demonstrated that in all the species studied staining was confined to the sperm nucleus, confirming that some polypeptides of the small nuclear ribonucleoprotein complex are present in the gamete.

  14. Proliferating cell nuclear antigen/cyclin in cultured human keratinocytes.

    PubMed

    Okada, N; Miyagawa, S; Steinberg, M L; Yoshikawa, K

    1990-09-01

    Expression of proliferating cell nuclear antigen (PCNA)/cyclin in cultured human keratinocytes was studied using an antibody from an SLE patient as the reagent. By indirect immunofluorescence staining, SV40-transformed human keratinocytes expressed PCNA/cyclin in 40-45% of the cells as a nulcear granular fluorescence. After synchronization of these cells, their nuclear distribution pattern during the S phase was sequential and showed a clear correlation with DNA synthesis. Primary cultured keratinocytes grown in high Ca+ medium expressed PCNA/cyclin in 10-15% of the cells with a similar staining pattern. These positively stained cells were confined to the basal and immediate suprabasal layers of the stratified culture sheet. The keratinocytes disaggregated by trypsin were separated according to cell size through a screen of Nitex monofilament cloth. The cells smaller than 15 microns in diameter synthesized abundant PCNA/cyclin, while the larger cells expressed very low levels. These results indicate that the expression of PCNA/cyclin correlates with DNA synthesis in cultured keratinocytes, but is not associated with their differentiation process.

  15. Antibodies against lytic and latent Kaposi's sarcoma-associated herpes virus antigens and lymphoma in the European EpiLymph case–control study

    PubMed Central

    Benavente, Y; Mbisa, G; Labo, N; Casabonne, D; Becker, N; Maynadie, M; Foretova, L; Cocco, P L; Nieters, A; Staines, A; Bofetta, P; Brennan, P; Whitby, D; de Sanjosé, S

    2011-01-01

    Background: Kaposi's sarcoma-associated herpes virus is associated with primary effusion lymphoma and multicentric Castleman's disease. Methods: Seropositivity to lytic and latent Kaposi's sarcoma herpes virus (KSHV) antigens were examined in 2083 lymphomas and 2013 controls from six European countries. Results: Antibodies against KSHV latent and lytic antigens were detectable in 4.5% and 3.4% of controls, respectively, and 3.6% of cases (P>0.05). The KSHV seropositivity was associated with splenic marginal zone lymphoma (SMZL) (odds ratio (OR)=4.11, 95% confidence interval (CI)=1.57–10.83) and multiple myeloma (OR=0.31, 95% CI=0.11–0.85). Conclusion: The KSHV is unlikely to contribute importantly to lymphomagenesis among immunocompetent subjects. However, the observed association with SMZL may underline a chronic antigen mechanism in its aetiology. PMID:21952625

  16. HLA-DR antigens in systemic lupus erythematosus: association with specificity of autoantibody responses to nuclear antigens.

    PubMed Central

    Smolen, J S; Klippel, J H; Penner, E; Reichlin, M; Steinberg, A D; Chused, T M; Scherak, O; Graninger, W; Hartter, E; Zielinski, C C

    1987-01-01

    HLA-DR antigens and autoantibodies to the nuclear or cytoplasmic antigens Ro/SSA, La/SSB, Sm, and RNP were determined in North American and Austrian patients with systemic lupus erythematosus (SLE). Analysis of the association of antibodies to these ribonucleic acid (RNA)-protein antigens with HLA-DR antigens showed that HLA-DR3 was related to the presence of anti-Ro/SSA or anti-La/SSB, or both. In contrast, anti-Sm or anti-RNP, or both were associated with HLA-DR4. HLA-DR5 was associated with absence of these autoantibodies. The data extend evidence for the complexity and heterogeneity of SLE. Moreover, they indicate that, in SLE, genes linked to those coding for HLA-DR antigens, are related to the specificity of autoantibody responses rather than to the primary immunological abnormalities of this disorder. PMID:3498447

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

    SciTech Connect

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih; Zhao, Bo; Kieff, Elliott; Peng, Chih-Wen

    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.

  18. Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

    SciTech Connect

    Rahayu, Retno; Ohsaki, Eriko; Omori, Hiroko; Ueda, Keiji

    2016-09-15

    In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres, and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. - Highlights: • This is the first report showing LANA dots on mitotic chromosomes by fluorescent microscopy followed by electron microscopy. • LANA dots localized randomly on condensed chromosomes other than centromere/pericentromere and telomere/peritelomre. • Cellular mitotic checkpoint should not be always involved in the segregation of KSHV genomes in the latency.

  19. Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells

    PubMed Central

    Niimi, Atsuko; Brown, Stephanie; Sabbioneda, Simone; Kannouche, Patricia L.; Scott, Andrew; Yasui, Akira; Green, Catherine M.; Lehmann, Alan R.

    2008-01-01

    After exposure to DNA-damaging agents that block the progress of the replication fork, monoubiquitination of proliferating cell nuclear antigen (PCNA) mediates the switch from replicative to translesion synthesis DNA polymerases. We show that in human cells, PCNA is monoubiquitinated in response to methyl methanesulfonate and mitomycin C, as well as UV light, albeit with different kinetics, but not in response to bleomycin or camptothecin. Cyclobutane pyrimidine dimers are responsible for most of the PCNA ubiquitination events after UV-irradiation. Failure to ubiquitinate PCNA results in substantial sensitivity to UV and methyl methanesulfonate, but not to camptothecin or bleomycin. PCNA ubiquitination depends on Replication Protein A (RPA), but is independent of ATR-mediated checkpoint activation. After UV-irradiation, there is a temporal correlation between the disappearance of the deubiquitinating enzyme USP1 and the presence of PCNA ubiquitination, but this correlation was not found after chemical mutagen treatment. By using cells expressing photolyases, we are able to remove the UV lesions, and we show that PCNA ubiquitination persists for many hours after the damage has been removed. We present a model of translesion synthesis behind the replication fork to explain the persistence of ubiquitinated PCNA. PMID:18845679

  20. Diminished Systemic and Antigen-Specific Type 1, Type 17, and Other Proinflammatory Cytokines in Diabetic and Prediabetic Individuals With Latent Mycobacterium tuberculosis Infection

    PubMed Central

    Kumar, Nathella Pavan; George, Parakkal Jovvian; Kumaran, Paul; Dolla, Chandra Kumar; Nutman, Thomas B.; Babu, Subash

    2014-01-01

    Background. Diabetes mellitus type 2 (DM) is known to be a major risk factor for the development of active tuberculosis, although its influence on latent Mycobacterium tuberculosis infection (hereafter, “latent infection”) remains poorly characterized. Methods. We examined circulating plasma cytokine levels in individuals with latent infection with DM or pre-DM (ie, intermediate hyperglycemia) and compared them to levels in patients with latent infection and normal glycemic control. Results. In persons with DM or pre-DM, latent infection is characterized by diminished circulating levels of type 1 (interferon γ, interleukin 2, and tumor necrosis factor α) and type 17 (interleukin 17F) cytokines. This was associated with decreased systemic levels of other proinflammatory cytokines (interleukin 1β and interleukin 18) and the antiinflammatory cytokine interleukin 10 but not with decreased systemic levels of type 2 cytokines. Moreover, latently infected individuals with DM had diminished levels of spontaneous and M. tuberculosis antigen–specific levels of type 1 and type 17 cytokines when antigen-stimulated whole blood was examined. Finally, there was no significant correlation between the levels of any of the cytokines measured (with the exception of interleukin 22) with hemoglobin A1c levels. Conclusions. Our data reveal that latent infection in the presence of DM or pre-DM, is characterized by diminished production of cytokines, implicated in the control of M. tuberculosis activation, allowing for a potential immunological mechanism that could account for the increased risk of active tuberculosis in latently infected individuals with DM. PMID:24907382

  1. The Functional Response of B Cells to Antigenic Stimulation: A Preliminary Report of Latent Tuberculosis

    PubMed Central

    du Plessis, Willem J.; Kleynhans, Léanie; du Plessis, Nelita; Stanley, Kim; Malherbe, Stephanus T.; Maasdorp, Elizna; Ronacher, Katharina; Chegou, Novel N.; Walzl, Gerhard; Loxton, Andre G.

    2016-01-01

    Mycobacterium tuberculosis (M.tb) remains a successful pathogen, causing tuberculosis disease numbers to constantly increase. Although great progress has been made in delineating the disease, the host-pathogen interaction is incompletely described. B cells have shown to function as both effectors and regulators of immunity via non-humoral methods in both innate and adaptive immune settings. Here we assessed specific B cell functional interaction following stimulation with a broad range of antigens within the LTBI milieu. Our results indicate that B cells readily produce pro- and anti-inflammatory cytokines (including IL-1β, IL-10, IL-17, IL-21 and TNF-α) in response to stimulation. TLR4 and TLR9 based stimulations achieved the greatest secreted cytokine-production response and BCG stimulation displayed a clear preference for inducing IL-1β production. We also show that the cytokines produced by B cells are implicated strongly in cell-mediated communication and that plasma (memory) B cells (CD19+CD27+CD138+) is the subset with the greatest contribution to cytokine production. Collectively our data provides insight into B cell responses, where they are implicated in and quantifies responses from specific B cell phenotypes. These findings warrant further functional B cell research with a focus on specific B cell phenotypes under conditions of active TB disease to further our knowledge about the contribution of various cell subsets which could have implications for future vaccine development or refined B cell orientated treatment in the health setting. PMID:27050308

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

    SciTech Connect

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

    2010-03-15

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

  3. Epstein-Barr virus latent genes.

    PubMed

    Kang, Myung-Soo; Kieff, Elliott

    2015-01-23

    Latent Epstein-Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized.

  4. Surface swarming motility by Pectobacterium atrosepticum is a latent phenotype that requires O antigen and is regulated by quorum sensing.

    PubMed

    Bowden, Steven D; Hale, Nicola; Chung, Jade C S; Hodgkinson, James T; Spring, David R; Welch, Martin

    2013-11-01

    We describe a previously cryptic phenotype associated with the opportunistic phytopathogen Pectobacterium atrosepticum (Pca): surface swarming. We found that when Pca was spotted onto plates containing <0.5% (w/v) agar, the culture produced copious amounts of extracellular matrix material containing highly motile cells. Once produced, this 'slime layer' spread rapidly across the plate either as an advancing front or as tendrils. Transposon mutagenesis was used to identify mutants that were affected in swarming. Hypo-swarmer mutants mostly carried insertions in a horizontally acquired island (HAI5), which encodes a cluster of genes involved in O antigen biosynthesis. Hyper-swarmer mutants mostly carried insertions in hexY, a known antagonist of the class I flagellar master regulator, FlhD4C2. In addition, we found that the nucleoid protein, histone-like nuclear structuring protein 2 (H-NS2), also regulated swarming behaviour. A mutant in which hns2 was overexpressed displayed a hyper-swarming phenotype, whereas a mutant in which the hns2 ORF was inactivated had a hypo-swarming phenotype. Swarming was also regulated by quorum sensing (QS) and by the carbon source being utilized. We show, using a range of epistasis experiments, that optimal swarming requires both motility and O antigen biosynthesis, and that H-NS2 and QS both promote swarming through their effects on motility.

  5. CD4+ and CD8+ T-Cell Responses to Latent Antigen EBNA-1 and Lytic Antigen BZLF-1 during Persistent Lymphocryptovirus Infection of Rhesus Macaques

    PubMed Central

    Leskowitz, R. M.; Zhou, X. Y.; Villinger, F.; Fogg, M. H.; Kaur, A.; Lieberman, P. M.; Wang, F.

    2013-01-01

    Epstein-Barr virus (EBV) infection leads to lifelong viral persistence through its latency in B cells. EBV-specific T cells control reactivations and prevent the development of EBV-associated malignancies in most healthy carriers, but infection can sometimes cause chronic disease and malignant transformation. Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies and is a promising target for a therapeutic vaccine. Here, we studied the EBNA-1-specific immune response using the EBV-homologous rhesus lymphocryptovirus (rhLCV) infection in rhesus macaques. We assessed the frequency, phenotype, and cytokine production profiles of rhLCV EBNA-1 (rhEBNA-1)-specific T cells in 15 rhesus macaques and compared them to the lytic antigen of rhLCV BZLF-1 (rhBZLF-1). We were able to detect rhEBNA-1-specific CD4+ and/or CD8+ T cells in 14 of the 15 animals screened. In comparison, all 15 animals had detectable rhBZLF-1 responses. Most peptide-specific CD4+ T cells exhibited a resting phenotype of central memory (TCM), while peptide-specific CD8+ T cells showed a more activated phenotype, belonging mainly to the effector cell subset. By comparing our results to the human EBV immune response, we demonstrate that the rhLCV model is a valid system for studying chronic EBV infection and for the preclinical development of therapeutic vaccines. PMID:23698300

  6. Epstein-Barr Virus Nuclear Antigen 3C Interacts with and Enhances the Stability of the c-Myc Oncoprotein▿

    PubMed Central

    Bajaj, Bharat G.; Murakami, Masanao; Cai, Qiliang; Verma, Subhash C.; Lan, Ke; Robertson, Erle S.

    2008-01-01

    Epstein-Barr virus (EBV) was the first human DNA virus to be associated with cancer. Its oncogenic potential was further demonstrated by its ability to transform primary B lymphocytes in vitro. EBV nuclear antigen 3C (EBNA3C) is one of a small subset of latent antigens critical for the transformation of human primary B lymphocytes. Although EBNA3C has been shown to modulate several cellular functions, additional targets involved in cellular transformation remain to be explored. EBNA3C can recruit key components of the SCFSkp2 ubiquitin ligase complex. In this report, we show that EBNA3C residues 130 to 190, previously shown to bind to the SCFSkp2 complex, also can strongly associate with the c-Myc oncoprotein. Additionally, the interaction of EBNA3C with c-Myc was mapped to the region of c-Myc that includes the highly conserved Skp2 binding domain. Skp2 has been shown to regulate c-Myc stability and also has been shown to function as a coactivator of transcription for c-Myc target genes. We now show that the EBV latent oncoprotein EBNA3C can stabilize c-Myc and that the recruitment of both c-Myc and its cofactor Skp2 to c-Myc-dependent promoters can enhance c-Myc-dependent transcription. This same region of EBNA3C also recruits and modulates the activity of retinoblastoma and p27, both major regulators of the mammalian cell cycle. The inclusion of c-Myc in the group of cellular targets modulated by this domain further accentuates the importance of these critical residues of EBNA3C in bypassing the cell cycle checkpoints. PMID:18256156

  7. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    SciTech Connect

    Kim, Sun Young; Song, Kyung-A; Kieff, Elliott; Kang, Myung-Soo

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. Black-Right-Pointing-Pointer A small molecule and a peptide as EBNA1 dimerization inhibitors identified. Black-Right-Pointing-Pointer Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. Black-Right-Pointing-Pointer Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-J{kappa} binding to the J{kappa} site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with

  8. Expression of B7 (CD80) and CD40 antigens and the CD40 ligand in Hodgkin's disease is independent of latent Epstein—Barr virus infection

    PubMed Central

    Murray, P G; Oates, J; Reynolds, G M; Crocker, J; Young, L S

    1995-01-01

    Aim—To examine the expression of CD40 and B7 (CD80) antigens and the CD40 ligand in Hodgkin's disease. Methods—Antigen and ligand expression was studied in 17 cases of Hodgkin's disease using immunohistochemistry. The study included 11 cases of Hodgkin's disease in which latent Epstein-Barr virus (EBV) infection could be demonstrated within tumour cells by in situ hybridisation for the EBV encoded early RNAs (EBERs). Results—In all cases, irrespective of EBV status, Reed-Sternberg cells and their variants (HRS cells) showed strong expression of both B7 and CD40 antigens. CD40 ligand expression was not shown in HRS cells but was confined to a subset of small lymphocytes some of which were seen to be in intimate contact with HRS cells. Paraffin wax sections from a further 60 cases of Hodgkin's disease were examined for CD40 and EBER expression alone. The CD40 antigen was identified in HRS cells in all of these cases irrespective of EBER expression. Conclusions—As CD40 and B7 expression are features of professional antigen presenting cells, these results provide further evidence that HRS cells may have antigen presenting properties and that this may contribute to the characteristic recruitment and activation of non-malignant lymphocytes which is a feature of Hodgkin's disease. The ability of HRS cells to activate Th cells may in turn contribute to their own survival through the induction of the gp39/CD40 pathway. Images PMID:16695980

  9. Antigenic variation in African trypanosomes: the importance of chromosomal and nuclear context in VSG expression control.

    PubMed

    Glover, Lucy; Hutchinson, Sebastian; Alsford, Sam; McCulloch, Richard; Field, Mark C; Horn, David

    2013-12-01

    African trypanosomes are lethal human and animal parasites that use antigenic variation for evasion of host adaptive immunity. To facilitate antigenic variation, trypanosomes dedicate approximately one third of their nuclear genome, including many minichromosomes, and possibly all sub-telomeres, to variant surface glycoprotein (VSG) genes and associated sequences. Antigenic variation requires transcription of a single VSG by RNA polymerase I (Pol-I), with silencing of other VSGs, and periodic switching of the expressed gene, typically via DNA recombination with duplicative translocation of a new VSG to the active site. Thus, telomeric location, epigenetic controls and monoallelic transcription by Pol-I at an extranucleolar site are prominent features of VSGs and their expression, with telomeres, chromatin structure and nuclear organization all making vitally important contributions to monoallelic VSG expression control and switching. We discuss VSG transcription, recombination and replication control within this chromosomal and sub-nuclear context.

  10. Salivary production of IgA and IgG to human herpes virus 8 latent and lytic antigens by patients in whom Kaposi's sarcoma has regressed.

    PubMed

    Mbopi-Keou, Francois-Xavier; Legoff, Jerome; Piketty, Christophe; Hocini, Hakim; Malkin, Jean-Elie; Inoue, Naoki; Scully, Crispian M; Porter, Stephen R; Teo, Chong-Gee; Belec, Laurent

    2004-01-23

    IgG and IgA antibodies with specificities to a latent and a lytic antigen of human herpes virus 8 (HHV-8) were detectable in the saliva and serum of eight patients whose Kaposi's sarcoma had regressed, seven of whom were HIV-1 infected. The measurement of antibody-specific activity and secretion rate, and the detection of secretory IgA all indicate anti-HHV-8 antibody activity in saliva. The specific humoral responses possibly influence mucosal replication of HHV-8, and in turn, that of HIV.

  11. Antibodies specific for Epstein-Barr virus nuclear antigen-1 cross-react with human heterogeneous nuclear ribonucleoprotein L

    PubMed Central

    Lindsey, J. William; deGannes, Samantha L.; Pate, Kimberly A.; Zhao, Xiurong

    2015-01-01

    Epstein-Barr virus (EBV) is associated with multiple sclerosis (MS), and antibodies to the EBV nuclear antigen-1 (EBNA-1) are consistently increased in MS patients. The hypothesis of this study is that anti-EBNA-1 antibodies cross-react with a self antigen in MS patients. We affinity purified anti-EBNA-1 antibodies from human plasma, used the anti-EBNA-1 to immunoprecipitate antigens from human brain, and identified bound antigens with mass spectrometry. Anti-EBNA-1 consistently bound heterogeneous nuclear ribonucleoprotein L (HNRNPL). We expressed both the long and short isoforms of this protein, and verified with Western blots and ELISA that the long isoform cross-reacts with EBNA-1. Immunohistochemistry demonstrated that anti-EBNA-1 bound to an antigen in the nucleus of cultured rat central nervous system cells. ELISA demonstrated the presence of antibodies to HNRNPL in the plasma of both healthy controls and MS patients, but anti-HNRNPL was not increased in MS patients. We conclude that HNRNPL is an autoantigen which cross-reacts with EBNA-1. The relevance of this autoantigen to MS and other autoimmune diseases remains to be investigated. PMID:26637929

  12. Antibodies specific for Epstein-Barr virus nuclear antigen-1 cross-react with human heterogeneous nuclear ribonucleoprotein L.

    PubMed

    Lindsey, J William; deGannes, Samantha L; Pate, Kimberly A; Zhao, Xiurong

    2016-01-01

    Epstein-Barr virus (EBV) is associated with multiple sclerosis (MS), and antibodies to the EBV nuclear antigen-1 (EBNA-1) are consistently increased in MS patients. The hypothesis of this study is that anti-EBNA-1 antibodies cross-react with a self antigen in MS patients. We affinity purified anti-EBNA-1 antibodies from human plasma, used the anti-EBNA-1 to immunoprecipitate antigens from human brain, and identified bound antigens with mass spectrometry. Anti-EBNA-1 consistently bound heterogeneous nuclear ribonucleoprotein L (HNRNPL). We expressed both the long and short isoforms of this protein, and verified with Western blots and ELISA that the long isoform cross-reacts with EBNA-1. Immunohistochemistry demonstrated that anti-EBNA-1 bound to an antigen in the nucleus of cultured rat central nervous system cells. ELISA demonstrated the presence of antibodies to HNRNPL in the plasma of both healthy controls and MS patients, but anti-HNRNPL was not increased in MS patients. We conclude that HNRNPL is an autoantigen which cross-reacts with EBNA-1. The relevance of this autoantigen to MS and other autoimmune diseases remains to be investigated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Latency-Associated Nuclear Antigen E3 Ubiquitin Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation.

    PubMed

    Cerqueira, Sofia A; Tan, Min; Li, Shijun; Juillard, Franceline; McVey, Colin E; Kaye, Kenneth M; Simas, J Pedro

    2016-09-01

    Viruses have evolved mechanisms to hijack components of cellular E3 ubiquitin ligases, thus modulating the ubiquitination pathway. However, the biological relevance of such mechanisms for viral pathogenesis in vivo remains largely unknown. Here, we utilized murid herpesvirus 4 (MuHV-4) infection of mice as a model system to address the role of MuHV-4 latency-associated nuclear antigen (mLANA) E3 ligase activity in gammaherpesvirus latent infection. We show that specific mutations in the mLANA SOCS box (V199A, V199A/L202A, or P203A/P206A) disrupted mLANA's ability to recruit Elongin C and Cullin 5, thereby impairing the formation of the Elongin BC/Cullin 5/SOCS (EC5S(mLANA)) complex and mLANA's E3 ligase activity on host NF-κB and Myc. Although these mutations resulted in considerably reduced mLANA binding to viral terminal repeat DNA as assessed by electrophoretic mobility shift assay (EMSA), the mutations did not disrupt mLANA's ability to mediate episome persistence. In vivo, MuHV-4 recombinant viruses bearing these mLANA SOCS box mutations exhibited a deficit in latency amplification in germinal center (GC) B cells. These findings demonstrate that the E3 ligase activity of mLANA contributes to gammaherpesvirus-driven GC B cell proliferation. Hence, pharmacological inhibition of viral E3 ligase activity through targeting SOCS box motifs is a putative strategy to control gammaherpesvirus-driven lymphoproliferation and associated disease. The gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause lifelong persistent infection and play causative roles in several human malignancies. Colonization of B cells is crucial for virus persistence, and access to the B cell compartment is gained by virus-driven proliferation in germinal center (GC) B cells. Infection of B cells is predominantly latent, with the viral genome persisting as a multicopy episome and expressing only a small subset of viral genes. Here, we focused on

  14. Analysis of speckled fluorescent antinuclear antibody test antisera using electrofocused nuclear antigens.

    PubMed

    Okarma, T B; Krueger, J A; Holman, H R

    1982-08-01

    Antibodies to different components of the extractable nuclear antigen (ENA) have been thought to be serological markers for clinical subsets of rheumatic diseases. However, incomplete characterization and standardization of antigenic components such as ribonucleoprotein (RNP), Sm, and SS-B (Ha), and the multiplicity of autoantibodies produced by different patients have confounded correlations between autoantibody specificity and disease subsets. This study describes the preparative separation of the antigens Sm, RNP, and Ss-B (Ha) by electrofocusing and their use in a rocket electrophoretic assay that in one step identifies and quantifies the multiple reactivities of patient sera exhibiting the speckled FANA pattern. Preparative electrofocusing generates milligram quantities of these antigens with retention of their immunologic and biochemical characteristics, facilitating further study of their biological properties and relationships to disease subsets.

  15. Epigenotypes of latent herpesvirus genomes.

    PubMed

    Minarovits, J

    2006-01-01

    Epigenotypes are modified cellular or viral genotypes which differ in transcriptional activity in spite of having an identical (or nearly identical) DNA sequence. Restricted expression of latent, episomal herpesvirus genomes is also due to epigenetic modifications. There is no virus production (lytic viral replication, associated with the expression of all viral genes) in tight latency. In vitro experiments demonstrated that DNA methylation could influence the activity of latent (and/or crucial lytic) promoters of prototype strains belonging to the three herpesvirus subfamilies (alpha-, beta-, and gamma-herpesviruses). In vivo, however, DNA methylation is not a major regulator of herpes simplex virus type 1 (HSV-1, a human alpha-herpesvirus) latent gene expression in neurons of infected mice. In these cells, the promoter/enhancer region of latency-associated transcripts (LATs) is enriched with acetyl histone H3, suggesting that histone modifications may control HSV-1 latency in terminally differentiated, quiescent neurons. Epstein-Barr virus (EBV, a human gamma-herpesvirus) is associated with a series of neoplasms. Latent, episomal EBV genomes are subject to host cell-dependent epigenetic modifications (DNA methylation, binding of proteins and protein complexes, histone modifications). The distinct viral epigenotypes are associated with distinct EBV latency types, i.e., cell type-specific usage of latent EBV promoters controlling the expression of latent, growth transformation-associated EBV genes. The contribution of major epigenetic mechanisms to the regulation of latent EBV promoters is variable. DNA methylation contributes to silencing of Wp and Cp (alternative promoters for transcripts coding for the nuclear antigens EBNA 1-6) and LMP1p, LMP2Ap, and LMP2Bp (promoters for transcripts encoding transmembrane proteins). DNA methylation does not control, however, Qp (a promoter for EBNA1 transcripts only) in lymphoblastoid cell lines (LCLs), although in vitro

  16. Increased expression of nuclear envelope gp210 antigen in small bile ducts in primary biliary cirrhosis.

    PubMed

    Nakamura, Minoru; Takii, Yasushi; Ito, Masahiro; Komori, Atsumasa; Yokoyama, Terufumi; Shimizu-Yoshida, Yuki; Koyabu, Makiko; Matsuyama, Mutsumi; Mori, Tsuyoshi; Kamihira, Takashi; Daikoku, Manabu; Migita, Kiyoshi; Yatsuhashi, Hiroshi; Nozaki, Naohito; Shimoda, Shinji; Ishibashi, Hiromi

    2006-03-01

    The sustained antibody response to nuclear envelope gp210 antigen indicates a group of primary biliary cirrhosis (PBC) patients at high risk for the progression to end-stage hepatic failure. To address this issue, we immunohistochemically studied the expression of gp210 antigen in needle liver biopsy specimens from PBC patients using a monoclonal antibody specific for gp210 antigen. The specimens from autoimmune hepatitis (AIH), chronic viral hepatitis B (CHB) and C (CHC) patients served as disease controls. The expression of gp210 antigen was apparently increased on the nuclear envelope of biliary epithelial cells (BECs) of small bile ducts in almost all specimens from PBC. In contrast, the expression of gp210 antigen was negative in BECs of small bile ducts in normal liver, while relatively weak anti-gp210 immunostaining was observed in AIH, CHC and CHB. In addition, the degree of gp210 expression in BECs of small bile ducts was positively correlated to that of portal inflammation, interface hepatitis and lobular inflammation in PBC. These results indicate that the increased expression of gp210 in small bile ducts, which is probably associated with damage to BECs by inflammation, is possibly involved in autoimmune response to gp210 leading to the progression to end-stage hepatic failure in PBC.

  17. Conserved cell cycle regulatory properties within the amino terminal domain of the Epstein-Barr virus nuclear antigen 3C

    SciTech Connect

    Sharma, Nikhil; Knight, Jason S.; Robertson, Erle S. . E-mail: erle@mail.med.upenn.edu

    2006-03-15

    The gammaherpesviruses Rhesus lymphocryptovirus (LCV) and Epstein-Barr virus (EBV) are closely related phylogenetically. Rhesus LCV efficiently immortalizes Rhesus B cells in vitro. However, despite a high degree of conservation between the Rhesus LCV and EBV genomes, Rhesus LCV fails to immortalize human B cells in vitro. This species restriction may, at least in part, be linked to the EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), known to be essential for B cell transformation. We compared specific properties of EBNA3C, a well-characterized and essential EBV protein, with its Rhesus counterpart to determine whether EBNA3C phenotypes which contribute to cell cycle regulation are conserved in the Rhesus LCV. We show that both EBNA3C and Rhesus EBNA3C bind to a conserved region of mammalian cyclins, regulate pRb stability, and modulate SCF{sup Skp2}-dependent ubiquitination. These results suggest that Rhesus LCV restriction from human B cell immortalization is independent of the conserved cell cycle regulatory functions of the EBNA3C protein.

  18. Regulation and autoregulation of the promoter for the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Jeong, Joseph H; Orvis, Joshua; Kim, Jong Wook; McMurtrey, Curtis P; Renne, Rolf; Dittmer, Dirk P

    2004-04-16

    Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 has been established as the etiological agent of Kaposi's sarcoma and certain AIDS-associated lymphomas. KSHV establishes latent infection in these tumors, invariably expressing high levels of the viral latency-associated nuclear antigen (LANA) protein. LANA is necessary and sufficient to maintain the KSHV episome. It also modulates viral and cellular transcription and has been implicated directly in oncogenesis because of its ability to bind to the p53 and pRb tumor suppressor proteins. Previously, we identified the LANA promoter (LANAp) and showed that it was positively regulated by LANA itself. Here, we present a detailed mutational analysis and define cis-acting elements and trans-acting factors for the core LANAp. We found that a downstream promoter element, TATA box, and GC box/Sp1 site at -29 are all individually required for activity. This architecture places LANAp into the small and unusual group of eukaryotic promoters that contain both the downstream promoter element and TATA element but lack a defined initiation site. Furthermore, we demonstrate that LANA regulates its own promoter via its C-terminal domain and does bind to a defined site within the core promoter.

  19. G-quadruplexes regulate Epstein-Barr virus–encoded nuclear antigen 1 mRNA translation

    PubMed Central

    Murat, Pierre; Zhong, Jie; Lekieffre, Lea; Cowieson, Nathan P; Clancy, Jennifer L; Preiss, Thomas; Balasubramanian, Shankar; Khanna, Rajiv; Tellam, Judy

    2014-01-01

    Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs. PMID:24633353

  20. Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1).

    PubMed

    Lu, Fang; Wikramasinghe, Priyankara; Norseen, Julie; Tsai, Kevin; Wang, Pu; Showe, Louise; Davuluri, Ramana V; Lieberman, Paul M

    2010-10-07

    The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites.

  1. Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1)

    PubMed Central

    2010-01-01

    The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites. PMID

  2. Immunological detection of nucleic acids and antibodies to nucleic acids and nuclear antigens by counterimmunoelectrophoresis

    PubMed Central

    Schur, P. H.; De Angelis, Diane; Jackson, Jean M.

    1974-01-01

    A counterimmunoelectrophoresis (CIEP) technique has been developed for the rapid, simple, specific detection of nucleic acids as antigens, or for the detection of precipitating antibodies to nucleic acids or nuclear antigens. The majority of precipitins could be detected within 1 hr. As little as 0·0015 μg of antigen per ml (e.g. poly A: poly U) could be detected. Specificity of rabbit antisera to nucleic acids was demonstrated by selective reactions using a panel of polynucleotides. 1091 patient sera were examined for precipitins to DNA, single-stranded DNA, nucleoprotein and calf thymus nucleoprotein. Precipitins to DNA were found in 42% of systemic lupus erythematosus sera, 9% of rheumatoid arthritis sera and 4% of juvenile rheumatoid arthritis sera. Results with the CIEP method showed equal sensitivity as results obtained by complement fixation or binding assays, but were more sensitive than double diffusion in agar (Ouchterlony). PMID:4549570

  3. High temperature antigen retrieval and loss of nuclear morphology: a comparison of microwave and autoclave techniques.

    PubMed Central

    Hunt, N C; Attanoos, R; Jasani, B

    1996-01-01

    The use of high temperature antigen retrieval methods has been of major importance in increasing the diagnostic utility of immunocytochemistry. However, these techniques are not without their problems and in this report attention is drawn to a loss of nuclear morphological detail, including mitotic figures, following microwave antigen retrieval. This was not seen with an equivalent autoclave technique. This phenomenon was quantified using image analysis in a group of B cell lymphomas stained with the antibody L26. Loss of nuclear morphological detail may lead to difficulty in identifying cells accurately, which is important in the diagnostic setting-for example, when trying to distinguish a malignant lymphoid infiltrate within a mixed cell population. In such cases it would clearly be wise to consider the use of alternative high temperature retrieval methods and accept their slightly lower staining enhancement capability compared with the microwave technique. Images PMID:9038766

  4. Proliferating cell nuclear antigen immunostaining in breast carcinoma and its relationship to clinical and pathological variables.

    PubMed

    Agarwal, S; Jain, R; Rusia, U; Gupta, R L

    1997-01-01

    Tumour proliferative activity of 74 breast lesions was assessed by determining mitotic index and immunostaining for proliferative cell nuclear antigen using Peroxidase antiperoxidase method. The indices were correlated with histomorphology and clinical stage of the disease. Positively stained nuclei and mitotic figures were counted per 1000 cells to calculate Proliferating Cell Nuclear Antigen (PCNA) and mitotic index respectively. Sixty four cases stained positive for PCNA. The index ranged between 0 to 98. PCNA index was significantly low in benign lesions as compared to malignant lesions (p < 0.0002). There was a linear correlation between the mitotic index and PCNA index. PCNA index also showed significant correlation with tumour size and histologic grade; however, it had no correlation with axillary lymph node status.

  5. Magnesium Presence Prevents Removal of Antigenic Nuclear-Associated Proteins from Bovine Pericardium for Heart Valve Engineering.

    PubMed

    Dalgliesh, Ailsa J; Liu, Zhi Zhao; Griffiths, Leigh G

    2017-03-10

    Current heart valve prostheses are associated with significant complications, including aggressive immune response, limited valve life expectancy, and inability to grow in juvenile patients. Animal derived "tissue" valves undergo glutaraldehyde fixation to mask tissue antigenicity; however, chronic immunological responses and associated calcification still commonly occur. A heart valve formed from an unfixed bovine pericardium (BP) extracellular matrix (ECM) scaffold, in which antigenic burden has been eliminated or significantly reduced, has potential to overcome deficiencies of current bioprostheses. Decellularization and antigen removal methods frequently use sequential solutions extrapolated from analytical chemistry approaches to promote solubility and removal of tissue components from resultant ECM scaffolds. However, the extent to which such prefractionation strategies may inhibit removal of antigenic tissue components has not been explored. We hypothesize that presence of magnesium in prefractionation steps causes DNA precipitation and reduces removal of nuclear-associated antigenic proteins. Keeping all variables consistent bar the addition or absence of magnesium (2 mM magnesium chloride hexahydrate), residual BP ECM scaffold antigenicity and removed antigenicity were assessed, along with residual and removed DNA content, ECM morphology, scaffold composition, and recellularization potential. Furthermore, we used proteomic methods to determine the mechanism by which magnesium presence or absence affects scaffold residual antigenicity. This study demonstrates that absence of magnesium from antigen removal solutions enhances solubility and subsequent removal of antigenic nuclear-associated proteins from BP. We therefore conclude that the primary mechanism of action for magnesium removal during antigen removal processes is avoidance of DNA precipitation, facilitating solubilization and removal of nuclear-associated antigenic proteins. Future studies are

  6. Expression patterns of proliferating cell nuclear antigen in trichloroacetic acid peeled skin.

    PubMed

    Yamamoto, Yuki; Uede, Koji; Yonei, Nozomi; Furukawa, Fukumi

    2007-02-01

    This study was designed to investigate whether trichloroacetic acid (TCA) peeling induces cellular proliferation in human skin using an immunohistochemical method. A 40% TCA peel resulted in a greater number of proliferating cell nuclear antigen (PCNA)-immunopositive cells in the whole epidermis as compared with 60% TCA or phenol peels. This finding suggests that long-term and frequent TCA peelings of low concentration would require special attention for unexpected cutaneous lesions such as skin tumors.

  7. Constitutive expression of Epstein-Barr virus-encoded RNAs and nuclear antigen during latency and after induction of Epstein-Barr virus replication.

    PubMed Central

    Weigel, R; Fischer, D K; Heston, L; Miller, G

    1985-01-01

    We examined the fate of two major products of latency as Epstein-Barr virus was induced to replicate. We studied a superinducible clone of HR-1 cells in the presence and absence of induction by phorbol ester, and we analyzed the X50-7 line with and without superinfection by an HR-1 viral variant which disrupts latency. The two methods of induction yielded qualitatively similar results. After induction, there was abundant synthesis of viral transcripts, amplification of viral DNA, and the appearance of many new viral polypeptides. Nonetheless, there were no changes in the cytoplasmic abundance of Epstein-Barr virus-encoded RNAs and no alteration in the level of Epstein-Barr virus nuclear antigen mRNA or polypeptide. Thus, under conditions in which numerous other Epstein-Barr virus gene products are activated, the two major latent gene products are expressed at a constitutive level. Expression of Epstein-Barr virus-encoded RNAs and nuclear antigen must therefore be regulated in a manner completely different from expression of replicative functions. Images PMID:2981344

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

    PubMed

    Kumar, Amit; Sahu, Sushil Kumar; Mohanty, Suchitra; Chakrabarti, Sudipta; Maji, Santanu; Reddy, R Rajendra; Jha, Asutosh K; Goswami, Chandan; Kundu, Chanakya N; Rajasubramaniam, Shanmugam; Verma, Subhash C; Choudhuri, Tathagata

    2014-01-01

    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.

  9. Identification of a nuclear export signal in the KSHV latent protein LANA2 mediating its export from the nucleus

    SciTech Connect

    Munoz-Fontela, C.; Collado, M.; Rodriguez, E.; Garcia, M.A.; Alvarez-Barrientos, A.; Arroyo, J.; Nombela, C.; Rivas, C. . E-mail: mdcrivas@farm.ucm.es

    2005-11-15

    LANA2 is a latent protein detected in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected B cells that inhibits p53-dependent transcriptional transactivation and apoptosis and PKR-dependent apoptosis, suggesting an important role in the transforming activity of the virus. It has been reported that LANA2 localizes into the nucleus of both KSHV-infected B cells and transiently transfected HeLa cells. In this study, we show that LANA2 is a nucleocytoplasmic shuttling protein that requires a Rev-type nuclear export signal located in the C-terminus to direct the protein to the cytoplasm, through an association with the export receptor CRM1. In addition, a functional protein kinase B (PKB)/Akt phosphorylation motif partially overlapping with the nuclear export signal was identified. Nuclear exclusion of LANA2 was negatively regulated by the phosphorylation of threonine 564 by Akt. The ability of LANA2 to shuttle between nucleus and cytoplasm has implications for the function of this viral protein.

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

    SciTech Connect

    Alves, Carolina; Freitas, Natalia; Cunha, Celso

    2008-01-05

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

  11. Assay Development and High-Throughput Screening for Inhibitors of Kaposi's Sarcoma-Associated Herpesvirus N-Terminal Latency-Associated Nuclear Antigen Binding to Nucleosomes.

    PubMed

    Beauchemin, Chantal; Moerke, Nathan J; Faloon, Patrick; Kaye, Kenneth M

    2014-07-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies, especially in immunocompromised hosts. KSHV latently infects tumor cells and persists as an extrachromosomal episome (plasmid). KSHV latency-associated nuclear antigen (LANA) mediates KSHV episome persistence. LANA binds specific KSHV sequence to replicate viral DNA. In addition, LANA tethers KSHV genomes to mitotic chromosomes to efficiently segregate episomes to daughter nuclei after mitosis. N-terminal LANA (N-LANA) binds histones H2A and H2B to attach to chromosomes. Currently, there are no specific inhibitors of KSHV latent infection. To enable high-throughput screening (HTS) of inhibitors of N-LANA binding to nucleosomes, here we develop, miniaturize, and validate a fluorescence polarization (FP) assay that detects fluorophore-labeled N-LANA peptide binding to nucleosomes. We also miniaturize a counterscreen to identify DNA intercalators that nonspecifically inhibit N-LANA binding to nucleosomes, and also develop an enzyme-linked immunosorbent assay to assess N-LANA binding to nucleosomes in the absence of fluorescence. HTS of libraries containing more than 350,000 compounds identified multiple compounds that inhibited N-LANA binding to nucleosomes. No compounds survived all counterscreens, however. More complex small-molecule libraries will likely be necessary to identify specific inhibitors of N-LANA binding to histones H2A and H2B; these assays should prove useful for future screens.

  12. Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference inhibits proliferation of EBV-positive Burkitt's lymphoma cells.

    PubMed

    Hong, Mei; Murai, Yoshihiro; Kutsuna, Tomohiko; Takahashi, Hiroyuki; Nomoto, Kazuhiro; Cheng, Chun-Mei; Ishizawa, Shin; Zhao, Qing-Li; Ogawa, Ryohei; Harmon, Brian V; Tsuneyama, Koichi; Takano, Yasuo

    2006-01-01

    Epstein-Barr virus (EBV) is associated with the development of several lymphoid and epithelial malignancies, including Burkitt's lymphoma. The EBV latent protein, EBV Nuclear Antigen 1 (EBNA1), is detectable in almost all types of EBV-associated tumors and is essential for replication and maintenance of the latent episome of EBV. We here examined whether the RNA interference (RNAi) technique could be employed to suppress expression of EBNA1 in EBV-positive Burkitt's lymphoma cells. A Raji cell line expressing small hairpin RNAs (shRNAs) against EBNA1 was established and EBNA1 mRNA level was determined by real-time RT-PCR analysis. We investigated the effects of EBNA1 silence on lymphoma cell growth and cell cycle progression. Transfection of an EBNA1 RNAi plasmid resulted in substantial loss of EBNA1 mRNA and significantly inhibited proliferation of Raji cells relative to the control plasmid case. Suppression of EBNA1 was also associated with downregulation of EBV oncogene EBNA2, a decreased PCNA labeling index and increased G0/G1 fraction in cell cycle analysis. These findings point to potential therapeutic applications for vector-mediated siRNA delivery to control EBV-associated malignant disorders.

  13. Transcriptional activation by the Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen is facilitated by an N-terminal chromatin-binding motif.

    PubMed

    Wong, Lai-Yee; Matchett, Gerald A; Wilson, Angus C

    2004-09-01

    In immunocompromised patients, infection with Kaposi's sarcoma-associated herpesvirus (KSHV) can give rise to Kaposi's sarcoma and several lymphoproliferative disorders. In these tumors, KSHV establishes a latent infection in many of the rapidly proliferating and morphologically abnormal cells. Only a few viral gene products are expressed by the latent virus, and one of the best characterized is the latency-associated nuclear antigen (LANA), a nuclear protein required for the maintenance of viral episomal DNA in the dividing host cell. LANA can also activate or repress an assortment of cellular and viral promoters and may contribute to pathogenesis by allowing the proliferation and survival of host cells. Here we show that activation of the human E2F1 and cyclin-dependent kinase-2 (CDK2) promoters requires elements from both the N- and C-terminal regions of LANA. Deletion of the first 22 amino acids, which are necessary for episome tethering, does not affect nuclear localization but significantly reduces transactivation. Within the deleted peptide, we have identified a short sequence, termed the chromatin-binding motif (CBM), that binds tightly to interphase and mitotic chromatin. A second chromatin-binding activity resides in the C terminus but is not sufficient for optimal transactivation. Alanine substitutions within the CBM reveal a close correlation between the transactivation and chromatin binding activities, implying a mechanistic link. In contrast to promoter activation, we find that the 223 amino acids of the LANA C terminus are sufficient to inhibit p53-mediated activation of the human BAX promoter, indicating that the CBM is not required for all transcription-related functions.

  14. Immunolocalization of the Epstein-Barr nuclear antigen-1 in conjunctival squamous carcinomas and dysplasias.

    PubMed

    Restelli, Marcela; Grinstein, Saul; Gattuso, Paolo; Preciado, M Victoria; Brunzini, Mario A; Zarate, Jorge; Mosquera, Juan-Miguel; Gould, Victor E

    2005-04-01

    Epstein-Barr virus (EBV) has been linked etiologically to infectious mononucleosis, some non-Hodgkin as well as Hodgkin lymphomas, and lymphoepithelioma-like carcinomas. Moreover, various EBV antigens have been identified by a variety of techniques in a number of visceral carcinomas including breast, prostate, colon and lung primaries. We have now demonstrated by immunohistochemistry the presence of EBV nuclear antigen-1 (EBNA-1) in 4 of 15 cases of conjuntival squamous carcinomas and related dysplasias. At present, there is no significant evidence linking etiologically EVB to this type of tumor and dysplasia. However, our findings merit further investigation given the growing evidence that EBV may enhance proliferation and aggressiveness of tumor systems as well as the immortalization of non-neoplastic cells.

  15. Cytoplasmic Inheritance of Transplantation Antigens In Animals Produced by Nuclear Transfer

    PubMed Central

    Hanekamp, John S.; Okumi, Masayoshi; Tena, Aseda; Arn, Scott; Yamada, Kazuhiko; Sachs, David H.

    2010-01-01

    Background Nuclear transfer has been utilized as a means of selectively modifying the mammalian genome. One possible consequence of this technology is that the oocytes used in nuclear transfer may provide additional antigens via cytoplasmic inheritance of maternally derived, mitochondrial DNA. These studies examine the potential consequences of such inheritance in a large animal transplantation model. Methods Renal transplants were performed between MHC-identical animals differing only in the source of their maternally derived cytoplasmic DNA, using a protocol which uniformly leads to tolerance within standard MHC-inbred lines. In an attempt to correlate transplant results with a putative marker for disparities in cytoplasmically inherited minor histocompatibility antigens, we examined one hypervariable region of mitochondrial DNA (mtDNA), designated HV1. Results The mtDNA sequence of the HV1 region was found to be invariant among MGH miniature swine of different haplotypes, despite twenty years of selective breeding of the sublines of this colony. In contrast, swine derived by nuclear transfer into outbred oocytes differed in the HV1 region sequence from each other and from MGH swine. Renal transplants from standard, inbred MGH swine to their MHC-identical knockout counterparts derived from outbred oocytes were rejected within two weeks, while transplants in the reverse direction were accepted for over 30 days. Conclusions The HV1 sequence of mtDNA may serve as a marker for the level of diversity of mtDNA. These transplant data are consistent with the existence of mtDNA-encoded mitochondrial minor antigens with a similar level of diversity that can influence the outcome of renal transplantation. PMID:19584677

  16. Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products

    PubMed Central

    1990-01-01

    A cDNA clone encoding full-length human proliferating cell nuclear antigen (PCNA) was used to generate a panel of in vitro translated labeled protein products with COOH-terminal deletions and to construct a set of fusion proteins with COOH- and NH2-terminal deletions. A rabbit antiserum raised against an NH2-terminal peptide, a well- characterized murine monoclonal antibody (mAb), and 14 human lupus sera with autoantibody to PCNA were analyzed for their reactivity with the constructs using both immunoprecipitation and immunoblotting techniques. The rabbit antiserum reacted in immunoprecipitation and immunoblotting with constructs containing the appropriate NH2-terminal sequence and mAb reacted with a sequence from the midregion of PCNA. These experimentally induced antibodies also reacted with 15-mer synthetic peptides in enzyme-linked immunosorbent assay (ELISA). In contrast, none of the lupus sera reacted with synthetic peptides in ELISA. 9 of the 14 lupus sera also failed to react in Western immunoblotting with any recombinant fusion protein, although they all immunoprecipitated in vitro translated full-length protein. Four of the nine had variable patterns of immunoprecipitation with shorter constructs. The remaining five lupus sera were able to immunoprecipitate translation products as well as Western blot recombinant fusion proteins. From analysis of the patterns of reactivity of human lupus sera, it was deduced that the apparent heterogeneity of human autoantibodies to PCNA could be explained by immune response to highly conformational epitopes. These observations demonstrate that there might be special features in "native" epitopes of intranuclear antigens that are recognized by autoantibodies, and that these special features of native epitopes might not be present in prepared antigen used for experimental immunization. These features may be related to protein folding or to association of the antigen with other intranuclear proteins or nucleic acids, as

  17. Antibodies to extractable nuclear antigens. Has technological drift affected clinical interpretation?

    PubMed

    Lock, R J; Unsworth, D J

    2001-03-01

    Precipitating antibodies to extractable nuclear antigens are important in the diagnosis of connective tissue diseases. Disease associations are defined using gel based techniques. Alternative technologies have been introduced, including passive haemagglutination, enzyme linked immunosorbent assay, and western blotting. This leader contains a review of the literature on the clinical usefulness of these assays, together with knowledge gained from personal experience. Using the example of systemic lupus erythematosus, the sensitivity, specificity, and positive predictive value of the assays for disease is discussed, as is their differences in performance. The conclusion drawn is that disease specificity is method dependent. Validation and audit of performance of the method selected by the investigation laboratory is essential.

  18. Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells

    PubMed Central

    Freund, Guillaume; Sibler, Annie-Paule; Desplancq, Dominique; Oulad-Abdelghani, Mustapha; Vigneron, Marc; Gannon, Julian; Van Regenmortel, Marc H.; Weiss, Etienne

    2013-01-01

    Antibodies are valuable tools for functional studies in vitro, but their use in living cells remains challenging because they do not naturally cross the cell membrane. Here, we present a simple and highly efficient method for the intracytoplasmic delivery of any antibody into cultured cells. By following the fate of monoclonal antibodies that bind to nuclear antigens, it was possible to image endogenous targets and to show that inhibitory antibodies are able to induce cell growth suppression or cell death. Our electrotransfer system allowed the cancer cells we studied to be transduced without loss of viability and may have applications for a variety of intracellular immuno-interventions. PMID:23765067

  19. Autoantibodies against nuclear, nucleolar, and mitochondrial antigens in systemic sclerosis (scleroderma).

    PubMed

    Reimer, G

    1990-02-01

    One of the most characteristic serologic features of systemic sclerosis (scleroderma) is the occurrence of autoantibodies against nuclear and most notably against nucleolar antigens. This humoral autoimmune response is one of best studied immunologic phenomena in scleroderma. Detailed molecular information on the structure and function, as well as on reactive epitopes of autoantigens targeted by specific serum antibodies, has been revealed by clinical, immunologic, and biochemic studies in several laboratories. Autoantigens such as DNA topoisomerase I (Scl-70), centromere proteins, RNA polymerase I, U3 RNP-associated fibrillarin, PM-Scl, and 7-2 RNP antigens were shown to be specific targets of scleroderma patients and were observed to have clinical correlates within the scleroderma disease spectrum. Therefore, autoantibodies in scleroderma are not only valuable diagnostic tools but also prognosticators of the disease. Although autoantibodies in scleroderma do not appear to play a pathogenetic role in the disease process, the knowledge of the structure and function of their reactive antigens may help in answering questions concerning the etiology of the disease.

  20. Crystallization and X-ray diffraction studies of crustacean proliferating cell nuclear antigen

    PubMed Central

    Carrasco-Miranda, Jesus S.; Cardona-Felix, Cesar S.; Lopez-Zavala, Alonso A.; de-la-Re-Vega, Enrique; De la Mora, Eugenio; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.; Brieba, Luis G.

    2012-01-01

    Proliferating cell nuclear antigen (PCNA), a member of the sliding clamp family of proteins, interacts specifically with DNA replication and repair proteins through a small peptide motif called the PCNA-interacting protein or PIP box. PCNA is recognized as one of the key proteins involved in DNA metabolism. In the present study, the recombinant PCNA from Litopenaeus vannamei (LvPCNA) was heterologously overexpressed and purified using metal ion-affinity chromatography. Crystals suitable for diffraction grew overnight using the hanging-drop vapour-diffusion method. LvPCNA crystals belong to space group C2 with unit-cell parameters a = 144.6, b = 83.4, c = 74.3 Å, β = 117.6°. One data set was processed to 3 Å resolution, with an overall R meas of 0.09 and a completeness of 93.3%. Initial phases were obtained by molecular replacement using a homology model of LvPCNA as the search model. Refinement and structural analysis are underway. This report is the first successful crystallographic analysis of a marine crustacean decapod shrimp (L. vannamei) proliferating cell nuclear antigen. PMID:23143251

  1. Crystallization and X-ray diffraction studies of crustacean proliferating cell nuclear antigen.

    PubMed

    Carrasco-Miranda, Jesus S; Cardona-Felix, Cesar S; Lopez-Zavala, Alonso A; de-la-Re-Vega, Enrique; De la Mora, Eugenio; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R; Brieba, Luis G

    2012-11-01

    Proliferating cell nuclear antigen (PCNA), a member of the sliding clamp family of proteins, interacts specifically with DNA replication and repair proteins through a small peptide motif called the PCNA-interacting protein or PIP box. PCNA is recognized as one of the key proteins involved in DNA metabolism. In the present study, the recombinant PCNA from Litopenaeus vannamei (LvPCNA) was heterologously overexpressed and purified using metal ion-affinity chromatography. Crystals suitable for diffraction grew overnight using the hanging-drop vapour-diffusion method. LvPCNA crystals belong to space group C2 with unit-cell parameters a=144.6, b=83.4, c=74.3 Å, β=117.6°. One data set was processed to 3 Å resolution, with an overall Rmeas of 0.09 and a completeness of 93.3%. Initial phases were obtained by molecular replacement using a homology model of LvPCNA as the search model. Refinement and structural analysis are underway. This report is the first successful crystallographic analysis of a marine crustacean decapod shrimp (L. vannamei) proliferating cell nuclear antigen.

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

    PubMed

    Nakada, Ryohei; Hirano, Hidemi; Matsuura, Yoshiyuki

    2017-02-26

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

  3. Cytoplasmic proliferating cell nuclear antigen connects glycolysis and cell survival in acute myeloid leukemia

    PubMed Central

    Ohayon, Delphine; De Chiara, Alessia; Chapuis, Nicolas; Candalh, Céline; Mocek, Julie; Ribeil, Jean-Antoine; Haddaoui, Lamya; Ifrah, Norbert; Hermine, Olivier; Bouillaud, Frédéric; Frachet, Philippe; Bouscary, Didier; Witko-Sarsat, Véronique

    2016-01-01

    Cytosolic proliferating cell nuclear antigen (PCNA), a scaffolding protein involved in DNA replication, has been described as a key element in survival of mature neutrophil granulocytes, which are non-proliferating cells. Herein, we demonstrated an active export of PCNA involved in cell survival and chemotherapy resistance. Notably, daunorubicin-resistant HL-60 cells (HL-60R) have a prominent cytosolic PCNA localization due to increased nuclear export compared to daunorubicin-sensitive HL-60 cells (HL-60S). By interacting with nicotinamide phosphoribosyltransferase (NAMPT), a protein involved in NAD biosynthesis, PCNA coordinates glycolysis and survival, especially in HL-60R cells. These cells showed a dramatic increase in intracellular NAD+ concentration as well as glycolysis including increased expression and activity of hexokinase 1 and increased lactate production. Furthermore, this functional activity of cytoplasmic PCNA was also demonstrated in patients with acute myeloid leukemia (AML). Our data uncover a novel pathway of nuclear export of PCNA that drives cell survival by increasing metabolism flux. PMID:27759041

  4. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    SciTech Connect

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-04-01

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone lambdaHB''-1 from a phage lambdagt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambdaHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone lambdaHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambdaHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambdaHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

  5. A Latent Markov Modelling Approach to the Evaluation of Circulating Cathodic Antigen Strips for Schistosomiasis Diagnosis Pre- and Post-Praziquantel Treatment in Uganda

    PubMed Central

    Koukounari, Artemis; Donnelly, Christl A.; Moustaki, Irini; Tukahebwa, Edridah M.; Kabatereine, Narcis B.; Wilson, Shona; Webster, Joanne P.; Deelder, André M.; Vennervald, Birgitte J.; van Dam, Govert J.

    2013-01-01

    Regular treatment with praziquantel (PZQ) is the strategy for human schistosomiasis control aiming to prevent morbidity in later life. With the recent resolution on schistosomiasis elimination by the 65th World Health Assembly, appropriate diagnostic tools to inform interventions are keys to their success. We present a discrete Markov chains modelling framework that deals with the longitudinal study design and the measurement error in the diagnostic methods under study. A longitudinal detailed dataset from Uganda, in which one or two doses of PZQ treatment were provided, was analyzed through Latent Markov Models (LMMs). The aim was to evaluate the diagnostic accuracy of Circulating Cathodic Antigen (CCA) and of double Kato-Katz (KK) faecal slides over three consecutive days for Schistosoma mansoni infection simultaneously by age group at baseline and at two follow-up times post treatment. Diagnostic test sensitivities and specificities and the true underlying infection prevalence over time as well as the probabilities of transitions between infected and uninfected states are provided. The estimated transition probability matrices provide parsimonious yet important insights into the re-infection and cure rates in the two age groups. We show that the CCA diagnostic performance remained constant after PZQ treatment and that this test was overall more sensitive but less specific than single-day double KK for the diagnosis of S. mansoni infection. The probability of clearing infection from baseline to 9 weeks was higher among those who received two PZQ doses compared to one PZQ dose for both age groups, with much higher re-infection rates among children compared to adolescents and adults. We recommend LMMs as a useful methodology for monitoring and evaluation and treatment decision research as well as CCA for mapping surveys of S. mansoni infection, although additional diagnostic tools should be incorporated in schistosomiasis elimination programs. PMID:24367250

  6. IFN-γ and IL-2 Responses to Recombinant AlaDH against ESAT-6/CFP-10 Fusion Antigens in the Diagnosis of Latent versus Active Tuberculosis Infection

    PubMed Central

    Movahedi, Bahram; Mokarram, Pooneh; Hemmati, Mina; Mosavari, Nader; Zare, Razie; Ardekani, Leila Safaee; Mostafavi-Pour, Zohreh

    2017-01-01

    Background: Discriminating latent tuberculosis infection (LTBI) from active TBI may be challenging. The objective of this study was to produce the recombinant L-alanine dehydrogenase (AlaDH) antigen and evaluate individuals with LTBI, those with active TBI, and uninfected individuals by enzyme-linked immunospot assay (ELISPOT) in order to distinguish LTBI from active TBI. Methods: This exploratory study was performed in the Iranian city of Shiraz from 2014 to 2015. The study population (N=99) was divided into 3 groups: individuals with newly diagnosed active TBI (n=33), their household contacts (n=33), and controls (n=33). AlaDH was produced through PCR and cloning methods. The diagnostic characteristics of AlaDH vs. ESAT-6/CFP-10 were evaluated in responses to interferon-γ (IFN-γ) and interleukin-2 (IL-2) with ELISPOT. Differences between the groups were assessed with the Kruskal–Wallis and Mann–Whitney tests for nonparametric data analysis. The statistical analyses were performed with SPSS, version 16. Results: IFN-γ responses to both ESAT-6/CFP-10 (P=0.81) and AlaDH (P=0.18) revealed that there were no significant differences between the individuals with LTBI and those with active TBI. The same results were determined for IL-2 responses to ESAT-6/CFP-10 between the 2 groups, while significantly higher IL-2 responses to AlaDH were observed in LTBI than in active TBI. According to the ROC curve analysis, a cutoff value of 275 SFC showed sensitivity of 75.8% and specificity of 78.8% for distinguishing LTBI from active TBI by IL-2 responses to AlaDH. Conclusion: The current study suggests that it may be possible to discriminate LTBI from active TBI by IL-2 responses to AlaDH. PMID:28533576

  7. Antibodies to components of extractable nuclear antigen. Clinical characteristics of patients.

    PubMed

    Farber, S J; Bole, G G

    1976-04-01

    Forty-four patients with antibodies to ribonuclease-sensitive extractable nuclear antigen (ENA), ribonuclease-resistant ENA, or both, are described. Most patients with antiribonucleoprotein (anti-RNP) antibodies have overlapping features of systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), and polymyositis, and have a low incidence of nephritis. Most patients with antibody solely to ribonuclease-insensitive ENA have SLE; these patients with SLE are typical of the general SLE population, except that they demonstrate an increased incidence of Raynaud phenomenon. Furthermore, it is shown that antibody to ENA may occur in other rheumatic and nonrheumatic diseases, and that not all patients who have a clinical overlap of SLE and PSS that is suggestive of mixed connective tissue disease have anti-RNP antibody.

  8. Proliferating cell nuclear antigen uses two distinct modes to move along DNA.

    PubMed

    Kochaniak, Anna B; Habuchi, Satoshi; Loparo, Joseph J; Chang, Debbie J; Cimprich, Karlene A; Walter, Johannes C; van Oijen, Antoine M

    2009-06-26

    Proliferating cell nuclear antigen (PCNA) plays an important role in eukaryotic genomic maintenance by topologically binding DNA and recruiting replication and repair proteins. The ring-shaped protein forms a closed circle around double-stranded DNA and is able to move along the DNA in a random walk. The molecular nature of this diffusion process is poorly understood. We use single-molecule imaging to visualize the movement of individual, fluorescently labeled PCNA molecules along stretched DNA. Measurements of diffusional properties as a function of viscosity and protein size suggest that PCNA moves along DNA using two different sliding modes. Most of the time, the clamp moves while rotationally tracking the helical pitch of the DNA duplex. In a less frequently used second mode of diffusion, the movement of the protein is uncoupled from the helical pitch, and the clamp diffuses at much higher rates.

  9. Homogeneous assay for detection of active Epstein-Barr nuclear antigen 1 by thrombin activity modulation.

    PubMed

    Garai-Ibabe, Gaizka; Grinyte, Ruta; Canaan, Allon; Pavlov, Valeri

    2012-07-17

    Epstein-Barr virus (EBV) has been associated with several malignancies as Burkitt's lymphoma, nasopharyngeal carcinoma, and Hodgkin's disease. In those diseases, Epstein-Barr nuclear antigen 1 (EBNA-1) is constitutively expressed. Here, we reported an innovative system to detect active EBNA-1 protein in a homogeneous assay. The system is based on the modulation of thrombin activity by a self-complementary single stranded DNA (scssDNA), which was designed and synthesized to mimic the palindromic target sites of EBNA-1 in the EBV genome. This model system showed a limit of detection of 3.75 ng mL(-1) of active EBNA-1 protein with a dynamic detection range from 3.75 to 250 ng mL(-1) with a correlation coefficient of 0.997. This new homogeneous assay for active EBNA-1 protein detection and quantification provides a very useful tool for rapid screening of EBNA-1 blockers in biomedical research.

  10. Structure and biochemical characterization of proliferating cellular nuclear antigen from a parasitic protozoon

    SciTech Connect

    Cardona-Felix, Cesar S.; Lara-Gonzalez, Samuel; Brieba, Luis G.

    2012-02-08

    Proliferating cellular nuclear antigen (PCNA) is a toroidal-shaped protein that is involved in cell-cycle control, DNA replication and DNA repair. Parasitic protozoa are early-diverged eukaryotes that are responsible for neglected diseases. In this work, a PCNA from a parasitic protozoon was identified, cloned and biochemically characterized and its crystal structure was determined. Structural and biochemical studies demonstrate that PCNA from Entamoeba histolytica assembles as a homotrimer that is able to interact with and stimulate the activity of a PCNA-interacting peptide-motif protein from E. histolytica, EhDNAligI. The data indicate a conservation of the biochemical mechanisms of PCNA-mediated interactions between metazoa, yeast and parasitic protozoa.

  11. Epstein-Barr Virus Nuclear Antigen 1 Hijacks the Host Kinase CK2 To Disrupt PML Nuclear Bodies▿

    PubMed Central

    Sivachandran, Nirojini; Cao, Jennifer Yinuo; Frappier, Lori

    2010-01-01

    Latent Epstein-Barr virus (EBV) infection is an important causative factor in the development of several cancers, including nasopharyngeal carcinoma (NPC). The one EBV protein expressed in the nucleus of NPC cells, EBNA1, has been shown to disrupt promyelocitic leukemia (PML) nuclear bodies (NBs) by inducing the degradation of PML proteins, leading to impaired DNA repair and increased cell survival. Although EBNA1-mediated PML disruption is likely to be an important factor in the development of NPC, little is known about its mechanism. We now show that an interaction between EBNA1 and the host CK2 kinase is crucial for EBNA1 to disrupt PML bodies and degrade PML proteins. EBNA1 increases the association of CK2 with PML proteins, thereby increasing the phosphorylation of PML proteins by CK2, a modification that is known to trigger the polyubiquitylation and degradation of PML. The interaction between EBNA1 and CK2 is direct and occurs through the β regulatory subunit of CK2 and EBNA1 amino acids 387 to 394. The binding of EBNA1 to the host ubiquitin specific protease USP7 has also been shown to be important for EBNA1-mediated PML disruption. We show that EBNA1 also increases the occupancy of USP7 at PML NBs and that CK2 and USP7 bind independently and simultaneously to EBNA1 to form a ternary complex. The combined results indicate that EBNA1 usurps two independent cellular pathways to trigger the loss of PML NBs. PMID:20719947

  12. Labeling and use of monoclonal antibodies in immunofluorescence: protocols for cytoskeletal and nuclear antigens.

    PubMed

    Bauer, Christoph R

    2014-01-01

    Antibodies are widely used to target and label specifically extra- or intracellular antigens within cells and tissues. Most protocols follow an indirect approach implying the successive incubation with primary and secondary antibodies. In these protocols the primary antibodies are specifically targeted against the antigen in question and are normally not labeled. The secondary antibodies come from a different species and are in contrast fluorescently labeled. The idea is that the primary antibodies specifically bind to their targets but cannot be visualized directly. Only binding of the secondary (fluorescent) antibodies to the constant region of the primary antibodies allows consecutively the visualization in a fluorescent microscope.Primary antibodies can be either of monoclonal (normally produced in mouse) or of polyclonal origin (normally produced in rabbit, goat, sheep, or donkey). Using (primary) monoclonal antibodies has the clear advantage that all antibodies used are identical in origin and behavior and should thus give a more clear-cut labeling result. On the other hand the demands towards labeling protocols might be concomitantly higher: Binding of primary antibodies will only occur if fixation and labeling protocols preserve the antigen sufficiently to keep its specific and unique target structure available. One could imagine that for polyclonal antibodies this demand is slightly lower as there is a pool of antibodies with varying specificities against multiple parts of their target antigens. Certain fractions of this pool might thus tolerate a larger variety of conditions, and consequently a larger variety of protocols might still result in successful labeling.Each step in a labeling protocol can be decisive for the outcome of an experiment especially if monoclonal antibodies are used. Especially critical are choice of buffer and fixation and permeabilization parameters of the protocol.In this chapter we discuss and detail proven protocols using

  13. Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

    PubMed Central

    Wang, Anqi; Welch, Rene; Zhao, Bo; Ta, Tram; Keleş, Sündüz

    2015-01-01

    ABSTRACT Latent infection of B lymphocytes by Epstein-Barr virus (EBV) in vitro results in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator, which targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our chromatin immunoprecipitation with deep sequencing (ChIP-seq) analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrated that EBNA3A, EBNA3B, and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30 to 40% of EBNA3-bound sites colocalize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near EBNA3A- or EBNA3C-regulated genes is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A-, EBNA3B-, and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A- and EBNA3C-bound sites and revealed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-negative BJAB cells, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B, binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors. IMPORTANCE Epstein-Barr virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell lines in vitro. EBV nuclear antigens (EBNAs) and membrane proteins constitutively activate pathways important for

  14. RelB nuclear translocation regulates B cell MHC molecule, CD40 expression, and antigen-presenting cell function

    PubMed Central

    O'Sullivan, Brendan J.; MacDonald, Kelli P. A.; Pettit, Allison R.; Thomas, Ranjeny

    2000-01-01

    Mice with targeted RelB mutations demonstrated an essential role for RelB in immune responses and in myeloid dendritic cell differentiation. Human studies suggested a more global transcriptional role in antigen presentation. Burkitt lymphoma cell lines were used as a model to examine the role of RelB in antigen presentation. After transient transfection of BJAB with RelB, strong nuclear expression of RelB-p50 heterodimers was associated with increased APC function and expression of CD40 and MHC class I. Antisense RelB in DG75 reduced antigen-presenting capacity and CD40-mediated up-regulation of MHC molecules. The data indicate that RelB transcriptional activity directly affects antigen presentation and CD40 synthesis. Stimulation of RelB transcriptional activity may provide a positive feedback loop for facilitating productive APC/T cell interactions. PMID:11027342

  15. Amino acid chloramine damage to proliferating cell nuclear antigen in mammalian cells.

    PubMed

    Salama, Samir A; Snapka, Robert M

    2012-01-01

    Amino acid chloramines (AACLs) are reactive secondary products of activated neutrophils. To understand AACL damage in cell nuclei, we exploited proliferating cell nuclear antigen (PCNA) as a nuclear protein damage reporter, using western blotting and mass spectrometry. Chloramines of proline, arginine, and glycine caused significant damage to PCNA in cells. Chloramines of taurine and histidine caused slight damage to PCNA in cells. Other AACLs caused no PCNA damage in intact cells. Evidence supports a sulfonamide, sulfinamide, or sulfenamide crosslinking mechanism involving cysteine 148 at the PCNA subunit interface, methionine sulfoxide formation as the basis of electrophoretic mobility shifting, and tyrosine and/or methionine residues as the likely targets of AACL damage to the PCNA antibody epitope. An interstitial fluid model experiment showed that physiological amino acids can mediate HOCl damage to PCNA in the presence of proteins that would otherwise completely quench the HOCl. PCNA is a sensitive biomarker of AACL damage in cell nuclei. Arginine chloramine and proline chloramine, or reactive species derived from them, were shown to enter cells and damage PCNA. Amino acids were shown to have at least two different mechanisms for suppressing PCNA damage in cells by their corresponding AACLs. Cysteine 148 was shown to be essential for PCNA subunit crosslinking by AACLs, and a crosslinking mechanism was proposed.

  16. Nuclear export signal-interacting protein forms complexes with lamin A/C-Nups to mediate the CRM1-independent nuclear export of large hepatitis delta antigen.

    PubMed

    Huang, Cheng; Jiang, Jia-Yin; Chang, Shin C; Tsay, Yeou-Guang; Chen, Mei-Ru; Chang, Ming-Fu

    2013-02-01

    Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.

  17. The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence.

    PubMed

    Li, Shijun; Tan, Min; Juillard, Franceline; Ponnusamy, Rajesh; Correia, Bruno; Simas, J Pedro; Carrondo, Maria A; McVey, Colin E; Kaye, Kenneth M

    2015-11-20

    Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.

  18. Mitotic chromosome interactions of Epstein-Barr nuclear antigen 1 (EBNA1) and human EBNA1-binding protein 2 (EBP2).

    PubMed

    Nayyar, Vipra Kapur; Shire, Kathy; Frappier, Lori

    2009-12-01

    The Epstein-Barr nuclear antigen 1 (EBNA1) protein enables the stable persistence of Epstein-Barr virus episomal genomes during latent infection, in part by tethering the episomes to the cellular chromosomes in mitosis. A host nucleolar protein, EBNA1-binding protein 2 (EBP2), has been shown to be important for interactions between EBNA1 and chromosomes in metaphase and to associate with metaphase chromosomes. Here, we examine the timing of the chromosome associations of EBNA1 and EBP2 through mitosis and the regions of EBNA1 that mediate the chromosome interactions at each stage of mitosis. We show that EBP2 is localized to the nucleolus until late prophase, after which it relocalizes to the chromosome periphery, where it remains throughout telophase. EBNA1 is associated with chromosomes early in prophase through to telophase and partially colocalizes with chromosomal EBP2 in metaphase through to telophase. Using EBNA1 deletion mutants, the chromosome association of EBNA1 at each stage of mitosis was found to be mediated mainly by a central glycine-arginine region, and to a lesser degree by N-terminal sequences. These sequence requirements for chromosome interaction mirrored those for EBP2 binding. Our results suggest that interactions between EBNA1 and chromosomes involve at least two stages, and that the contribution of EBP2 to these interactions occurs in the second half of mitosis.

  19. Small Molecule Inhibition of Epstein - Barr Virus Nuclear Antigen-1 DNA Binding Activity Interferes with Replication and Persistence of the Viral Genome

    PubMed Central

    Noh, Ka-Won; Joo, Eun Hye; Zhao, Bo; Kieff, Elliott; Kang, Myung-Soo

    2014-01-01

    The replication and persistence of extra chromosomal Epstein-Barr virus (EBV) episome in latently infected cells are primarily dependent on the binding of EBV-encoded nuclear antigen 1 (EBNA1) to the cognate EBV oriP element. In continuation of the previous study, herein we characterized EBNA1 small molecule inhibitors (H20, H31) and their underlying inhibitory mechanisms. In silico docking analyses predicted that H20 fits into a pocket in the EBNA1 DNA binding domain (DBD). However, H20 did not significantly affect EBNA1 binding to its cognate sequence. A limited structure-relationship study of H20 identified a hydrophobic compound H31, as an EBNA1 inhibitor. An in vitro EBNA1 EMSA and in vivo EGFP-EBNA1 confocal microscopy analysis showed that H31 inhibited EBNA1-dependent oriP sequence-specific DNA binding activity, but not sequence-nonspecific chromosomal association. Consistent with this, H31 repressed the EBNA1-dependent transcription, replication, and persistence of an EBV oriP plasmid. Furthermore, H31 induced progressive loss of EBV episome. In addition, H31 selectively retarded the growth of EBV-infected LCL or Burkitt’s lymphoma cells. These data indicate that H31 inhibition of EBNA1-dependent DNA binding decreases transcription from and persistence of EBV episome in EBV-infected cells. These new compounds might be useful probes for dissecting EBNA1 functions in vitro and in vivo. PMID:24486954

  20. Epstein-Barr virus nuclear antigen 1 (EBNA1) induced cytotoxicity in epithelial cells is associated with EBNA1 degradation and processing.

    PubMed

    Jones, Richard J; Smith, Laura J; Dawson, Christopher W; Haigh, Tracy; Blake, Neil W; Young, Lawrence S

    2003-09-01

    Epstein-Barr virus nuclear antigen 1 (EBNA1) has a central role in the maintenance and segregation of the Epstein-Barr virus (EBV) episome and by virtue of a glycine-alanine repeat domain is prevented from being endogenously processed for recognition by HLA class I restricted cytotoxic T lymphocytes (CTLs). We found that EBNA1 expression resulted in growth inhibition and a G2/M arrest in human squamous epithelial cell lines (SCC12F, SVK) but not epithelial cell lines of glandular origin (Hela, Ad/AH). The cytotoxicity of EBNA1 was associated with EBNA1 degradation and both these effects were blocked in SCC12F cells expressing either the anti-apoptotic bcl-2 protein or the EBV homolog of bcl-2, BHRF1. The endogenous degradation of EBNA1 in SVK epithelial cells was associated with specific CTL recognition, an effect not evident in EBNA1-expressing Hela cells. Consistent with the inability of SVK cells to tolerate EBNA1 expression, studies with a recombinant EBV demonstrated that SVK cells are unable to maintain stable virus infection, whereas Hela cells are able to efficiently establish latent EBV infection. These data have important implications for both the cellular requirements necessary to sustain a stable EBV infection and for the possible role of CTL responses in controlling EBV infection of epithelial cells.

  1. A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter.

    PubMed

    Hayashi, Y; Yamagishi, M; Nishimoto, Y; Taguchi, O; Matsukage, A; Yamaguchi, M

    1999-12-03

    The Drosophila proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including upstream regulatory element (URE), DNA replication-related element, E2F recognition sites, and three common regulatory factor for DNA replication and DNA replication-related element-binding factor genes recognition sites. In nuclear extracts of Drosophila embryos, we detected a protein factor, the URE-binding factor (UREF), that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies revealed that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allowed isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (GRH/NTF-1). The nucleotide sequence required for binding to GRH was indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to GRH reacted with UREF in embryo nuclear extracts. From these results we conclude that GRH is identical to UREF. Although GRH has been thought to be involved in regulation of differentiation-related genes, this study demonstrates, for the first time, involvement of a GRH-binding site in regulation of the DNA replication-related proliferating cell nuclear antigen gene.

  2. Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

    SciTech Connect

    Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong; Chung, Hyun Joo; Kim, Myoung Hee

    2010-02-19

    Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna was similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.

  3. Comparative analyses of the two proliferating cell nuclear antigens from the hyperthermophilic archaeon, Thermococcus kodakarensis.

    PubMed

    Kuba, Yumani; Ishino, Sonoko; Yamagami, Takeshi; Tokuhara, Masahiro; Kanai, Tamotsu; Fujikane, Ryosuke; Daiyasu, Hiromi; Atomi, Haruyuki; Ishino, Yoshizumi

    2012-11-01

    The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen (PCNA) is the sliding clamp. The ring-shaped PCNA encircles double-stranded DNA within its central hole and tethers other proteins on DNA. The majority of Crenarchaeota, a subdomain of Archaea, have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. In contrast, most organisms in Euryarchaeota, the other major subdomain, have a single PCNA forming a homotrimeric ring structure. Among the Euryarchaeota whose genome is sequenced, Thermococcus kodakarensis is the only species with two genes encoding PCNA homologues on its genome. We cloned the two genes from the T. kodakarensis genome, and the gene products, PCNA1 and PCNA2, were characterized. PCNA1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T. kodakarensis in vitro. PCNA2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA2, whereas gene disruption for PCNA1 was not possible, suggesting that PCNA1 is essential for DNA replication. The sensitivities of the Δpcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate (MMS) and mitomycin C (MMC) were indistinguishable from those of the wild-type strain.

  4. A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein

    PubMed Central

    Li, Zhuo; Huang, Richard Y.-C.; Yopp, Daniel C.; Hileman, Travis H.; Santangelo, Thomas J.; Hurwitz, Jerard; Hudgens, Jeffrey W.; Kelman, Zvi

    2014-01-01

    Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA. PMID:24728986

  5. Validating the disruption of proliferating cell nuclear antigen interactions in the development of targeted cancer therapeutics

    PubMed Central

    Smith, Shanna J.; Hickey, Robert J.; Malkas, Linda H.

    2016-01-01

    ABSTRACT Human DNA replication and repair is a highly coordinated process involving the specifically timed actions of numerous proteins and enzymes. Many of these proteins require interaction with proliferating cell nuclear antigen (PCNA) for activation within the process. The interdomain connector loop (IDCL) of PCNA provides a docking site for many of those proteins, suggesting that this region is critically important in the regulation of cellular function. Previous work in this laboratory has demonstrated that a peptide mimicking a specific region of the IDCL (caPeptide) has the ability to disrupt key protein-protein interactions between PCNA and its binding partners, thereby inhibiting DNA replication within the cells. In this study, we confirm the ability of the caPeptide to disrupt DNA replication function using both intact cell and in vitro DNA replication assays. Further, we were able to demonstrate that treatment with caPeptide results in a decrease of polymerase δ activity that correlates with the observed decrease in DNA replication. We have also successfully developed a surface plasmon resonance (SPR) assay to validate the disruption of the PCNA-pol δ interaction with caPeptide. PMID:26889573

  6. Proliferating cell nuclear antigen prevents trinucleotide repeat expansions by promoting repeat deletion and hairpin removal

    PubMed Central

    Beaver, Jill M.; Lai, Yanhao; Rolle, Shantell J.; Liu, Yuan

    2017-01-01

    DNA base lesions and base excision repair (BER) within trinucleotide repeat (TNR) tracts modulate repeat instability through the coordination among the key BER enzymes DNA polymerase β, flap endonuclease 1 (FEN1) and DNA ligase I (LIG I). However, it remains unknown whether BER cofactors can also alter TNR stability. In this study, we discovered that proliferating cell nuclear antigen (PCNA), a cofactor of BER, promoted CAG repeat deletion and removal of a CAG repeat hairpin during BER in a duplex CAG repeat tract and CAG hairpin loop, respectively. We showed that PCNA stimulated LIG I activity on a nick across a small template loop during BER in a duplex (CAG)20 repeat tract promoting small repeat deletions. Surprisingly, we found that during BER in a hairpin loop, PCNA promoted reannealing of the upstream flap of a double-flap intermediate, thereby facilitating the formation of a downstream flap and stimulating FEN1 cleavage activity and hairpin removal. Our results indicate that PCNA plays a critical role in preventing CAG repeat expansions by modulating the structures of dynamic DNA via cooperation with BER enzymes. We provide the first evidence that PCNA prevents CAG repeat expansions during BER by promoting CAG repeat deletion and removal of a TNR hairpin. PMID:27793507

  7. Proliferating cell nuclear antigen (PCNA) immunostaining--a prognostic factor in ovarian cancer?

    PubMed Central

    Thomas, H.; Nasim, M. M.; Sarraf, C. E.; Alison, M. R.; Love, S.; Lambert, H. E.; Price, P.

    1995-01-01

    The measurement of tumour cell proliferation is becoming increasingly recognised in defining prognostic groups. Proliferating cell nuclear antigen (PCNA) immunolocalisation can be used as an index of cell proliferation and may define the extent of departure from normal growth control. The monoclonal antibody PC10 stains PCNA in archival paraffin-embedded tissue. This study investigates its potential as a prognostic marker in early and advanced ovarian cancer. A three-stage immunoperoxidase technique was developed to detect the monoclonal antibody PC10. Archival paraffin-embedded tissue from 19 stage I ovarian tumours (13 malignant and six borderline) and 79 advanced (stage IIb-IV) ovarian tumours (patients entered into the Third North-West Thames Ovarian Cancer Trial) was immunostained with PC10. PC10 immunostaining was performed successfully in 91.8% of cases. The PC10 labelling index (PC10 LI) ranged from 1.5% to 88% with a mean value of 47.4%. Stage I borderline tumours had significantly lower PCNA labelling indexes than stage I malignant tumours (P < 0.048). In advanced disease there was an inverse correlation between PC10 and overall survival, and in those patients who underwent good debulking surgery (37 patients with disease < 2 cm diameter) a low PC10 value (< 36.5%) correlated with improved survival (log-rank trend test for survival, chi 2 = 5.75, P = 0.017). PCNA immunostaining defines a good prognostic subgroup in adequately debulked patients with ovarian cancer. Images Figure 1 PMID:7841053

  8. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    PubMed Central

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; Robinson, Howard; Sallman, Zahur F.; Marino, John P.; Kelman, Zvi

    2016-01-01

    Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP. PMID:27141962

  9. Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein

    SciTech Connect

    Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio )

    1991-10-01

    A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

  10. Ribosome Protein L4 is essential for Epstein–Barr Virus Nuclear Antigen 1 function

    PubMed Central

    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

    2016-01-01

    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

  11. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    SciTech Connect

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; Robinson, Howard; Sallman, Zahur F.; Marino, John P.; Kelman, Zvi

    2016-05-03

    Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.

  12. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    DOE PAGES

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; ...

    2016-05-03

    Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain amore » canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

  13. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    SciTech Connect

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; Robinson, Howard; Sallman, Zahur F.; Marino, John P.; Kelman, Zvi

    2016-05-03

    Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.

  14. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    SciTech Connect

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; Robinson, Howard; Sallman, Zahur F.; Marino, John P.; Kelman, Zvi

    2016-05-03

    Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.

  15. RAD5a ubiquitin ligase is involved in ubiquitination of Arabidopsis thaliana proliferating cell nuclear antigen.

    PubMed

    Strzalka, Wojciech; Bartnicki, Filip; Pels, Katarzyna; Jakubowska, Agata; Tsurimoto, Toshiki; Tanaka, Katsunori

    2013-02-01

    The proliferating cell nuclear antigen (PCNA) is post-translationally modified by ubiquitin in yeast and mammalian cells. It is widely accepted that in yeast mono- and polyubiquitinated PCNA is involved in distinct pathways of DNA postreplication repair. This study showed an interaction between plant ubiquitin and PCNA in the plant cell. Using different approaches, it was demonstrated that Arabidopsis RAD5a ubiquitin ligase is involved in the post-translational modification of plant PCNA. A detailed analysis of the properties of selected Arabidopsis ubiquitin-conjugating enzymes (AtUBC) has shown that a plant homologue of yeast RAD6 (AtUBC2) is sufficient to monoubiquitinate AtPCNA in the absence of ubiquitin ligase. Using different combinations of selected AtUBC proteins together with AtRAD5a, it was demonstrated that plants have potential to use different pathways to ubiquitinate PCNA. The analysis of Arabidopsis PCNA1 and PCNA2 did not demonstrate substantial differences in the ubiquitination pattern between these two proteins. The major ubiquitination target of Arabidopsis PCNA, conserved in eukaryotes, is lysine 164. Taken together, the presented results clearly demonstrate the involvement of Arabidopsis UBC and RAD5a proteins in the ubiquitination of plant PCNA at lysine 164. The data show the complexity of the plant ubiquitination system and open new questions about its regulation in the plant cell.

  16. Asymmetric Arginine dimethylation of Epstein-Barr virus nuclear antigen 2 promotes DNA targeting

    SciTech Connect

    Gross, Henrik; Barth, Stephanie; Mamiani, Alfredo; Zimber-Strobl, Ursula; West, Michelle J.; Kremmer, Elisabeth; Graesser, Friedrich A.

    2010-02-20

    The Epstein-Barr virus (EBV) growth-transforms B-lymphocytes. The virus-encoded nuclear antigen 2 (EBNA2) is essential for transformation and activates gene expression by association with DNA-bound transcription factors such as RBPJkappa (CSL/CBF1). We have previously shown that EBNA2 contains symmetrically dimethylated Arginine (sDMA) residues. Deletion of the RG-repeat results in a reduced ability of the virus to immortalise B-cells. We now show that the RG repeat also contains asymmetrically dimethylated Arginines (aDMA) but neither non-methylated (NMA) Arginines nor citrulline residues. We demonstrate that only aDMA-containing EBNA2 is found in a complex with DNA-bound RBPJkappa in vitro and preferentially associates with the EBNA2-responsive EBV C, LMP1 and LMP2A promoters in vivo. Inhibition of methylation in EBV-infected cells results in reduced expression of the EBNA2-regulated viral gene LMP1, providing additional evidence that methylation is a prerequisite for DNA-binding by EBNA2 via association with the transcription factor RBPJkappa.

  17. Characterization of X-ray-induced immunostaining of proliferating cell nuclear antigen in human diploid fibroblasts

    SciTech Connect

    Miura, Masahiko; Sasaki, Takehito; Takasaki, Yoshinari

    1996-01-01

    The repair of X-ray-induced DNA damage related to the proliferating cell nuclear antigen (PCNA) was characterized in human diploid fibroblasts by an indirect immunofluorescence method. PCNA staining induced by X rays was lost after DNase I treatment but not after RNase treatment. The staining was not induced when ATP was depleted or the temperature was lowered to 0{degrees}C during the X irradiation. When cells were incubated at 37{degrees}C after X irradiation, PCNA staining diminished gradually and was almost entirely absent 12-15 h later. On the other hand, PCNA staining persisted during aphidicolin treatment even 20 h after X irradiation. Induction of PCNA staining was not affected by the aphidicolin treatment. Cycloheximide treatment did not affect induction of the staining either, but did inhibit the disappearance of the staining. There was no difference in the staining pattern and time course of PCNA staining after X irradiation between normal and xeroderma pigmentosum group A (XP-A) cells. These results imply that PCNA-dependent, aphidicolin-sensitive DNA polymerases may be involved in repair of X-ray-induced DNA damage in vivo, but the repair initiation step could be different from that of nucleotide excision repair initiated by XP proteins. 39 refs., 6 figs.

  18. Clinical Significance of Antinuclear and Antiextractable Nuclear Antigen Antibody in Childhood Immune Thrombocytopenia.

    PubMed

    Liu, Qihui; Xu, Hongzhen; Guan, Xianmin; Shen, Yali; Wen, Xianhao; Guo, Yuxia; Yu, Jie; Su, Yongchun

    2017-09-01

    This study aims to determine the clinical significance of positive antinuclear/antiextractable nuclear antigen (ANA/A-ENA) antibody on manifestation and therapeutic response of childhood immune thrombocytopenia (ITP). Overall, 1,330 patients aged between 1 and 15.6 years diagnosed with primary ITP were retrospectively analyzed, excluding those with secondary ITP. Bleeding manifestations were recorded. All patients underwent autoantibody testing and follow-up for 32 months on average (range: 23-54 months). Steroid response was also assessed. Response rates were compared between ANA/A-ENA-positive and ANA/A-ENA-negative patients. Of all the patients enrolled, 84 tested positive only for ANA, 102 tested positive for A-ENA, 54 tested positive for both ANA and A-ENA, and 1,090 tested negative for both. Patients who were ANA/A-ENA positive were more likely to be female and older than 10 years. Patients who were A-ENA positive were more likely to have either persistent or chronic disease and suffer from life-threatening bleeding as well as poor short-term therapeutic response. We conclude that autoantibody testing is important to determine the short-term prognosis of ITP patients. Females, patients older than 10 years of age, and patients with either mixed positivity or A-ENA positivity should be more closely monitored. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  19. Three proliferating cell nuclear antigen homologues from Metallosphaera sedula form a head-to-tail heterotrimer

    PubMed Central

    Iwata, Fumiya; Hirakawa, Hidehiko; Nagamune, Teruyuki

    2016-01-01

    Proliferating cell nuclear antigen (PCNA) is a sliding clamp that plays a key role in DNA metabolism. Genome sequence analysis has revealed that some crenarchaea possess three PCNA genes in their genome, but it has been reported that three PCNAs do not always form a unique heterotrimer composed of one of each molecule. The thermoacidophilic archaeon, Metallosphaera sedula, has three PCNA homologue genes. Here, we demonstrated that the three PCNA homologues, MsePCNA1, MsePCNA2 and MsePCNA3, exclusively form a heterotrimer in a stepwise fashion; MsePCNA1 and MsePCNA2 form a heterodimer, and then MsePCNA3 binds to the heterodimer. We determined that the dissociation constants between MsePCNA1 and MsePCNA2, and between MsePCNA3 and the MsePCNA1:MsePCNA2 heterodimer are 0.29 and 43 nM, respectively. Moreover, the MsePCNA1, MsePCNA2 and MsePCNA3 heterotrimer stimulated M. sedula DNA ligase 1 activity, suggesting that the heterotrimer works as a DNA sliding clamp in the organism. The stable and stepwise heterotrimerization of M. sedula PCNA homologues would be useful to generate functional protein-based materials such as artificial multi-enzyme complexes, functional hydrogels and protein fibres, which have recently been achieved by protein self-assembly. PMID:27228945

  20. Validating the disruption of proliferating cell nuclear antigen interactions in the development of targeted cancer therapeutics.

    PubMed

    Smith, Shanna J; Hickey, Robert J; Malkas, Linda H

    2016-01-01

    Human DNA replication and repair is a highly coordinated process involving the specifically timed actions of numerous proteins and enzymes. Many of these proteins require interaction with proliferating cell nuclear antigen (PCNA) for activation within the process. The interdomain connector loop (IDCL) of PCNA provides a docking site for many of those proteins, suggesting that this region is critically important in the regulation of cellular function. Previous work in this laboratory has demonstrated that a peptide mimicking a specific region of the IDCL (caPeptide) has the ability to disrupt key protein-protein interactions between PCNA and its binding partners, thereby inhibiting DNA replication within the cells. In this study, we confirm the ability of the caPeptide to disrupt DNA replication function using both intact cell and in vitro DNA replication assays. Further, we were able to demonstrate that treatment with caPeptide results in a decrease of polymerase δ activity that correlates with the observed decrease in DNA replication. We have also successfully developed a surface plasmon resonance (SPR) assay to validate the disruption of the PCNA-pol δ interaction with caPeptide.

  1. The presence of antibodies against extractable nuclear antigens in serum: a comparison of immunoblotting versus radial immunodiffusion.

    PubMed

    Uyttenbroeck, W; Cooreman, W; Scharpe, S

    1993-01-01

    A commercial immunoblotting kit has recently been introduced to determine auto-antibodies against extractable nuclear antigens. We compared this new test with radial immunodiffusion for its usefulness in the routine laboratory procedures of a general hospital. Antigen preparation in immunoblotting includes a protein denaturation step prior to electrophoretic separation of the different proteins. In this way antigenic determinants that depend heavily on the protein superstructure are lost. In theory, auto-antibodies against these epitopes may be missed. In our series of 100 samples that had tested positively for antinuclear antibodies, radial immunodiffusion was able to detect one SSA positive sample that was negative by immunoblotting. However, 47 samples positive in immunoblotting, 37 positive for UBP, seven for anti-SSA, are for anti-Jo-1, one for anti-RNP and one for anti-Sm were missed by radial immunodiffusion. Most of these samples had low antinuclear antibody titres (1/80 or 1/160).

  2. Tight coevolution of proliferating cell nuclear antigen (PCNA)-partner interaction networks in fungi leads to interspecies network incompatibility.

    PubMed

    Zamir, Lyad; Zaretsky, Marianna; Fridman, Yearit; Ner-Gaon, Hadas; Rubin, Eitan; Aharoni, Amir

    2012-02-14

    The structure and connectivity of protein-protein interaction (PPI) networks are maintained throughout evolution by coordinated changes (coevolution) of network proteins. Despite extensive research, relatively little is known regarding the molecular basis and functional implications of the coevolution of PPI networks. Here, we used proliferating cell nuclear antigen, a hub protein that mediates DNA replication and repair in eukaryotes, as a model system to study the coevolution of PPI networks in fungi. Using a combined bioinformatics and experimental approach, we discovered that PCNA-partner interactions tightly coevolved in fungal species, leading to specific modes of recognition. We found that fungal proliferating cell nuclear antigen-partner interaction networks diverged into two distinct groups as a result of such coevolution and that hybrid networks of these groups are functionally noncompatible in Saccharomyces cerevisiae. Our results indicate that the coevolution of PPI networks can form functional barriers between fungal species, and thus can promote and fix speciation.

  3. The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen.

    PubMed Central

    Rihs, H P; Jans, D A; Fan, H; Peters, R

    1991-01-01

    We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport. Images PMID:1848177

  4. Efficiency of different strategies to detect autoantibodies to extractable nuclear antigens.

    PubMed

    Almeida González, Delia; Cabrera de León, Antonio; Rodríguez Pérez, María Del Cristo; Brito Díaz, Buenaventura; González Hernández, Ana; García García, Diego; Vázquez Moncholi, Carmen; Aguirre Jaime, Armando

    2010-08-31

    Autoantibodies to extractable nuclear antigens (anti-ENA) are identified mainly in samples positive for antinuclear antibodies (ANA). Although the method of choice for ANA screening is indirect immunofluorescence (IIF), several techniques are available to detect anti-ENA. The aim of this study was to compare the efficiency of five different strategies to determine anti-ENA. During a 2-year period we screened ANA in 30375 samples with IIF, and the 4475 samples ANA positive were tested for anti-ENA by double immune diffusion screening or fluoroenzymeimmunoassay (Screening FI); anti-ENA specificities were then determined by line immunoassay (LIA) or fluoroenzymeimmunoassay (FI). We compared five strategies that involved FI or LIA identification of anti-ENA with or without prior screening, or an algorithm that combined fluorescence pattern, number of anti-ENA specificities requested by the clinician and ANA dilution titer. One cost unit (CU) was defined as the cost of 1 test of ANA determination. We detected 553 anti-ENA positive samples. The most efficient strategy was the algorithm, at a cost of 3.3 CU per sample processed, the second most efficient strategy was screening plus FI identification (cost=3.8 CU), and the third most efficient strategy was screening plus LIA identification (cost=3.9 CU). The fourth most efficient strategy was FI identification without prior screening (13.3 CU per sample) and the least efficient was LIA identification without prior screening (13.6 CU per sample). In conclusion, an algorithm that combined techniques for detection, ANA titer, fluorescence pattern and number of specificities requested was the most efficient strategy for determining anti-ENA. 2010 Elsevier B.V. All rights reserved.

  5. Structure of a mutant form of proliferating cell nuclear antigen that blocks translesion DNA synthesis †

    PubMed Central

    Freudenthal, Bret D.; Ramaswamy, S.; Hingorani, Manju M.; Washington, M. Todd

    2009-01-01

    Proliferating cell nuclear antigen (PCNA) is a homotrimeric protein that functions as a sliding clamp during DNA replication. Several mutant forms of PCNA that block translesion DNA synthesis have been identified in genetic studies in yeast. One such mutant protein (encoded by the rev6-1 allele) is a glycine to serine substitution at residue 178, located at the subunit interface of PCNA. To better understand how this substitution interferes with translesion synthesis, we have determined the X-ray crystal structure of the G178S PCNA mutant protein. This substitution has little effect on the structure of the domain in which the substitution occurs. Instead, significant, local structural changes are observed in the adjacent subunit. The most notable difference between mutant and wild-type structures is in a single, extended loop (comprising amino acid residues 105-110), which we call loop J. In the mutant protein structure, loop J adopts a very different conformation in which the atoms of the protein backbone have moved by as much as 6.5 Å from their positions in the wild-type structure. To better understand the functional consequences of this structural change, we have examined the ability of this mutant protein to stimulate nucleotide incorporation by DNA polymerase eta (pol η). Steady state kinetic studies show that while wild-type PCNA stimulates incorporation by pol η opposite an abasic site, the mutant PCNA protein actually inhibits incorporation opposite this DNA lesion. These results show that the position of loop J in PCNA plays an essential role in facilitating translesion synthesis. PMID:19053247

  6. Isolation and characterization of proliferating cell nuclear antigen from the dinoflagellate Pfiesteria piscicida.

    PubMed

    Zhang, Huan; Hou, Yubo; Lin, Senjie

    2006-01-01

    Proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerases delta and epsilon, is essential for DNA replication and repair. Understanding the structure and expression characteristics of this gene in dinoflagellates would enable us to gain insights into how the cell cycle in these enigmatic eukaryotes is regulated and whether this gene can be a growth marker of these ecologically important organisms. We analyzed pcna and its encoded protein from Pfiesteria piscicida (Ppi_PCNA). Using reverse transcription-polymerase chain reaction (RT-PCR) and RNA ligase mediated-rapid amplification of cDNA ends (RLM-RACE) methods, Ppi_pcna cDNA was isolated; it contained a coding region for 258 amino acid residues (aa) preceded by various 5'- and 3'-untranslated ends. The deduced protein length was similar to that of typical vertebrate and plant PCNA. PCR using genomic DNA as the template yielded multiple products whose sequences revealed multiple copies of pcna in tandem repeats separated by an unknown sequence. Using real-time PCR, we estimated 41+/-7 copies of this gene in each P. piscicida cell. Reverse transcription real-time PCR indicated a similar pcna mRNA level between the exponential and the stationary growth phases. Western blot analysis revealed a slightly higher PCNA level (<2-fold) in the exponential than in the stationary growth phases. We conclude that (1) P. piscicida possesses a typical eukaryote PCNA; (2) unlike in other eukaryotes, pcna in P. piscicida occurs in multiple copies arranged in tandem; and (3) regulation of P. piscicida PCNA probably lies in post-translational modification.

  7. Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation.

    PubMed

    Strzalka, Wojciech; Ziemienowicz, Alicja

    2011-05-01

    PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome. This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution. Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.

  8. Epstein–Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines

    PubMed Central

    Zhao, Bo; Mar, Jessica C.; Maruo, Seiji; Lee, Sungwook; Gewurz, Benjamin E.; Johannsen, Eric; Holton, Kristina; Rubio, Renee; Takada, Kenzo; Quackenbush, John; Kieff, Elliott

    2011-01-01

    EBV nuclear antigen 3C (EBNA3C) is an essential transcription factor for EBV transformed lymphoblast cell line (LCL) growth. To identify EBNA3C-regulated genes in LCLs, microarrays were used to measure RNA abundances in each of three different LCLs that conditionally express EBNA3C fused to a 4-OH-Tamoxifen–dependent estrogen receptor hormone binding domain (EBNA3CHT). At least three RNAs were assayed for each EBNA3CHT LCL under nonpermissive conditions, permissive conditions, and nonpermissive conditions with wild-type EBNA3C transcomplementation. Using a two-way ANOVA model of EBNA3C levels, we identified 550 regulated genes that were at least 1.5-fold up- or down-regulated with false discovery rates < 0.01. EBNA3C-regulated genes overlapped significantly with genes regulated by EBNA2 and EBNA3A consistent with coordinated effects on cell gene transcription. Of the 550 EBNA3C-regulated genes, 106 could be placed in protein networks. A seeded Bayesian network analysis of the 80 most significant EBNA3C-regulated genes suggests that RAC1, LYN, and TNF are upstream of other EBNA3C-regulated genes. Gene set enrichment analysis found enrichment for MAP kinase signaling, cytokine–cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecules, implicating these pathways in EBNA3C effects on LCL growth or survival. EBNA3C significantly up-regulated the CXCL12 ligand and its CXCR4 receptor and increased LCL migration. CXCL12 up-regulation depended on EBNA3C's interaction with the cell transcription factor, RBPJ, which is essential for LCL growth. EBNA3C also up-regulated MYC 1.3-fold and down-regulated CDKN2A exons 2 and 3, shared by p16 and p14, 1.4-fold, with false discovery rates < 5 × 10−4. PMID:21173222

  9. Nuclear accumulation of epidermal growth factor receptor and acceleration of G1/S stage by Epstein-Barr-encoded oncoprotein latent membrane protein 1

    SciTech Connect

    Tao Yongguang; Song Xing; Deng Xiyun; Xie Daxin; Lee, Leo M.; Liu Yiping; Li Wei; Li Lili; Deng Lin; Wu Qiao; Gong Jianping; Cao Ya . E-mail: ycao98@public.cs.hn.cn

    2005-02-15

    Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV-encoded proteins and has always been the core of the oncogenic mechanism of EBV. Advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly improved our knowledge of the biological function of cell surface receptors. In this study, we used the Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1-integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 which could be regulated by the Tet system. We found that LMP1 could regulate the nuclear accumulation of EGFR in a dose-dependent manner quantitatively and qualitatively. We also demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation and EGFR in the nucleus could bind to the promoters of cyclinD1 and cyclinE, respectively. We further demonstrated that EGFR is involved in the acceleration of the G1/S phase transition by LMP1 through binding to cyclinD1 and cyclinE directly. These findings provided a novel view that the acceleration of LMP1 on the G1/S transition via the nuclear accumulation of EGFR was critical in the process of nasopharyngeal carcinoma.

  10. Epstein-Barr virus nuclear antigen 3C targets p53 and modulates its transcriptional and apoptotic activities

    SciTech Connect

    Yi Fuming; Saha, Abhik; Murakami, Masanao; Kumar, Pankaj; Knight, Jason S.; Cai Qiliang; Choudhuri, Tathagata; Robertson, Erle S.

    2009-06-05

    The p53 tumor suppressor gene is one of the most commonly mutated genes in human cancers and the corresponding encoded protein induces apoptosis or cell-cycle arrest at the G1/S checkpoint in response to DNA damage. To date, previous studies have shown that antigens encoded by human tumor viruses such as SV40 large T antigen, adenovirus E1A and HPV E6 interact with p53 and disrupt its functional activity. In a similar fashion, we now show that EBNA3C, one of the EBV latent antigens essential for the B-cell immortalization in vitro, interacts directly with p53. Additionally, we mapped the interaction of EBNA3C with p53 to the C-terminal DNA-binding and the tetramerization domain of p53, and the region of EBNA3C responsible for binding to p53 was mapped to the N-terminal domain of EBNA3C (residues 130-190), previously shown to interact with a number of important cell-cycle components, specifically SCF{sup Skp2}, cyclin A, and cMyc. Furthermore, we demonstrate that EBNA3C substantially represses the transcriptional activity of p53 in luciferase based reporter assays, and rescues apoptosis induced by ectopic p53 expression in SAOS-2 (p53{sup -/-}) cells. Interestingly, we also show that the DNA-binding ability of p53 is diminished in the presence of EBNA3C. Thus, the interaction between the p53 and EBNA3C provides new insights into the mechanism(s) by which the EBNA3C oncoprotein can alter cellular gene expression in EBV associated human cancers.

  11. Structure and binding studies of proliferating cell nuclear antigen from Leishmania donovani.

    PubMed

    Yadav, Satya Prakash; Singh, Prashant Kumar; Sharma, Pradeep; Iqbal, Naseer; Kaur, Punit; Sharma, Sujata; Singh, Tej P

    2017-11-01

    Proliferating cell nuclear antigen (PCNA) acts as a sliding clamp to support DNA replication and repair. The structure of PCNA from Leishmania donovani (LdPCNA) has been determined at 2.73Å resolution. Structure consists of six crystallographically independent molecules which form two trimeric rings. The pore diameter of the individual trimeric ring is of the order of 37Å. The two rings are stacked through their front to front faces. In order to gain a stable packing, the rings are rotated by 42° about the pore axis and shifted by 7Å and tilted by 16° along the perpendicular direction to pore axis. This form of stacking reduced the effective diameter of the pore to 32Å. The sequence of LdPCNA consists of a long segment of 41 amino acid residues (186-Gly-Val-Ser-Asp-Arg-Ser-Thr-Lys-Ser-Glu-Val-Lys-Ala-Glu-Val-Lys-Ala-Glu-Ala-Arg-Asp-Asp-Asp-Glu-Glu-Pro-Leu-Ser-Arg-Lys-Tyr-Gly-Lys-Ala-Asp-Ser-Ser-Ala-Asn-Ala-Ile-226) whereas the corresponding segments in other PCNAs contain only eight residues corresponding to 186-Gly-Val-Ser-Asp-Arg------224-Asn-Ala-Ile-226. The enhanced length of this segment in LdPCNA may influence its mode of interaction with DNA and other proteins. The dissociation constants obtained using real time binding studies with surface plasmon resonance (SPR) for two peptides, Lys-Arg-Arg-Gln-Thr-Ser-Met-Thr-Asp-Phe-Tyr-His (P1) from human cyclin-dependent kinase inhibitor-1(CKI-1) and Lys-Thr-Gln-Gly-Arg-Leu-Asp-Ser-Phe-Phe-Thr-Val (P2) from flap endonuclease 1 (Fen-1) as well as with two small molecule inhibitors, (S)-4-(4-(2-amino-3-hydroxypropyl)-2, 6-diiodophenoxy) phenol hydrochloride (ADPH) and N-(3-methylthiophene-2-carboxylicacid)-N'-((3-hydroxy-2-naphthalenyl) methylene) hydrazide (MCMH) are 0.29±0.09μM, 0.37±0.08μM, 0.35±0.09μM and 1.20±0.08μM respectively. The corresponding values obtained using fluorescence spectroscopic methods were 0.22±0.06μM, 0.68±0.07μM, 0.44±0.07μM and 0.75±0.05μM respectively. Copyright © 2017

  12. Enzyme-linked immunosorbent assay of antibodies to Epstein-Barr virus nuclear and early antigens in patients with infectious mononucleosis and nasopharyngeal carcinoma.

    PubMed

    Halprin, J; Scott, A L; Jacobson, L; Levine, P H; Ho, J H; Niederman, J C; Hayward, S D; Milman, G

    1986-03-01

    A sensitive enzyme-linked immunosorbent assay was used to measure titers of IgG antibodies against bacterially synthesized Epstein-Barr virus nuclear antigen and early antigen in sera from 100 healthy North Americans, 40 North American patients with infectious mononucleosis, and 48 Asian patients with nasopharyngeal carcinoma. All healthy persons previously infected with Epstein-Barr virus had antibodies to nuclear antigen, and 70% had very low but detectable antibody titers to early antigen. In contrast, patients with mononucleosis had nondetectable or very low levels of antibodies to nuclear antigen and high antibody levels to early antigen. High levels of antibody to early antigen also were seen in patients with nasopharyngeal carcinoma, and a decrease in this response during the first 12 months after diagnosis and treatment was a significant prognostic indicator of survival. The probability of survival was 75% for patients whose antibody concentration to early antigen remained constant or decreased, and near 0% for patients with increasing levels of antibody.

  13. Detection of anti-extractable nuclear antigens in connective tissue diseases: comparison between passive hemagglutination, counterimmunoelectrophoresis and double immunodiffusion.

    PubMed

    Siracusano, A; Agelli, M; Ioppolo, S; Quintieri, F; Bombardieri, S

    1985-01-01

    Antibodies to the three major components of the complex called soluble extractable nuclear antigen (ENA) were detected by passive hemagglutination (HA), counterimmunoelectrophoresis (CIE) and double immunodiffusion (DI) in 256 patients with connective tissue diseases. Anti-ENA antibodies were demonstrated by all the three employed methods in only 44.9% of the cases. These methods were not able to detect all antibodies to these antigens or any single specificity; CIE was however the most sensitive method for anti-RNP and HA for anti-Sm antibodies, while DI was the most suitable technique for serum samples with multiple anti-ENA specificities. Only in less than 50% of the cases the specificity detected by HA was comparable with that given by CIE or DI. Hence, for detecting anti-ENA antibodies a combination of these methods should be maintained, at least until more precise and reliable methods will become available.

  14. Accumulation of Heterochromatin Components on the Terminal Repeat Sequence of Kaposi's Sarcoma-Associated Herpesvirus Mediated by the Latency-Associated Nuclear Antigen

    PubMed Central

    Sakakibara, Shuhei; Ueda, Keiji; Nishimura, Ken; Do, Eunju; Ohsaki, Eriko; Okuno, Toshiomi; Yamanishi, Koichi

    2004-01-01

    In the latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), its 160-kb circularized episomal DNA is replicated and maintained in the host nucleus. KSHV latency-associated nuclear antigen (LANA) is a key factor for maintaining viral latency. LANA binds to the terminal repeat (TR) DNA of the viral genome, leading to its localization to specific dot structures in the nucleus. In such an infected cell, the expression of the viral genes is restricted by a mechanism that is still unclear. Here, we found that LANA interacts with SUV39H1 histone methyltransferase, a key component of heterochromatin formation, as determined by use of a DNA pull-down assay with a biotinylated DNA fragment that contained a LANA-specific binding sequence and a maltose-binding protein pull-down assay. The diffuse localization of LANA on the chromosomes of uninfected cells changed to a punctate one with the introduction of a bacterial artificial chromosome containing most of the TR region, and SUV39H1 clearly colocalized with the LANA-associated dots. Thus, the LANA foci in KSHV-infected cells seemed to include SUV39H1 as well as heterochromatin protein 1. Furthermore, a chromatin immunoprecipitation assay revealed that the TR and the open reading frame (ORF) K1 and ORF50/RTA genes, but not the ORF73/LANA gene, lay within the heterochromatin during KSHV latency. Taken together, these observations indicate that LANA recruits heterochromatin components to the viral genome, which may lead to the establishment of viral latency and govern the transcription program. PMID:15220403

  15. Berberine inhibits the proliferation of human nasopharyngeal carcinoma cells via an Epstein-Barr virus nuclear antigen 1-dependent mechanism.

    PubMed

    Wang, Chao; Wang, Huan; Zhang, Yaqian; Guo, Wei; Long, Cong; Wang, Jingchao; Liu, Limei; Sun, Xiaoping

    2017-04-01

    Nasopharyngeal carcinoma (NPC) is a malignancy derived from the epithelial cells of the nasopharynx cavity, and is closely associated with Epstein-Barr virus (EBV) infection. In addition to NPC, EBV causes various human malignancies, such as gastric cancer, hematological tumors and lymphoepithelioma-like carcinomas. Epstein-Barr nuclear antigen 1 (EBNA1) encoded by EBV is indispensable for replication, partition, transcription and maintenance of viral genomes. Berberine, a naturally occurring isoquinoline alkaloid, shows anti-inflammatory, anticholinergic, antioxidative, and anticancer activities. In the present study, the antitumor effect of berberine was studied. Cell Counting Kit-8 (CCK-8) assays were performed to demonstrate whether the proliferation of EBV-positive NPC cells was inhibited by berberine. Flow cytometric results revealed that berberine induced cell cycle arrest and apoptosis. Quantitative-PCR and western blotting results indicated that berberine decreased the expression of EBNA1 at both the mRNA and protein levels in the EBV-positive NPC cells. The function of EBNA1 promoter Qp which is to drive EBNA1 transcription in type Ⅱ latent infection was strongly suppressed by berberine. Overexpression of EBNA1 attenuated this inhibitory effect. Berberine also suppressed the activity of signal transducer and activator of transcription 3 which is a new therapeutic target in a series of malignancies, including NPC. Viral titer experiments demonstrated that berberine decreased the production of virions in HONE1 and HK1-EBV cells. In a mouse xenograft model of NPC induced by HONE1 cells, berberine significantly inhibited tumor formation. Altogether, these results indicate that berberine decreases the expression of EBNA1 and exhibits an antitumor effect against NPC both in vitro and in vivo.

  16. Immunoexpression of Ki67, proliferative cell nuclear antigen, and Bcl-2 proteins in a case of ameloblastic fibrosarcoma.

    PubMed

    Pontes, Hélder Antônio Rebelo; Pontes, Flávia Sirotheau Corrêa; Silva, Brunno Santos de Freitas; Cury, Sérgio Elias Vieira; Fonseca, Felipe Paiva; Salim, Rodrigo Alves; Pinto Júnior, Décio dos Santos

    2010-12-01

    Ameloblastic fibrosarcoma (AFS), regarded as the malignant counterpart of the benign ameloblastic fibroma, is an extremely rare odontogenic neoplasm with only 68 cases reported in the English literature up to 2009. It is composed of a benign odontogenic epithelium, resembling that of ameloblastoma, and a malignant mesenchymal part exhibiting features of fibrosarcoma. Due to the rarity of the lesion, little is known about its molecular pathogenesis; therefore, in the current study, we sought to evaluate the immunoexpression of Ki67, proliferative cell nuclear antigen, and Bcl-2 proteins in AFS, comparing the results obtained with its benign counterpart, as well as to report a new case of this rare entity affecting a 19-year-old female patient. The results obtained revealed that all the proteins evaluated were overexpressed in the malignant mesenchymal portion of AFS if compared with ameloblastic fibroma, suggesting that nuclear proliferative factors such as Ki67 and proliferative cell nuclear antigen, in association to histopathologic features, may be useful markers for identifying the malignancy and that, despite the lack of molecular analysis in the case reported, Bcl-2 alteration may play a role in AFS pathogenesis. Copyright © 2010 Elsevier Inc. All rights reserved.

  17. Identification of MEF2B, EBF1, and IL6R as Direct Gene Targets of Epstein-Barr Virus (EBV) Nuclear Antigen 1 Critical for EBV-Infected B-Lymphocyte Survival

    PubMed Central

    Tempera, Italo; De Leo, Alessandra; Kossenkov, Andrew V.; Cesaroni, Matteo; Song, Hui; Dawany, Noor; Showe, Louise; Lu, Fang; Wikramasinghe, Priyankara

    2015-01-01

    ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the EBV-encoded nuclear antigen and sequence-specific DNA binding protein required for viral origin binding and episome maintenance during latency. EBNA1 can also bind to numerous sites in the cellular genome and can provide a host cell survival function, but it is not yet known how EBNA1 sequence-specific binding is responsible for host cell survival. Here, we integrate EBNA1 chromatin immunoprecipitation sequencing (ChIP-Seq) with transcriptome sequencing (RNA-Seq) after EBNA1 depletion to identify cellular genes directly regulated by EBNA1 that are also essential for B-cell survival. We first compared EBNA1 ChIP-Seq patterns in four different EBV-positive cell types, including Burkitt lymphoma (BL) cells, nasopharyngeal carcinoma (NPC) cells, and lymphoblastoid cell lines (LCLs). EBNA1 binds to ∼1,000 sites that are mostly invariant among cell types and share a consensus recognition motif. We found that a large subset of EBNA1 binding sites are located proximal to transcription start sites and correlate genome-wide with transcription activity. EBNA1 bound to genes of high significance for B-cell growth and function, including MEF2B, IL6R, and EBF1. EBNA1 depletion from latently infected LCLs results in the loss of cell proliferation and the loss of gene expression for some EBNA1-bound genes, including MEF2B, EBF1, and IL6R. Depletion of MEF2B, EBF1, or IL6R partially phenocopies EBNA1 depletion by decreasing the cell growth and viability of cells latently infected with EBV. These findings suggest that EBNA1 binds to a large cohort of cellular genes important for cell viability and implicates EBNA1 as a critical regulator of transcription of host cell genes important for enhanced survival of latently infected cells. IMPORTANCE Epstein-Barr virus (EBV) latent infection is responsible for a variety of lymphoid and epithelial cell malignancies. EBNA1 is the EBV-encoded nuclear antigen that is

  18. Evolution of the levels of human leukocyte antigen G (HLA-G) in Beninese infant during the first year of life in a malaria endemic area: using latent class analysis.

    PubMed

    d'Almeida, Tania C; Sadissou, Ibrahim; Cottrell, Gilles; Tahar, Rachida; Moreau, Philippe; Favier, Benoit; Moutairou, Kabirou; Donadi, Eduardo A; Massougbodji, Achille; Rouass-Freiss, Nathalie; Courtin, David; Garcia, André

    2016-02-09

    HLA-G, a non-classical HLA class I antigen, is of crucial interest during pregnancy by inhibiting maternal immune response. Its role during infections is discussed, and it has been described that high levels of soluble HLA-G during childhood increase the risk of malaria. To explore more precisely interactions between soluble HLA-G and malaria, latent class analysis was used to test whether distinct sub-populations of children, each with distinctive soluble HLA-G evolutions may suggest the existence of groups presenting variable malaria susceptibility. A study was conducted in Benin from 2010 to 2013 and 165 children were followed from birth to 12 months. Evolution of soluble HLA-G was studied by the latent class method. Three groups of children were identified: one with consistently low levels of soluble HLA-G during follow-up, a second with very high levels and a last intermediate group. In all groups, low birth weight, high number of malaria infections and high exposure to malaria transmission were associated with high level of soluble HLA-G. Placental malaria was not. Presence of soluble HLA-G in cord blood increased the probability of belonging to the highest trajectory. These results, together with previous ones, confirm the important role of HLA-G in the individual susceptibility to malaria. Assaying soluble HLA-G at birth could be a good indicator of newborns more fragile and at risk of infections during childhood.

  19. Complementary serum test of antibodies to Epstein-Barr virus nuclear antigen-1 and early antigen: a possible alternative for primary screening of nasopharyngeal carcinoma.

    PubMed

    Chang, Kai-Ping; Hsu, Cheng-Lung; Chang, Yu-Liang; Tsang, Ngan-Ming; Chen, Chin-Kuo; Lee, Ta-Jen; Tsao, Kuo-Chien; Huang, Chung-Guei; Chang, Yu-Sun; Yu, Jau-Song; Hao, Sheng-Po

    2008-08-01

    This hospital-based cohort study evaluated the efficacy of three Epstein-Barr virus (EBV) - associated assays for nasopharyngeal carcinoma (NPC) primary screening and monitoring treatment outcome. Five hundred and seventeen consecutive subjects, including 156 NPC patients, 264 healthy volunteers and 97 patients with head and neck squamous cell carcinoma (HNSCC) were enrolled. The sensitivity and specificity of EBV IgAs to viral capsid antigen (VCA), complementary EBV IgAs to early antigen and nuclear antigen-1 (EA+EBNA-1), and EBV DNA load were examined by immunofluorescent assays, enzyme-linked immunosorbent assays, and quantitative real-time PCR, respectively. After constructing the receiver operating characteristics to demonstrate screening efficacy, EBV EA+EBNA-1 IgA (AUC: 0.952; 95% CI, 0.930-0.974) was proved superior to EBV VCA IgA (AUC: 0.888; 95% CI, 0.854-0.922) or EBV DNA load (AUC: 0.893; 95% CI, 0.854-0.932) in differentiating NPC patients from controls. Comparison of screening efficacy between NPC patients and HNSCC patients revealed EBV EA+EBNA-1 IgA (AUC: 0.964; 95% CI, 0.943-0.985) still outperformed EBV VCA IgA (AUC: 0.884; 95% CI, 0.845-0.923). In subjects with higher serum titer or level equal to or above 1:80 and 6 EU/ml for EBV VCA IgA and EA+EBNA-1 IgA, the specificity reached as high as 99.2% and 95.1%, respectively, in the control groups. However, correlation of these three assays with clinicopathological manifestations of NPC, revealed only EBV DNA load significantly associated with N stage and overall stage in NPC patients. Additionally, EBV DNA load could be used to further raise the specificity of EBV EA+EBNA-1 IgA assays and was also the only assay to be consistently predictive of tumor relapse in post-treatment patients according to serial test results by time frame. Consequently, an EBV EA+EBNA-1 IgA-based protocol is recommended for mass screening, but EBV DNA load should be used solely for post-treatment monitoring for NPC in

  20. Expression of alpha V integrin is modulated by Epstein-Barr virus nuclear antigen 3C and the metastasis suppressor Nm23-H1 through interaction with the GATA-1 and Sp1 transcription factors

    SciTech Connect

    Choudhuri, Tathagata; Verma, Subhash C.; Lan, Ke; Robertson, Erle S. . E-mail: erle@mail.med.upenn.edu

    2006-07-20

    Epstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. A subset of latent EBV antigens is required for immortalization of primary B-lymphocytes. The metastatic suppressor Nm23-H1 which is downregulated in human invasive breast carcinoma reduces the migration and metastatic activity of breast carcinoma cells when expressed from a heterologous promoter. Interestingly, the EBV nuclear antigen 3C (EBNA3C) reverses these activities of Nm23-H1. The alpha V integrins recognize a variety of ligands for signaling and are involved in cell migration and proliferation and also serve as major receptors for extracellular-matrix-mediated cell adhesion and migration. The goal of this study was to determine if Nm23-H1 and EBNA3C can modulate alpha V integrin expression and downstream activities. The results of our studies indicate that Nm23-H1 downregulates alpha V intregrin expression in a dose responsive manner. In contrast, EBNA3C can upregulate alpha V integrin expression. Furthermore, the study showed that the association of the Sp1 and GATA transcription factors with Nm23-H1 is required for modulation of the alpha V integrin activity. Thus, these results suggest a direct correlation between the alpha V integrin expression and the interaction of Nm23-H1 with EBNA3C.

  1. Interference of fisetin with targets of the nuclear factor-κB signal transduction pathway activated by Epstein-Barr virus encoded latent membrane protein 1.

    PubMed

    Li, Rong; Liang, Hong-Ying; Li, Ming-Yong; Lin, Chun-Yan; Shi, Meng-Jie; Zhang, Xiu-Juan

    2014-01-01

    Fisetin is an effective compound extracted from lacquer which has been used in the treatment of various diseases. Preliminary data indicate that it also exerts specific anti-cancer effects. However, the manner in which fisetin regulates cancer growth remains unknown. In this study, we elucidated interference of fisetin with targets of the nuclear factorκB signal transduction pathway activated by Epstein-Barr virus encoding latent membrane protein 1 (LMP1)in nasopharyngeal carcinoma (NPC) cells, Results showed that fisetin inhibited the survival rate of CNE-LMP1 cells and NF-κB activation caused by LMP1. Fisetin also suppressed nuclear translocation of NF-κB (p65) and IκBα phosphorylation, while inhibiting CyclinD1, all key targets of the NF-κB signal transduction pathway. It was suggested that interference effects of fisetin with signal transduction activated by LMP1 encoded by the Epstein-Barr virus may play an important role in its anticancer potential.

  2. Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-alpha.

    PubMed Central

    Fontes, Marcos R M; Teh, Trazel; Toth, Gabor; John, Anna; Pavo, Imre; Jans, David A; Kobe, Bostjan

    2003-01-01

    The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser112 in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha. PMID:12852786

  3. Differentiation between antibodies to protamines and somatic nuclear antigens by means of a comparative fluorescence study on swollen nuclei of spermatozoa and somatic cells.

    PubMed

    Samuel, T

    1978-05-01

    The indirect immunofluorescence test on swollen nuclei of rat thymocytes, chicken red blood cells and human and salmon spermatozoa was found to be an easy and satisfactory method for the discrimination between antibodies to sperm-specific nuclear antigens and somatic nuclear antigens. This study shows that nuclear antibodies present in the sera of vasectomized men and in rabbit antisera to human protamines are directed against the human sperm-specific nuclear antigens (protamines), and that they may cross-react with salmon protamine. These sera do not react with somatic nuclear antigens. This comparative fluorescence study and a complement fixation study, performed with sera from diabetic patients, proved that the administration of insulin retard (protamine-zinc-insulin) may lead to the formation of antibodies to the fish protamine. These antibodies may reveal a weak cross reaction with human protamines. The results obtained in this study also prove that the nuclei of chicken red blood cells and human sperm do not contain, or contain very small amounts of, histone fraction H1, and that salmon sperm nuclei do not contain any of the histone fractions, and suggest that the nuclei of mature human spermatozoa contain smaller amounts of histones in comparison to somatic cell nuclei.

  4. Proteomic Profiling of EBNA1-Host Protein Interactions in Latent and Lytic Epstein-Barr Virus Infections

    PubMed Central

    Malik-Soni, Natasha

    2012-01-01

    The Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV) is expressed in both latent and lytic modes of EBV infection and contributes to EBV-associated cancers. Using a proteomics approach, we profiled EBNA1-host protein interactions in nasopharyngeal and gastric carcinoma cells in the context of latent and lytic EBV infection. We identified several interactions that occur in both modes of infection, including a previously unreported interaction with nucleophosmin and RNA-mediated interactions with several heterogeneous ribonucleoproteins (hnRNPs) and La protein. PMID:22496234

  5. Proteomic profiling of EBNA1-host protein interactions in latent and lytic Epstein-Barr virus infections.

    PubMed

    Malik-Soni, Natasha; Frappier, Lori

    2012-06-01

    The Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV) is expressed in both latent and lytic modes of EBV infection and contributes to EBV-associated cancers. Using a proteomics approach, we profiled EBNA1-host protein interactions in nasopharyngeal and gastric carcinoma cells in the context of latent and lytic EBV infection. We identified several interactions that occur in both modes of infection, including a previously unreported interaction with nucleophosmin and RNA-mediated interactions with several heterogeneous ribonucleoproteins (hnRNPs) and La protein.

  6. Tumor microenvironment contributes to Epstein-Barr virus anti-nuclear antigen-1 antibody production in nasopharyngeal carcinoma.

    PubMed

    Ai, Ping; Li, Zhiping; Jiang, Yong; Song, Changping; Zhang, Lin; Hu, Huaizhong; Wang, Tao

    2017-08-01

    Nuclear antigen-1 (NA1) protein of Epstein-Barr virus (EBV) is expressed in EBV-infected cells in the microenvironment of cancer. Since immune cells infiltrate abundantly in nasopharyngeal carcinoma (NPC) tumor tissues, we hypothesized that the local tumor microenvironment may perform an important role in the production of antibodies directed at NA1. Furthermore, we hypothesized that anti-NA1 antibody originating in the local microenvironment could be secreted into the saliva of patients with NPC. In the present study, 20 healthy controls and 39 patients with NPC treated with intensity-modulated radiation therapy were recruited for the study. Saliva and serum samples were collected from the NPC patients, and nasopharyngeal tissue samples from the patients with NPC. The titers of anti-NA1 antibody [immunoglobulin A (IgA)] were determined by ELISA. Expression of NA1, human leukocyte antigen-antigen D related (HLA-DR), cluster of differentiation (CD)80, CD86, CD3, CD4, CD19 and IgA was detected by immunohistochemical staining on paraffin-embedded nasopharyngeal tissue sections. Anti-NA1 antibodies were detected in the serum and saliva samples of the patients with NPC. In infiltrating cells, expression of HLA-DR, CD80, CD86, CD3, CD4, CD19 and IgA was detected, indicating that dendritic cells, T lymphocytes and B lymphocytes were all present in the local tumor tissues. Furthermore, expression of EBNA1 protein was detected on the membrane of the NPC tumor cells. Therefore, the NPC tumor microenvironment has the potential to initiate a humoral response to EBNA1 by producing IgA antibodies.

  7. The latency-associated nuclear antigen of Kaposi sarcoma–associated herpesvirus induces B cell hyperplasia and lymphoma

    PubMed Central

    Fakhari, Farnaz D.; Jeong, Joseph H.; Kanan, Yogita; Dittmer, Dirk P.

    2006-01-01

    Kaposi sarcoma–associated herpesvirus (KSHV) is a human lymphotropic herpesvirus. It is implicated in B cell neoplasias such as primary effusion lymphoma and multicentric Castleman disease in AIDS patients. The KSHV latency-associated nuclear antigen (LANA) is consistently expressed in all KSHV-associated tumor cells and was shown to bind the tumor suppressor proteins p53 and pRb. To test LANA’s contribution to lymphomagenesis in vivo we generated transgenic mice expressing LANA under the control of its own promoter, which is B cell specific. All of the transgenic mice developed splenic follicular hyperplasia due to an expansion of IgM+IgD+ B cells and showed increased germinal center formation. We also observed lymphomas, implying that LANA can activate B cells and provide the first step toward lymphomagenesis. PMID:16498502

  8. Current concepts and future directions for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies.

    PubMed

    Mahler, Michael; Meroni, Pier-Luigi; Bossuyt, Xavier; Fritzler, Marvin J

    2014-01-01

    The detection of autoantibodies that target intracellular antigens, commonly termed anti-nuclear antibodies (ANA), is a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD). Different methods are available for detection of ANA and all bearing their own advantages and limitations. Most laboratories use the indirect immunofluorescence (IIF) assay based on HEp-2 cell substrates. Due to the subjectivity of this diagnostic platform, automated digital reading systems have been developed during the last decade. In addition, solid phase immunoassays using well characterized antigens have gained widespread adoption in high throughput laboratories due to their ease of use and open automation. Despite all the advances in the field of ANA detection and its contribution to the diagnosis of SARD, significant challenges persist. This review provides a comprehensive overview of the current status on ANA testing including automated IIF reading systems and solid phase assays and suggests an approach to interpretation of results and discusses meeting the problems of assay standardization and other persistent challenges.

  9. Current Concepts and Future Directions for the Assessment of Autoantibodies to Cellular Antigens Referred to as Anti-Nuclear Antibodies

    PubMed Central

    Mahler, Michael; Meroni, Pier-Luigi; Bossuyt, Xavier; Fritzler, Marvin J.

    2014-01-01

    The detection of autoantibodies that target intracellular antigens, commonly termed anti-nuclear antibodies (ANA), is a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD). Different methods are available for detection of ANA and all bearing their own advantages and limitations. Most laboratories use the indirect immunofluorescence (IIF) assay based on HEp-2 cell substrates. Due to the subjectivity of this diagnostic platform, automated digital reading systems have been developed during the last decade. In addition, solid phase immunoassays using well characterized antigens have gained widespread adoption in high throughput laboratories due to their ease of use and open automation. Despite all the advances in the field of ANA detection and its contribution to the diagnosis of SARD, significant challenges persist. This review provides a comprehensive overview of the current status on ANA testing including automated IIF reading systems and solid phase assays and suggests an approach to interpretation of results and discusses meeting the problems of assay standardization and other persistent challenges. PMID:24868563

  10. Flow cytometry in the diagnosis of myelodysplastic syndromes (MDS) and the value of myeloid nuclear differentiation antigen (MNDA).

    PubMed

    Bellos, Frauke; Kern, Wolfgang

    2014-09-25

    Background: Confirming diagnosis of myelodysplastic syndromes (MDS) is often challenging. Standard diagnostic methods are cytomorphology (CM) and cytogenetics (CG). Multiparameter flow cytometry (MFC) is upcoming in MDS diagnostic work up, comparability and investigator experience are critical. Myeloid nuclear differentiation antigen (MNDA) in myelomonocytic cells might be expressed more weakly in patients with MDS. The analysis of MNDA may thus improve diagnostic capabilities of MFC in MDS. Methods: Staining methods and antibody combinations for MFC in MDS are outlined, giving details for interpretation of results in regard to dyspoiesis. MFC results are correlated with CM and CG and with survival data. Use of myeloid nuclear differentiation antigen (MNDA) in MDS diagnostics was evaluated in 239 patients with MDS, AML, other cytopenic conditions and in 30 negative controls. Results: Strong correlation between findings in CM and MFC was found; MFC results correlated well with those of CG. Patients with higher grades of dysplasia in MFC had shorter overall survival. Percentages of granulocytes and monocytes with diminished MNDA expression (%dimG, %dimM) were higher in patients with MDS and AML. Mean fluorescence intensity (MFI) of MNDA in monocytes was lower in MDS and AML. Cut-off values for %dimG (12%) and %dimM (22%) as well as for MFI in monocytes (72) were defined discriminating between MDS and non-MDS. Conclusion: MFC adds significant information on dyspoiesis in the diagnostic work up for MDS and provides prognostic information. MNDA expression can be assessed by MFC and may facilitate evaluation of dyspoiesis when added to MDS MFC panels. © 2014 Clinical Cytometry Society.

  11. Highly specific antibody to Rous sarcoma virus src gene product recognizes nuclear and nucleolar antigens in human cells.

    PubMed Central

    David-Pfeuty, T; Nouvian-Dooghe, Y

    1995-01-01

    An antiserum to the Rous sarcoma virus-transforming protein pp60v-src, raised in rabbits immunized with the bacterially produced protein alpha p60 serum (M. D. Resh and R. L. Erikson, J. Cell Biol. 100:409-417, 1985) previously reported to detect very specifically a novel population of pp60v-src and pp60c-src molecules associated with juxtareticular nuclear membranes in normal and Rous sarcoma virus-infected cells of avian and mammalian origin, was used here to investigate by immunofluorescence microscopy localization patterns of Src molecules in human cell lines, either normal or derived from spontaneous tumors. We found that the alpha p60 serum reveals nuclear and nucleolar concentrations of antigens in all the human cell lines tested and in two rat and mouse hepatoma cell lines derived from adult tumorous tissues but not in any established rat and mouse cell lines either untransformed or transformed by the src and ras oncogenes. Both the nuclear and nucleolar stainings can be totally extinguished by preincubation of the serum with highly purified chicken c-Src. We show also that the partitioning of the alpha p60-reactive proteins among the whole nucleus and the nucleolus depends mostly on two different parameters: the position in the cell cycle and the degree of cell confluency. Our observations raise the attractive possibility that, in differentiated cells, pp60c-src and related proteins might be involved not only in mediating the transduction of mitogenic signals at the plasma membrane level but also in controlling progression through the cell cycle and entry in mitosis by interacting with cell division cycle regulatory components at the nuclear level. PMID:7853507

  12. Nuclear location of an endogenously expressed antigen, EBNA1, restricts access to macroautophagy and the range of CD4 epitope display.

    PubMed

    Leung, Carol S; Haigh, Tracey A; Mackay, Laura K; Rickinson, Alan B; Taylor, Graham S

    2010-02-02

    Whereas exogenously acquired proteins are the major source of antigens feeding the MHC class II pathway in antigen-presenting cells, some endogenously expressed antigens also access that pathway but the rules governing such access are poorly understood. Here we address this using Epstein-Barr virus (EBV)-coded nuclear antigen EBNA1, a protein naturally expressed in EBV-infected B lymphoblastoid cell lines (LCLs) and a source of multiple CD4(+) T cell epitopes. Using CD4(+) T cell clones against three indicator epitopes, we find that two epitopes are weakly displayed on the LCL surface whereas the third is undetectable, a pattern of limited epitope presentation that is maintained even when nuclear expression of EBNA1 is induced to high supraphysiological levels. Inhibitor and siRNA studies show that, of the two epitopes weakly presented under these conditions, one involves macroautophagy, and the second involves antigen delivery to the MHC II pathway by another endogenous route. In contrast, when EBNA1 is expressed as a cytoplasmic protein, all three CD4 epitopes are processed and presented much more efficiently, and all involve macroautophagy. We conclude that EBNA1's nuclear location limits its accessibility to the macroautophagy pathway and, in consequence, limits the level and range of EBNA1 CD4 epitopes naturally displayed on the infected cell surface.

  13. Selection of buffer pH by the isoelectric point of the antigen for the efficient heat-induced epitope retrieval: re-appraisal for nuclear protein pathobiology.

    PubMed

    Kajiya, Hanako; Takekoshi, Susumu; Takei, Mao; Egashira, Noboru; Miyakoshi, Takashi; Serizawa, Akihito; Teramoto, Akira; Osamura, Robert Y

    2009-12-01

    Epitope retrieval (ER) using heating causes a dramatic improvement in the sensitivity of immunohistochemistry for formalin-fixed paraffin-embedded (FFPE) tissue sections. Here, the relationship between the pH of the retrieval buffer used for heat-induced epitope retrieval (HIER) and the isoelectric points (pI) of the antigen recognized by antibodies against nuclear proteins (mainly human pituitary transcription factors in this study) was investigated using FFPE tissue sections. A universal buffer, with a buffering capacity over a wide pH range from 2.0 to 12.0, was used for HIER. We found that the intensity of staining for most nuclear proteins after HIER depended simply on the pH of the buffer. Importantly, for efficient HIER, antigens with acidic pI required basic pH buffer conditions, while antigens with alkaline pI required acidic conditions. This implies that the electrostatic charge of the antigens contributed significantly to the efficiency of HIER. We conclude that appropriate selection of the pH of the buffer based on the pI of the individual antigens is of great importance for efficient ER. It is concluded that the mechanism of HEIR may, therefore, depend to a large extent on the pI of the antigen under investigation.

  14. Association of the level of IFN-γ produced by T cells in response to Mycobacterium tuberculosis-specific antigens with the size of skin test indurations among individuals with latent tuberculosis in a highly tuberculosis-endemic setting.

    PubMed

    Legesse, Mengistu; Ameni, Gobena; Mamo, Gezahegne; Medhin, Girmay; Bjune, Gunnar; Abebe, Fekadu

    2012-02-01

    There is growing evidence showing the potential of T-cell-based gamma interferon (IFN-γ) release assays (IGRAs) for predicting the risk of progression of Mycobacterium tuberculosis (Mtb) infection, though there is little information from tuberculosis (TB)-endemic settings. In this study, we assessed the association between the level of IFN-γ produced by T cells in response to Mtb-specific antigens and the size of skin test indurations in 505 adult individuals who were screened for latent tuberculosis infection (LTBI) using the QuantiFERON-TB Gold In Tube (QFTGIT) assay and tuberculin skin test (TST). There was a strong positive correlation between the level of IFN-γ induced by the specific antigens and the diameter of the skin indurations (Spearman's rho = 0.6, P < 0.001). Body mass index and parasitic infection were not associated with the level of IFN-γ production or the TST reaction. In linear regression analysis, the size of the skin test indurations was significantly associated with the mean level of IFN-γ [coefficient, 0.65; 95% confidence interval (CI), 0.47 to 0.82, P < 0.001]. Similarly, results from logistic regression analysis demonstrated that individuals who had skin test indurations ≥ 10 mm were 6.82 times more likely than individuals who had skin test indurations < 10 mm to have high levels of IFN-γ (i.e. positive QFTGIT result) (adjusted odd ratio = 6.82; 95% CI, 3.67 to 12.69, P < 0.001). In conclusion, the results of this study could provide indirect evidence for the prognostic use of the QFTGIT assay for progression of Mtb infection, though prospective follow-up studies are needed to provide direct evidence.

  15. IFN-γ/TNF-α ratio in response to immuno proteomically identified human T-cell antigens of Mycobacterium tuberculosis - The most suitable surrogate biomarker for latent TB infection.

    PubMed

    Prabhavathi, Maddineni; Pathakumari, Balaji; Raja, Alamelu

    2015-08-01

    The enormous reservoir of latent TB infection (LTBI) poses a major hurdle for global TB control. The existing Tuberculin skin test (TST) and IFN-γ release assays (IGRAs) are found to be suboptimal for LTBI diagnosis. Previously we had taken an immunoproteomic approach and identified 10 protein fractions (contains 16 proteins), which are solely recognized by LTBI. In a cohort of 40 pulmonary TB patients (PTB) and 35 healthy household contacts (HHC), IFN-γ and TNF-α response were measured against 16 antigens by using 1:10 diluted whole blood assay. Among all the antigens, IFN-γ response to Rv2626c has shown positivity of 88.57% in HHC and 7.5% in PTB group. IFN-γ response to combination of Rv2626c + Rv3716c has demonstrated 100% positivity in HHC and 17.5% positivity in PTB respectively. Compared to individual cytokines (i.e. IFN-γ and TNF-α), ratio of IFN-γ/TNF-α has shown promising results for diagnosis of LTBI. IFN-γ/TNF-α ratio against Rv3716c and TrxC has exhibited a positivity of 94.29% in HHC and 5% in PTB group. Accession of Rv2626c and Rv3716c may improve the diagnostic performance of existing QFT-GIT. Independent of QFT-GIT assay, ratio of IFN-γ/TNF-α in response to either Rv3716c or TrxC may acts as suitable surrogate biomarker for LTBI.

  16. Molecular characterisation and stage-specific expression of proliferating cell nuclear antigen (PCNA) from the malarial parasite, Plasmodium falciparum.

    PubMed

    Kilbey, B J; Fraser, I; McAleese, S; Goman, M; Ridley, R G

    1993-01-25

    The gene encoding the malarial homologue of proliferating cell nuclear antigen, PCNA, has been identified and characterised. It is located on chromosome 13. The coding sequence of 825 nucleotides predicts a protein of 30,586 Da. There are no introns and northern analysis reveals a transcript of approximately 1.6kb. The conserved residues which characterise the PCNAs of human, Drosophila, Saccharomyces and Xenopus are present in PfPCNA but the overall identity of PfPCNA with human and yeast PCNAs is low; 34% and 31% respectively. PfPCNA is longer than the PCNAs of these other species by about 16 amino acids, most of which are present in a block near the carboxy terminus. Antibodies against a purified PfPCNA-glutathione-S-transferase fusion protein recognise a single band in western blots of parasite extracts at 32kDa. The same antiserum has been used to demonstrate that the expression of PfPCNA is regulated during the intraerythrocytic development of the parasite. Expression increases dramatically in late trophozoites and is maintained during the subsequent nuclear divisions which produce schizonts.

  17. Proliferating cell nuclear antigen as a molecular biomarker for spermatogenesis in PTU-induced hypothyroidism of rats.

    PubMed

    Tousson, Ehab; Ali, Ehab M M; Ibrahim, Wafaa; Mansour, Mohammed A

    2011-07-01

    The thyroid hormone has few serious effects on the testes except during the neonatal stage. There is little knowledge concerning the prolonged effect of thyroid hormone deficiency throughout the rat's life span and its effect on spermatogenesis. Proliferating cell nuclear antigen (PCNA) is a nuclear matrix protein, which is essential for multiple cell cycle pathways. Here we used PCNA immunohistochemistry as a marker to differentiate between the testes of control and hypothyroid rats. About 20 rats were equally divided into 2 groups; the first group was the control group, while the second group was the experimental group in which rats were fed 0.05% 6-n-propyl thiouracil (PTU) in drinking water for 6 weeks. Immunohistochemistry, using an antibody against PCNA, showed at least 3 differences in the pattern of PCNA immunoreactivity (PCNA-ir). First, PCNA-ir was not detected in Sertoli and Leydig cells in the testes of control rats and detected in some of the hypothyroid rats. Second, in the control group more than 96% of spermatogonia were PCNA-positive cells; however, hypothyroidism caused the reduction to approximately 25% PCNA staining in spermatogonia. The third difference was in the abnormal distribution of spermatogonia seen in the hypothyroid rat testis, not in the control one. These results suggest that prepubertal hypothyroidism affects the proliferation of spermatogenic cells leading to impaired spermatogenesis and that PCNA index is a useful marker for assessing germ cell kinetics and spermatogenesis in prepubertal hypothyroidism.

  18. Ribosomal Protein S6 Interacts with the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus ▿

    PubMed Central

    Chen, Wuguo; Dittmer, Dirk P.

    2011-01-01

    The latency-associated nuclear antigen (LANA) is central to the maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) and to the survival of KSHV-carrying tumor cells. In an effort to identify interaction partners of LANA, we purified authentic high-molecular-weight complexes of LANA by conventional chromatography followed by immunoprecipitation from the BC-3 cell line. This is the first analysis of LANA-interacting partners that is not based on forced ectopic expression of LANA. Subsequent tandem mass spectrometry (MS/MS) analysis identified many of the known LANA-interacting proteins. We confirmed LANA's interactions with histones. Three classes of proteins survived our stringent four-step purification procedure (size, heparin, anion, and immunoaffinity chromatography): two heat shock proteins (Hsp70 and Hsp96 precursor), signal recognition particle 72 (SRP72), and 10 different ribosomal proteins. These proteins are likely involved in structural interactions within LANA high-molecular-weight complexes. Here, we show that ribosomal protein S6 (RPS6) interacts with LANA. This interaction is mediated by the N-terminal domain of LANA and does not require DNA or RNA. Depletion of RPS6 from primary effusion lymphoma (PEL) cells dramatically decreases the half-life of full-length LANA. The fact that RPS6 has a well-established nuclear function beyond its role in ribosome assembly suggests that RPS6 (and by extension other ribosomal proteins) contributes to the extraordinary stability of LANA. PMID:21734034

  19. Myeloid Cell Nuclear Differentiation Antigen (MNDA) Expression Distinguishes Extramedullary Presentations of Myeloid Leukemia From Blastic Plasmacytoid Dendritic Cell Neoplasm.

    PubMed

    Johnson, Ryan C; Kim, Jinah; Natkunam, Yasodha; Sundram, Uma; Freud, Aharon G; Gammon, Bryan; Cascio, Michael J

    2016-04-01

    Myeloid neoplasms constitute one of the most common malignancies in adults. In most cases these proliferations initially manifest in the blood and marrow; however, extramedullary involvement may precede blood or marrow involvement in a subset of cases, making a definitive diagnosis challenging by morphologic and immunohistochemical assessment alone. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive entity that frequently presents in extramedullary sites and can show morphologic and immunophenotypic overlap with myeloid neoplasms. Given that BPDCN and myeloid neoplasms may both initially present in extramedullary sites and that novel targeted therapies may be developed that exploit the unique molecular signature of BPDCN, new immunophenotypic markers that can reliably separate myeloid neoplasms from BPDCN are desirable. We evaluated the utility of myeloid cell nuclear differentiation antigen (MNDA) expression in a series of extramedullary myeloid leukemias (EMLs) and BPDCN. Forty biopsies containing EML and 19 biopsies containing BPDCN were studied by MNDA immunohistochemistry. The majority of myeloid neoplasms showed nuclear expression of MNDA (65%). In contrast, all cases of BPDCN lacked MNDA expression. These findings show that MNDA is expressed in the majority of EMLs and support the inclusion of MNDA immunohistochemistry in the diagnostic evaluation of blastic hematopoietic infiltrates, particularly when the differential diagnosis is between myeloid leukemia and BPDCN.

  20. Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

    PubMed

    Waraky, Ahmed; Lin, Yingbo; Warsito, Dudi; Haglund, Felix; Aleem, Eiman; Larsson, Olle

    2017-09-18

    We have previously shown that the insulin like growth factor 1 receptor (IGF1R) translocates to the cell nucleus, where it binds to enhancer like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF1R (nIGF1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer binding factor 1 (Lef1), histone H3, and Brahma related gene 1 proteins. In the present study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF1R binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA coincubated with the IGF1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF1R targets, and PCNA phosphorylation was followed by mono and poly ubiquitination. Coimmunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF1R or mutation of Tyr60, Tyr133, or Tyr250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF1R, externally induced DNA damage in IGF1R negative cells caused G1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF1R in DDT. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  1. Epstein-Barr virus nuclear antigen 1 interacts with regulator of chromosome condensation 1 dynamically throughout the cell cycle.

    PubMed

    Deschamps, Thibaut; Bazot, Quentin; Leske, Derek M; MacLeod, Ruth; Mompelat, Dimitri; Tafforeau, Lionel; Lotteau, Vincent; Maréchal, Vincent; Baillie, George S; Gruffat, Henri; Wilson, Joanna B; Manet, Evelyne

    2017-02-01

    The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a sequence-specific DNA-binding protein that plays an essential role in viral episome replication and segregation, by recruiting the cellular complex of DNA replication onto the origin (oriP) and by tethering the viral DNA onto the mitotic chromosomes. Whereas the mechanisms of viral DNA replication are well documented, those involved in tethering EBNA1 to the cellular chromatin are far from being understood. Here, we have identified regulator of chromosome condensation 1 (RCC1) as a novel cellular partner for EBNA1. RCC1 is the major nuclear guanine nucleotide exchange factor for the small GTPase Ran enzyme. RCC1, associated with chromatin, is involved in the formation of RanGTP gradients critical for nucleo-cytoplasmic transport, mitotic spindle formation and nuclear envelope reassembly following mitosis. Using several approaches, we have demonstrated a direct interaction between these two proteins and found that the EBNA1 domains responsible for EBNA1 tethering to the mitotic chromosomes are also involved in the interaction with RCC1. The use of an EBNA1 peptide array confirmed the interaction of RCC1 with these regions and also the importance of the N-terminal region of RCC1 in this interaction. Finally, using confocal microscopy and Förster resonance energy transfer analysis to follow the dynamics of interaction between the two proteins throughout the cell cycle, we have demonstrated that EBNA1 and RCC1 closely associate on the chromosomes during metaphase, suggesting an essential role for the interaction during this phase, perhaps in tethering EBNA1 to mitotic chromosomes.

  2. Multifaceted Histone H3 Methylation and Phosphorylation Readout by the Plant Homeodomain Finger of Human Nuclear Antigen Sp100C.

    PubMed

    Zhang, Xiaojie; Zhao, Dan; Xiong, Xiaozhe; He, Zhimin; Li, Haitao

    2016-06-10

    The decoding of histone post-translational modifications by chromatin-binding modules ("readers") constitutes one major mechanism of epigenetic regulation. Nuclear antigen Sp100 (SPECKLED, 100 kDa), a constitutive component of the promyelocytic leukemia nuclear bodies, plays key roles in intrinsic immunity and transcriptional repression. Sp100C, a splicing isoform specifically up-regulated upon interferon stimulation, harbors a unique tandem plant homeodomain (PHD) finger and bromodomain at its C terminus. Combining structural, quantitative binding, and cellular co-localization studies, we characterized Sp100C PHD finger as an unmethylated histone H3 Lys(4) (H3K4me0) reader that tolerates histone H3 Thr(3) phosphorylation (H3T3ph), histone H3 Lys(9) trimethylation (H3K9me3), and histone H3 Ser(10) phosphorylation (H3S10ph), hallmarks associated with the mitotic chromosome. In contrast, whereas H3K4me0 reader activity is conserved in Sp140, an Sp100C paralog, the multivalent tolerance of H3T3ph, H3K9me3, and H3S10ph was lost for Sp140. The complex structure determined at 2.1 Å revealed a highly coordinated lysine ϵ-amine recognition sphere formed by an extended N-terminal motif for H3K4me0 readout. Interestingly, reader pocket rigidification by disulfide bond formation enhanced H3K4me0 binding by Sp100C. An additional complex structure solved at 2.7 Å revealed that H3T3ph is recognized by the arginine residue, Arg(713), that is unique to the PHD finger of Sp100C. Consistent with a restrictive cellular role of Sp100C, these results establish a direct chromatin targeting function of Sp100C that may regulate transcriptional gene silencing and promyelocytic leukemia nuclear body-mediated intrinsic immunity in response to interferon stimulation.

  3. Transcription of Epstein-Barr virus-encoded nuclear antigen 1 promoter Qp is repressed by transforming growth factor-beta via Smad4 binding element in human BL cells.

    PubMed

    Liang, C L; Tsai, C N; Chung, P J; Chen, J L; Sun, C M; Chen, R H; Hong, J H; Chang, Y S

    2000-11-10

    In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp. Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity. Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta. The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody. The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment. This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis. Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells.

  4. Humoral markers of active Epstein-Barr virus infection associate with anti-extractable nuclear antigen autoantibodies and plasma galectin-3 binding protein in systemic lupus erythematosus.

    PubMed

    Rasmussen, N S; Nielsen, C T; Houen, G; Jacobsen, S

    2016-12-01

    We investigated if signs of active Epstein-Barr virus and cytomegalovirus infections associate with certain autoantibodies and a marker of type I interferon activity in patients with systemic lupus erythematosus. IgM and IgG plasma levels against Epstein-Barr virus early antigen diffuse and cytomegalovirus pp52 were applied as humoral markers of ongoing/recently active Epstein-Barr virus and cytomegalovirus infections, respectively. Plasma galectin-3 binding protein served as a surrogate marker of type I interferon activity. The measurements were conducted in 57 systemic lupus erythematosus patients and 29 healthy controls using ELISAs. Regression analyses and univariate comparisons were performed for associative evaluation between virus serology, plasma galectin-3 binding protein and autoantibodies, along with other clinical and demographic parameters. Plasma galectin-3 binding protein concentrations were significantly higher in systemic lupus erythematosus patients (P = 0.009) and associated positively with Epstein-Barr virus early antigen diffuse-directed antibodies and the presence of autoantibodies against extractable nuclear antigens in adjusted linear regressions (B = 2.02 and 2.02, P = 0.02 and P = 0.002, respectively). Furthermore, systemic lupus erythematosus patients with anti-extractable nuclear antigens had significantly higher antibody levels against Epstein-Barr virus early antigen diffuse (P = 0.02). Our study supports a link between active Epstein-Barr virus infections, positivity for anti-extractable nuclear antigens and increased plasma galectin-3 binding protein concentrations/type I interferon activity in systemic lupus erythematosus patients.

  5. Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Inhibits Major Histocompatibility Complex Class II Expression by Disrupting Enhanceosome Assembly through Binding with the Regulatory Factor X Complex

    PubMed Central

    Thakker, Suhani; Purushothaman, Pravinkumar; Gupta, Namrata; Challa, Shanthan; Cai, Qiliang

    2015-01-01

    a novel mechanism used by KSHV to downregulate the expressions of MHC-II genes. IMPORTANCE Kaposi's sarcoma-associated herpesvirus is the causative agent of multiple human malignancies. It establishes a lifelong latent infection and persists in infected cells without being detected by the host's immune surveillance system. Only a limited number of viral proteins are expressed during latency, and these proteins play a significant role in suppressing both the innate and adaptive immunities of the host. Latency-associated nuclear antigen (LANA) is one of the major proteins expressed during latent infection. Here, we show that LANA blocks MHC-II gene expression to subvert the host immune system by disrupting the MHC-II enhanceosome through binding with RFX transcription factors. Therefore, this study identifies a novel mechanism utilized by KSHV LANA to deregulate MHC-II gene expression, which is critical for CD4+ T cell responses in order to escape host immune surveillance. PMID:25740990

  6. Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet

    SciTech Connect

    Lo, Yuan-Hung; Ho, Po-Chun; Chen, Min-Shan; Hugo, Eric; Ben-Jonathan, Nira; Wang, Shao-Chun

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Proliferating Cell Nuclear Antigen (PCNA) is phosphorylated at Y114. Black-Right-Pointing-Pointer Phospho-Y114 of PCNA is not required for cell proliferation for normal growth. Black-Right-Pointing-Pointer MCE during adipogenesis is abolished in the lack of the phosphorylation. Black-Right-Pointing-Pointer Homozygous Y114F mice are resistant to high fat diet induced obesity. Black-Right-Pointing-Pointer Our results shed light on the interface between proliferation and differentiation. -- Abstract: Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA{sup F/F}) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA{sup F/F} MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT

  7. Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture.

    PubMed

    Calderón, Aingeru; Ortiz-Espín, Ana; Iglesias-Fernández, Raquel; Carbonero, Pilar; Pallardó, Federico Vicente; Sevilla, Francisca; Jiménez, Ana

    2017-04-01

    Thioredoxins (Trxs), key components of cellular redox regulation, act by controlling the redox status of many target proteins, and have been shown to play an essential role in cell survival and growth. The presence of a Trx system in the nucleus has received little attention in plants, and the nuclear targets of plant Trxs have not been conclusively identified. Thus, very little is known about the function of Trxs in this cellular compartment. Previously, we studied the intracellular localization of PsTrxo1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrxo1 we identified proliferating cellular nuclear antigen (PCNA) as a PsTrxo1 target by means of affinity chromatography techniques using purified nuclei from pea leaves. Such protein-protein interaction was corroborated by dot-blot and bimolecular fluorescence complementation (BiFC) assays, which showed that both proteins interact in the nucleus. Moreover, PsTrxo1 showed disulfide reductase activity on previously oxidized recombinant PCNA protein. In parallel, we studied the effects of PsTrxo1 overexpression on Tobacco Bright Yellow-2 (TBY-2) cell cultures. Microscopy and flow-cytometry analysis showed that PsTrxo1 overexpression increases the rate of cell proliferation in the transformed lines, with a higher percentage of the S phase of the cell cycle at the beginning of the cell culture (days 1 and 3) and at the G2/M phase after longer times of culture (day 9), coinciding with an upregulation of PCNA protein. Furthermore, in PsTrxo1 overexpressed cells there is a decrease in the total cellular glutathione content but maintained nuclear GSH accumulation, especially at the end of the culture, which is accompanied by a higher mitotic index, unlike non-overexpressing cells. These results suggest that Trxo1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA

  8. Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 in lymphoma cells containing the EBV P3HR1 genome induces expression of B-cell activation molecules CD21 and CD23.

    PubMed Central

    Cordier, M; Calender, A; Billaud, M; Zimber, U; Rousselet, G; Pavlish, O; Banchereau, J; Tursz, T; Bornkamm, G; Lenoir, G M

    1990-01-01

    A set of B-cell activation molecules, including the Epstein-Barr virus (EBV) receptor CR2 (CD21) and the B-cell activation antigen CD23 (Blast2/Fc epsilon RII), is turned on by infecting EBV-negative B-lymphoma cell lines with immortalizing strains of the viruslike B95-8 (BL/B95 cells). This up regulation may represent one of the mechanisms involved in EBV-mediated B-cell immortalization. The P3HR1 nonimmortalizing strain of the virus, which is deleted for the entire Epstein-Barr nuclear antigen 2 (EBNA2) protein open reading frame, is incapable of inducing the expression of CR2 and CD23, suggesting a crucial role for EBNA2 in the activation of these molecules. In addition, lymphoma cells containing the P3HR1 genome (BL/P3HR1 cells) do not express the viral latent membrane protein (LMP), which is regularly expressed in cells infected with immortalizing viral strains. Using electroporation, we have transfected the EBNA2 gene cloned in an episomal vector into BL/P3HR1 cells and have obtained cell clones that stably express the EBNA2 protein. In these clones, EBNA2 expression was associated with an increased amount of CR2 and CD23 steady-state RNAs. Of the three species of CD23 mRNAs described, the Fc epsilon RIIa species was preferentially expressed in these EBNA2-expressing clones. An increased cell surface expression of CR2 but not of CD23 was observed, and the soluble form of CD23 molecule (SCD23) was released. We were, however, not able to detect any expression of LMP in these cell clones. These data demonstrate that EBNA2 gene is able to complement P3HR1 virus latent functions to induce the activation of CR2 and CD23 expression, and they emphasize the role of EBNA2 protein in the modulation of cellular gene implicated in B-cell proliferation and hence in EBV-mediated B-cell immortalization. Nevertheless, EBNA2 expression in BL/P3HR1 cells is not able to restore the level of CR2 and CD23 expression observed in BL/B95 cells, suggesting that other cellular or viral

  9. Epstein-Barr virus nuclear antigen 2 transactivates the long terminal repeat of human immunodeficiency virus type 1.

    PubMed

    Scala, G; Quinto, I; Ruocco, M R; Mallardo, M; Ambrosino, C; Squitieri, B; Tassone, P; Venuta, S

    1993-05-01

    Human immunodeficiency virus type 1 (HIV-1)-infected subjects show a high incidence of Epstein-Barr virus (EBV) infection. This suggests that EBV may function as a cofactor that affects HIV-1 activation and may play a major role in the progression of AIDS. To test this hypothesis, we generated two EBV-negative human B-cell lines that stably express the EBNA2 gene of EBV. These EBNA2-positive cell lines were transiently transfected with plasmids that carry either the wild type or deletion mutants of the HIV-1 long terminal repeat (LTR) fused to the chloramphenicol acetyltransferase (CAT) gene. There was a consistently higher HIV-1 LTR activation in EBNA2-expressing cells than in control cells, which suggested that EBNA2 proteins could activate the HIV-1 promoter, possibly by inducing nuclear factors binding to HIV-1 cis-regulatory sequences. To test this possibility, we used CAT-based plasmids carrying deletions of the NF-kappa B (pNFA-CAT), Sp1 (pSpA-CAT), or TAR (pTAR-CAT) region of the HIV-1 LTR and retardation assays in which nuclear proteins from EBNA2-expressing cells were challenged with oligonucleotides encompassing the NF-kappa B or Sp1 region of the HIV-1 LTR. We found that both the NF-kappa B and the Sp1 sites of the HIV-1 LTR are necessary for EBNA2 transactivation and that increased expression resulted from the induction of NF-kappa B-like factors. Moreover, experiments with the TAR-deleted pTAR-CAT and with the tat-expressing pAR-TAT plasmids indicated that endogenous Tat-like proteins could participate in EBNA2-mediated activation of the HIV-1 LTR and that EBNA2 proteins can synergize with the viral tat transactivator. Transfection experiments with plasmids expressing the EBNA1, EBNA3, and EBNALP genes did not cause a significant HIV-1 LTR activation. Thus, it appears that among the latent EBV genes tested, EBNA2 was the only EBV gene active on the HIV-1 LTR. The transactivation function of EBNA2 was also observed in the HeLa epithelial cell line

  10. International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies.

    PubMed

    Agmon-Levin, Nancy; Damoiseaux, Jan; Kallenberg, Cees; Sack, Ulrich; Witte, Torsten; Herold, Manfred; Bossuyt, Xavier; Musset, Lucille; Cervera, Ricard; Plaza-Lopez, Aresio; Dias, Carlos; Sousa, Maria José; Radice, Antonella; Eriksson, Catharina; Hultgren, Olof; Viander, Markku; Khamashta, Munther; Regenass, Stephan; Andrade, Luis Eduardo Coelho; Wiik, Allan; Tincani, Angela; Rönnelid, Johan; Bloch, Donald B; Fritzler, Marvin J; Chan, Edward K L; Garcia-De La Torre, I; Konstantinov, Konstantin N; Lahita, Robert; Wilson, Merlin; Vainio, Olli; Fabien, Nicole; Sinico, Renato Alberto; Meroni, Pierluigi; Shoenfeld, Yehuda

    2014-01-01

    Anti-nuclear antibodies (ANA) are fundamental for the diagnosis of autoimmune diseases, and have been determined by indirect immunofluorescence assay (IIFA) for decades. As the demand for ANA testing increased, alternative techniques were developed challenging the classic IIFA. These alternative platforms differ in their antigen profiles, sensitivity and specificity, raising uncertainties regarding standardisation and interpretation of incongruent results. Therefore, an international group of experts has created recommendations for ANA testing by different methods. Two groups of experts participated in this initiative. The European autoimmunity standardization initiative representing 15 European countries and the International Union of Immunologic Societies/World Health Organization/Arthritis Foundation/Centers for Disease Control and Prevention autoantibody standardising committee. A three-step process followed by a Delphi exercise with closed voting was applied. Twenty-five recommendations for determining ANA (1-13), anti-double stranded DNA antibodies (14-18), specific antibodies (19-23) and validation of methods (24-25) were created. Significant differences between experts were observed regarding recommendations 24-25 (p<0.03). Here, we formulated recommendations for the assessment and interpretation of ANA and associated antibodies. Notably, the roles of IIFA as a reference method, and the importance of defining nuclear and cytoplasmic staining, were emphasised, while the need to incorporate alternative automated methods was acknowledged. Various approaches to overcome discrepancies between methods were suggested of which an improved bench-to-bedside communication is of the utmost importance. These recommendations are based on current knowledge and can enable harmonisation of local algorithms for testing and evaluation of ANA and related autoantibodies. Last but not least, new more appropriate terminologies have been suggested.

  11. Mimicry between the hepatitis C virus polyprotein and antigenic targets of nuclear and smooth muscle antibodies in chronic hepatitis C virus infection.

    PubMed

    Gregorio, G V; Choudhuri, K; Ma, Y; Pensati, P; Iorio, R; Grant, P; Garson, J; Bogdanos, D P; Vegnente, A; Mieli-Vergani, G; Vergani, D

    2003-09-01

    Autoantibodies to smooth muscle (SMA) and nuclear components (ANA) arise in the natural course of chronic infection with hepatitis C virus. In view of the growing evidence for 'molecular mimicry' as a mechanism of autoimmunity we investigated whether cross-reactive immune reactions between host smooth muscle/nuclear components and HCV antigens may contribute to the formation of SMA and ANA in chronic HCV infection. Computer-assisted protein database search methods were used to identify three smooth muscle (smoothelin698-717, myosin1035-1054, vimentin69-88) and three nuclear (matrin722-741, histone H2A11-30, replication protein A133-152) host antigens with the highest local sequence similarity to the HCV polyprotein and 20-mer peptides corresponding to these regions were constructed. Sera from 51 children with chronic HCV infection [median age: 8 (2-16); 27 boys], 26 SMA positive and five ANA positive, were tested for reactivity to the synthesized HCV peptides and their human homologues by enzyme linked immunosorbent assay (ELISA). Sera from patients with HBV infection and chronic liver disease of different aetiologies were used as controls. 'Double reactivity' to HCV peptides and smooth muscle/nuclear homologues was associated strongly with HCV infection (P < 0.001 for both). Humoral cross-reactivity was established as the basis for double recognition by competition ELISA. Double-reactivity to smooth muscle and HCV peptide antigens correlated with SMA positivity by indirect immunofluouresence (P = 0.05). Of 15 patients double-reactive to myosin1035-1054 and its HCV homologue, 13 recognized whole myosin by immunoblot. These results suggest that ANA and SMA in chronic HCV infection may arise, at least in part, as a consequence of cross-reactive immune responses to HCV and host smooth muscle/nuclear antigens.

  12. MHC II tetramers visualize human CD4+ T cell responses to Epstein–Barr virus infection and demonstrate atypical kinetics of the nuclear antigen EBNA1 response

    PubMed Central

    Long, Heather M.; Chagoury, Odette L.; Leese, Alison M.; Ryan, Gordon B.; James, Eddie; Morton, Laura T.; Abbott, Rachel J.M.; Sabbah, Shereen; Kwok, William

    2013-01-01

    Virus-specific CD4+ T cells are key orchestrators of host responses to viral infection yet, compared with their CD8+ T cell counterparts, remain poorly characterized at the single cell level. Here we use nine MHC II–epitope peptide tetramers to visualize human CD4+ T cell responses to Epstein–Barr virus (EBV), the causative agent of infectious mononucleosis (IM), a disease associated with large virus-specific CD8+ T cell responses. We find that, while not approaching virus-specific CD8+ T cell expansions in magnitude, activated CD4+ T cells specific for epitopes in the latent antigen EBNA2 and four lytic cycle antigens are detected at high frequencies in acute IM blood. They then fall rapidly to values typical of life-long virus carriage where most tetramer-positive cells display conventional memory markers but some, unexpectedly, revert to a naive-like phenotype. In contrast CD4+ T cell responses to EBNA1 epitopes are greatly delayed in IM patients, in line with the well-known but hitherto unexplained delay in EBNA1 IgG antibody responses. We present evidence from an in vitro system that may explain these unusual kinetics. Unlike other EBNAs and lytic cycle proteins, EBNA1 is not naturally released from EBV-infected cells as a source of antigen for CD4+ T cell priming. PMID:23569328

  13. MHC II tetramers visualize human CD4+ T cell responses to Epstein-Barr virus infection and demonstrate atypical kinetics of the nuclear antigen EBNA1 response.

    PubMed

    Long, Heather M; Chagoury, Odette L; Leese, Alison M; Ryan, Gordon B; James, Eddie; Morton, Laura T; Abbott, Rachel J M; Sabbah, Shereen; Kwok, William; Rickinson, Alan B

    2013-05-06

    Virus-specific CD4(+) T cells are key orchestrators of host responses to viral infection yet, compared with their CD8(+) T cell counterparts, remain poorly characterized at the single cell level. Here we use nine MHC II-epitope peptide tetramers to visualize human CD4(+) T cell responses to Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis (IM), a disease associated with large virus-specific CD8(+) T cell responses. We find that, while not approaching virus-specific CD8(+) T cell expansions in magnitude, activated CD4(+) T cells specific for epitopes in the latent antigen EBNA2 and four lytic cycle antigens are detected at high frequencies in acute IM blood. They then fall rapidly to values typical of life-long virus carriage where most tetramer-positive cells display conventional memory markers but some, unexpectedly, revert to a naive-like phenotype. In contrast CD4(+) T cell responses to EBNA1 epitopes are greatly delayed in IM patients, in line with the well-known but hitherto unexplained delay in EBNA1 IgG antibody responses. We present evidence from an in vitro system that may explain these unusual kinetics. Unlike other EBNAs and lytic cycle proteins, EBNA1 is not naturally released from EBV-infected cells as a source of antigen for CD4(+) T cell priming.

  14. Prognostic values of proliferating cell nuclear antigen (PCNA) and Ki-67 for radiotherapy of oesophageal squamous cell carcinomas

    PubMed Central

    Okuno, Y; Nishimura, Y; Kashu, I; Ono, K; Hiraoka, M

    1999-01-01

    The relationship of immunohistochemical indices of proliferating cell nuclear antigen (PCNA) and Ki-67 to local control and survival rates for patients with oesophageal squamous cell carcinomas treated by definitive radiotherapy (RT) was investigated. Biopsy materials before RT were obtained from 65 patients with oesophageal cancer. The median PCNA labelling index (LI) and the median Ki-67 LI were 52% and 45% respectively. The PCNA LI was independent of known prognostic factors on local control for oesophageal cancer, although Ki-67 LI correlated with several prognostic factors. In the univariate analysis, patients with the PCNA LI of < 52% or the Ki-67 LI of < 45% showed significantly higher local recurrence rates than those with higher LIs (bothP < 0.05). This difference in local control rate according to LIs was prominent for the patients treated with conventional fractionation. In the multivariate analysis, T-stage (P = 0.0056) and PCNA LI (P = 0.0332) were significant factors for local control in the final model using a stepwise regression procedure. In conclusion, PCNA LI and Ki-67 LI were significantly correlated with local control probabilities in oesophageal squamous cell carcinomas treated by definitive RT. © 1999 Cancer Research Campaign PMID:10408843

  15. Human DNA polymerase β, but not λ, can bypass a 2-deoxyribonolactone lesion together with proliferating cell nuclear antigen

    PubMed Central

    Crespan, Emmanuele; Pasi, Emanuela; Imoto, Shuhei; Hübscher, Ulrich; Greenberg, Marc M.; Maga, Giovanni

    2012-01-01

    The C1′-oxidized lesion 2-deoxyribonolactone (L) is induced by free radical attack of DNA. This lesion is mutagenic, inhibits base excision repair, and can lead to strand scission. In double stranded DNA L is repaired by long-patch base excision repair, but it induces replication fork arrest in a single-strand template. Translesion synthesis requires a specialized DNA polymerase (Pol). In E. coli, Pol V is responsible for bypassing L, while in yeast Pol ζ has been shown to be required for efficient bypass. Very little is known about the identity of human Pols capable of bypassing L. For instance, the activity of family X enzymes has never been investigated. We examined the ability of different family X Pols: Pols β, λ and TdT from human cells and Pol IV from S. cerevisiae to act on DNA containing an isolated 2-deoxyribonolactone, as well as when the lesion comprises the 5′-component of a tandem lesion. We show that Pol β, but not Pol λ, can bypass a single L lesion in the template, and its activity is increased by the auxiliary protein proliferating cell nuclear antigen (PCNA), while both enzymes were completely blocked by a tandem lesion. Yeast Pol IV was able to bypass the single L and the tandem lesion but with little nucleotide insertion specificity. Finally, L did not affect the polymerization activity of the template-independent enzyme TdT. PMID:23101935

  16. Oxidative DNA Damage Bypass in Arabidopsis thaliana Requires DNA Polymerase λ and Proliferating Cell Nuclear Antigen 2[W

    PubMed Central

    Amoroso, Alessandra; Concia, Lorenzo; Maggio, Caterina; Raynaud, Cécile; Bergounioux, Catherine; Crespan, Emmanuele; Cella, Rino; Maga, Giovanni

    2011-01-01

    The oxidized base 7,8-oxoguanine (8-oxo-G) is the most common DNA lesion generated by reactive oxygen species. This lesion is highly mutagenic due to the frequent misincorporation of A opposite 8-oxo-G during DNA replication. In mammalian cells, the DNA polymerase (pol) family X enzyme DNA pol λ catalyzes the correct incorporation of C opposite 8-oxo-G, together with the auxiliary factor proliferating cell nuclear antigen (PCNA). Here, we show that Arabidopsis thaliana DNA pol λ, the only member of the X family in plants, is as efficient in performing error-free translesion synthesis past 8-oxo-G as its mammalian homolog. Arabidopsis, in contrast with animal cells, possesses two genes for PCNA. Using in vitro and in vivo approaches, we observed that PCNA2, but not PCNA1, physically interacts with DNA pol λ, enhancing its fidelity and efficiency in translesion synthesis. The levels of DNA pol λ in transgenic plantlets characterized by overexpression or silencing of Arabidopsis POLL correlate with the ability of cell extracts to perform error-free translesion synthesis. The important role of DNA pol λ is corroborated by the observation that the promoter of POLL is activated by UV and that both overexpressing and silenced plants show altered growth phenotypes. PMID:21325140

  17. Epstein–Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1

    PubMed Central

    Bazot, Quentin; Deschamps, Thibaut; Tafforeau, Lionel; Siouda, Maha; Leblanc, Pascal; Harth-Hertle, Marie L.; Rabourdin-Combe, Chantal; Lotteau, Vincent; Kempkes, Bettina; Tommasino, Massimo; Gruffat, Henri; Manet, Evelyne

    2014-01-01

    The Epstein–Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1. PMID:25092922

  18. Structural insights into the adaptation of proliferating cell nuclear antigen (PCNA) from Haloferax volcanii to a high-salt environment

    SciTech Connect

    Morgunova, Ekaterina; Gray, Fiona C.; MacNeill, Stuart A.; Ladenstein, Rudolf

    2009-10-01

    The crystal structure of PCNA from the halophilic archaeon H. volcanii reveals specific features of the charge distribution on the protein surface that reflect adaptation to a high-salt environment and suggests a different type of interaction with DNA in halophilic PCNAs. The sliding clamp proliferating cell nuclear antigen (PCNA) plays vital roles in many aspects of DNA replication and repair in eukaryotic cells and in archaea. Realising the full potential of archaea as a model for PCNA function requires a combination of biochemical and genetic approaches. In order to provide a platform for subsequent reverse genetic analysis, PCNA from the halophilic archaeon Haloferax volcanii was subjected to crystallographic analysis. The gene was cloned and expressed in Escherichia coli and the protein was purified by affinity chromatography and crystallized by the vapour-diffusion technique. The structure was determined by molecular replacement and refined at 3.5 Å resolution to a final R factor of 23.7% (R{sub free} = 25%). PCNA from H. volcanii was found to be homotrimeric and to resemble other homotrimeric PCNA clamps but with several differences that appear to be associated with adaptation of the protein to the high intracellular salt concentrations found in H. volcanii cells.

  19. Cissus quadrangularis L. extract attenuates chronic ulcer by possible involvement of polyamines and proliferating cell nuclear antigen

    PubMed Central

    Jainu, Mallika; Vijaimohan, K.; Kannan, K.

    2010-01-01

    The present study was designed to investigate whether Cissus quandrangularis extract (CQE) had healing effects on gastric ulcer, through modulation of polyamines and proliferating cell nuclear antigen (PCNA) in rats. Administration of acetic acid (AA) was accompanied by reduced PCNA which was determined by immunohistochemical staining, 3H-thymidine incorporation using liquid scintillation spectrometry, mitochondrial marker enzymes, polyamine contents and transforming growth factor-alpha (TGF-α) expression in gastric mucosa of rats. Administration of CQE after the application of AA to the stomach enhanced the reduction of ulcer area in a dose-dependent manner which was confirmed by histoarchitecture. Moreover, CQE significantly increased the 3H-thymidine incorporation and the levels of polyamines such as putrescine, spermine and spermidine in ulcerated rats. In addition, the extract offers gastroprotection in the ulcerated area by increased expression of TGF-α and also reversed the changes in the gastric mucosa of ulcerated rats with significant elevation in mitochondrial tricarboxylic acid (TCA) cycle enzymes and PCNA levels. Based on these results, the healing effect of CQE on AA induced gastric mucosal injury in rats may be attributed to its growth promoting and cytoprotective actions, possibly involving an increase in tissue polyamine contents and cell proliferation. PMID:20931084

  20. Cissus quadrangularis L. extract attenuates chronic ulcer by possible involvement of polyamines and proliferating cell nuclear antigen.

    PubMed

    Jainu, Mallika; Vijaimohan, K; Kannan, K

    2010-07-01

    The present study was designed to investigate whether Cissus quandrangularis extract (CQE) had healing effects on gastric ulcer, through modulation of polyamines and proliferating cell nuclear antigen (PCNA) in rats. Administration of acetic acid (AA) was accompanied by reduced PCNA which was determined by immunohistochemical staining, (3)H-thymidine incorporation using liquid scintillation spectrometry, mitochondrial marker enzymes, polyamine contents and transforming growth factor-alpha (TGF-α) expression in gastric mucosa of rats. Administration of CQE after the application of AA to the stomach enhanced the reduction of ulcer area in a dose-dependent manner which was confirmed by histoarchitecture. Moreover, CQE significantly increased the (3)H-thymidine incorporation and the levels of polyamines such as putrescine, spermine and spermidine in ulcerated rats. In addition, the extract offers gastroprotection in the ulcerated area by increased expression of TGF-α and also reversed the changes in the gastric mucosa of ulcerated rats with significant elevation in mitochondrial tricarboxylic acid (TCA) cycle enzymes and PCNA levels. Based on these results, the healing effect of CQE on AA induced gastric mucosal injury in rats may be attributed to its growth promoting and cytoprotective actions, possibly involving an increase in tissue polyamine contents and cell proliferation.

  1. Immunohistochemical distribution of heat shock protein 70 and proliferating cell nuclear antigen in mouse placenta at different gestational stages.

    PubMed

    Ozaydin, Tugba; Sur, Emrah; Oznurlu, Yasemin; Celik, Ilhami; Uluisik, Deniz

    2016-04-01

    The aim of the present study was to investigate immunohistochemical distribution of heat shock protein 70 (Hsp70) and proliferating cell nuclear antigen (PCNA) in the mouse placenta at different gestational stages. For this purpose a total of 18 Swiss albino female mice at 12-14 weeks of age were used. Females were sacrificed on days 3 (early), 10 (mid-), and 17 (late) of pregnancy and the implantation sites of the pregnant uterus were sampled. The sections were made transversely through the central region of the implantation site and stained with hematoxylin and eosin for histological examination. PCNA and Hsp70 was stained immunohistochemically. Since the definitive placenta was not still formed on day 3 of pregnancy, Hsp70 and PCNA positivity were evaluated in only luminal epithelium and decidual-stromal cells. On days 10 and 17 of pregnancy, Hsp70 and PCNA positivity were evaluated in labyrinth zone, junctional zone and decidual layer of placenta. Hsp70 expression was observed trophoblast cells and decidual cells and was relatively constant throughout the pregnancy. This protein was strongly labeled in the trophoblast cells; while decidual cells were displayed moderate staining. In early pregnant mouse uteri, PCNA was mainly localized in decidual-stromal cells. The trophoblast cells and decidual cells displayed highly proliferative activity at the midgestational period. However there was a significant decrease in the percentage of PCNA positive cells in late gestation.

  2. The MutSα-Proliferating Cell Nuclear Antigen Interaction in Human DNA Mismatch Repair*S⃞♦

    PubMed Central

    Iyer, Ravi R.; Pohlhaus, Timothy J.; Chen, Sihong; Hura, Gregory L.; Dzantiev, Leonid; Beese, Lorena S.; Modrich, Paul

    2008-01-01

    We have examined the interaction parameters, conformation, and functional significance of the human MutSα· proliferating cell nuclear antigen (PCNA) complex in mismatch repair. The two proteins associate with a 1:1 stoichiometry and a KD of 0.7 μm in the absence or presence of heteroduplex DNA. PCNA does not influence the affinity of MutSα for a mismatch, and mismatch-bound MutSα binds PCNA. Small angle x-ray scattering studies have established the molecular parameters of the complex, which are consistent with an elongated conformation in which the two proteins associate in an end-to-end fashion in a manner that does not involve an extended unstructured tether, as has been proposed for yeast MutSα and PCNA (Shell, S. S., Putnam, C. D., and Kolodner, R. D. (2007) Mol. Cell26 ,565 -57817531814). MutSα variants lacking the PCNA interaction motif are functional in 3′- or 5′-directed mismatch-provoked excision, but display a partial defect in 5′-directed mismatch repair. This finding is consistent with the modest mutability conferred by inactivation of the MutSα PCNA interaction motif and suggests that interaction of the replication clamp with other repair protein(s) accounts for the essential role of PCNA in MutSα-dependent mismatch repair. PMID:18326858

  3. Boron inhibits the proliferating cell nuclear antigen index, molybdenum containing proteins and ameliorates oxidative stress in hepatocellular carcinoma.

    PubMed

    Zafar, Hina; Ali, Shakir

    2013-01-15

    Hepatocellular carcinoma (HCC) is a common malignancy and the main cause of mortality in patients with chronic liver diseases. This study reports the inhibitory effect of boron on HCC induced in rats by administering thioacetamide (TAA) (0.03%) in drinking water for 400days. Boron (4mg/kg body weight) was administered orally after induction of carcinoma. Treatment was continued for 122days, and cell proliferation, histology and biochemistry of treated and control group of rats were studied. Proliferating cell nuclear antigen (PCNA), and [(3)H]-thymidine incorporation, which increased in rats exposed to carcinogen, significantly decreased after boron treatment. PCNA index decreased from 80 in HCC rats to 32 after boron treatment. In the control group, it was 20. Boron caused a dose-dependent decrease in carcinogen-induced [(3)H]-thymidine uptake by the rat hepatocyte. It could partially reverse the activity of selected biochemical indicators of hepatic damage, oxidative stress, selenium and serum retinol, which are depleted in liver cancer, and improved overall health of animal. The study implicates the elevated levels of mammalian molybdenum Fe-S containing flavin hydroxylases, which increase the free radical production and oxidative stress, consequently causing increased hepatic cell proliferation in HCC, and reports boron to ameliorate these changes in liver cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Out-of-plane motions in open sliding clamps: Molecular dynamics simulations of eukaryotic and archaeal proliferating cell nuclear antigen

    PubMed Central

    Kazmirski, Steven L.; Zhao, Yanxiang; Bowman, Gregory D.; O'Donnell, Mike; Kuriyan, John

    2005-01-01

    Sliding clamps are ring-like multimeric proteins that encircle duplex DNA and serve as mobile DNA-bound platforms that are essential for efficient DNA replication and repair. Sliding clamps are placed on DNA by clamp loader complexes, in which the clamp-interacting elements are organized in a right-handed spiral assembly. To understand how the flat, ring-like clamps might interact with the spiral interaction surface of the clamp loader complex, we have performed molecular dynamics simulations of sliding clamps (proliferating cell nuclear antigen from the budding yeast, humans, and an archaeal species) in which we have removed one of the three subunits so as to release the constraint of ring closure. The simulations reveal significant structural fluctuations corresponding to lateral opening and out-of-plane distortions of the clamp, which result principally from bending and twisting of the β-sheets that span the intermolecular interfaces, with smaller but similar contributions from β-sheets that span the intramolecular interfaces within each subunit. With the integrity of these β-sheets intact, the predominant fluctuations seen in the simulations are oscillations between lateral openings and right-handed spirals. The tendency for clamps to adopt a right-handed spiral conformation implies that once opened, the conformation of the clamp can easily match the spiraling of clamp loader subunits, a feature that is intrinsic to the recognition of DNA and subsequent hydrolysis of ATP by the clamp-bound clamp loader complex. PMID:16169903

  5. The solution structure of functionally active human proliferating cell nuclear antigen determined by small-angle neutron scattering.

    PubMed

    Schurtenberger, P; Egelhaaf, S U; Hindges, R; Maga, G; Jónsson, Z O; May, R P; Glatter, O; Hübscher, U

    1998-01-09

    The function of proliferating cell nuclear antigen (PCNA) in DNA replication and repair is to form a sliding clamp with replication factor C (RF-C) tethering DNA polymerase delta or epsilon to DNA. In addition, PCNA has been found to interact directly with various proteins involved in cell cycle regulation. The crystal structure of yeast PCNA shows that the protein forms a homotrimeric ring lining a hole through which double-stranded DNA can thread, thus forming a moving platform for DNA synthesis. Human and yeast PCNA are highly conserved at a structural and functional level. We determined the solution structure of functionally active human PCNA by small-angle neutron scattering. Our measurements strongly support a trimeric ring-like structure of functionally active PCNA in solution, and the data are in good agreement with model calculations based on the crystal structure from yeast PCNA. The human PCNA used in the small-angle neutron scattering experiments was active before and after the measurements in a RF-C independent and a RF-C dependent assay suggesting that the trimeric structure is the in vivo functional form.

  6. Crystal structure of the shrimp proliferating cell nuclear antigen: structural complementarity with WSSV DNA polymerase PIP-box.

    PubMed

    Carrasco-Miranda, Jesus S; Lopez-Zavala, Alonso A; Arvizu-Flores, Aldo A; Garcia-Orozco, Karina D; Stojanoff, Vivian; Rudiño-Piñera, Enrique; Brieba, Luis G; Sotelo-Mundo, Rogerio R

    2014-01-01

    DNA replication requires processivity factors that allow replicative DNA polymerases to extend long stretches of DNA. Some DNA viruses encode their own replicative DNA polymerase, such as the white spot syndrome virus (WSSV) that infects decapod crustaceans but still require host replication accessory factors. We have determined by X-ray diffraction the three-dimensional structure of the Pacific white leg shrimp Litopenaeus vannamei Proliferating Cell Nuclear Antigen (LvPCNA). This protein is a member of the sliding clamp family of proteins, that binds DNA replication and DNA repair proteins through a motif called PIP-box (PCNA-Interacting Protein). The crystal structure of LvPCNA was refined to a resolution of 3 Å, and allowed us to determine the trimeric protein assembly and details of the interactions between PCNA and the DNA. To address the possible interaction between LvPCNA and the viral DNA polymerase, we docked a theoretical model of a PIP-box peptide from the WSSV DNA polymerase within LvPCNA crystal structure. The theoretical model depicts a feasible model of interaction between both proteins. The crystal structure of shrimp PCNA allows us to further understand the mechanisms of DNA replication processivity factors in non-model systems.

  7. Crystal Structure of the Shrimp Proliferating Cell Nuclear Antigen: Structural Complementarity with WSSV DNA Polymerase PIP-Box

    PubMed Central

    Carrasco-Miranda, Jesus S.; Lopez-Zavala, Alonso A.; Arvizu-Flores, Aldo A.; Garcia-Orozco, Karina D.; Stojanoff, Vivian; Rudiño-Piñera, Enrique; Brieba, Luis G.; Sotelo-Mundo, Rogerio R.

    2014-01-01

    DNA replication requires processivity factors that allow replicative DNA polymerases to extend long stretches of DNA. Some DNA viruses encode their own replicative DNA polymerase, such as the white spot syndrome virus (WSSV) that infects decapod crustaceans but still require host replication accessory factors. We have determined by X-ray diffraction the three-dimensional structure of the Pacific white leg shrimp Litopenaeus vannamei Proliferating Cell Nuclear Antigen (LvPCNA). This protein is a member of the sliding clamp family of proteins, that binds DNA replication and DNA repair proteins through a motif called PIP-box (PCNA-Interacting Protein). The crystal structure of LvPCNA was refined to a resolution of 3 Å, and allowed us to determine the trimeric protein assembly and details of the interactions between PCNA and the DNA. To address the possible interaction between LvPCNA and the viral DNA polymerase, we docked a theoretical model of a PIP-box peptide from the WSSV DNA polymerase within LvPCNA crystal structure. The theoretical model depicts a feasible model of interaction between both proteins. The crystal structure of shrimp PCNA allows us to further understand the mechanisms of DNA replication processivity factors in non-model systems. PMID:24728082

  8. Kaposi's sarcoma herpesvirus-encoded latency-associated nuclear antigen stabilizes intracellular activated Notch by targeting the Sel10 protein.

    PubMed

    Lan, Ke; Verma, Subhash C; Murakami, Masanao; Bajaj, Bharat; Kaul, Rajeev; Robertson, Erle S

    2007-10-09

    Deregulation of the evolutionarily conserved Notch signaling is highly correlated with oncogenesis. Intracellular activated Notch (ICN) is a protooncogene linked to the transcription activation of a number of cellular genes involved in cell cycle regulation, differentiation, and proliferation. Stability of ICN is tightly regulated by the Sel10-mediated ubiquitin-proteasome pathway. Sel10 can function as a negative regulator of Notch and exhibits activities of a tumor-suppressor protein. This article shows that the Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) directly interacts with Sel10 and forms a complex in KSHV-infected cells. This results in suppression of ICN ubiquitination and degradation. The carboxyl terminus of LANA interacts with the F-box and WD40 domains of Sel10 and competes with ICN for binding to Sel10. This elevated level of ICN is also critical for maintaining the enhanced proliferation of KSHV-infected tumor cells. These findings describe a mechanism by which the KSHV-encoded LANA protein regulates ubiquitination of ICN mediated by the F-box component of the E3 ligase Sel10, leading to proliferation of the virus-infected cells.

  9. Post-translational modifications of proliferating cell nuclear antigen: A key signal integrator for DNA damage response (Review).

    PubMed

    Zhu, Qiong; Chang, Yuxiao; Yang, Jin; Wei, Quanfang

    2014-05-01

    Previous studies have shown that the post-translational modifications of proliferating cell nuclear antigen (PCNA) may be crucial in influencing the cellular choice between different pathways, such as the cell cycle checkpoint, DNA repair or apoptosis pathways, in order to maintain genomic stability. DNA damage leads to replication stress and the subsequent induction of PCNA modification by small ubiquitin (Ub)-related modifiers and Ub, which has been identified to affect multiple biological processes of genomic DNA. Thus far, much has been learned concerning the behavior of modified PCNA as a key signal integrator in response to DNA damage. In humans and yeast, modified PCNA activates DNA damage bypass via an error-prone or error-free pathway to prevent the breakage of DNA replication forks, which may potentially induce double-strand breaks and subsequent chromosomal rearrangements. However, the exact mechanisms by which these pathways work and by what means the modified PCNA is involved in these processes remain elusive. Thus, the improved understanding of PCNA modification and its implications for DNA damage response may provide us with more insight into the mechanisms by which human cells regulate aberrant recombination events, and cancer initiation and development. The present review focuses on the post-translational modifications of PCNA and its important functions in mediating mammalian cellular response to different types of DNA damage.

  10. Molecular modeling and expression of the Litopenaeus vannamei proliferating cell nuclear antigen (PCNA) after white spot syndrome virus shrimp infection.

    PubMed

    de-la-Re-Vega, Enrique; Muhlia-Almazan, Adriana; Arvizu-Flores, Aldo A; Islas-Osuna, Maria A; Yepiz-Plascencia, Gloria; Brieba, Luis G; Sotelo-Mundo, Rogerio R

    2011-01-01

    Proliferating cell nuclear antigen (PCNA) is the eukaryotic sliding clamp that tethers DNA polymerase to DNA during replication. The full-length cDNA of the Pacific white shrimp Litopenaeus vannamei PCNA (LvPCNA) was cloned and encoded a protein of 260 amino acids that is highly similar to other Crustacean PCNAs. The theoretical shrimp PCNA structure has all the domains that are necessary for its interaction with template DNA and DNA polymerase. RT-PCR analysis showed that LvPCNA is expressed mainly in muscle and hemocytes and much less in hepatopancreas and gills. LvPCNA mRNA levels are not statistically different in muscle from healthy and challenged shrimp with the white spot syndrome virus (WSSV). In contrast, the mRNA levels of the viral DNA polymerase show a biphasic pattern with expression at 6 h post-infection and later at 24 and 48 h. These results suggest that in shrimp muscle LvPCNA levels are steadily kept to allow viral replication and that WSSV DNA polymerase (WSSV-DNApol) is more responsive towards later stages of infection. More knowledge of the DNA replication machinery would result in a better understanding of the mechanism and components of viral replication, since the WSSV genome does not have all the components required for assembly of a fully functional replisome.

  11. Epstein-Barr virus nuclear antigen 1 (EBNA1) protein induction of epithelial-mesenchymal transition in nasopharyngeal carcinoma cells.

    PubMed

    Wang, Lu; Tian, Wen-Dong; Xu, Xia; Nie, Biao; Lu, Juan; Liu, Xiong; Zhang, Bao; Dong, Qi; Sunwoo, John B; Li, Gang; Li, Xiang-Ping

    2014-02-01

    The Epstein-Barr virus (EBV)-encoded EB nuclear antigen 1 (EBNA1) protein is required for maintenance and transmission of the viral episome in EBV-infected cells. The objective of this study was to investigate the role of EBNA1 protein in nasopharyngeal carcinoma (NPC). Tissue samples from 48 patients with NPC and 12 patients with chronic nasopharyngitis were subjected to immunohistochemical analysis of EBNA1 expression. EBNA1 combinational DNA was used to overexpress EBNA1 protein in NPC cell lines to assess tumor cell epithelial-mesenchymal transition (EMT), colony formation, migration and invasion, and gene expression. EBNA1 protein was highly expressed in NPC tissue specimens, and its expression was associated with NPC lymph node metastasis. EBNA1 expression affected NPC cell morphology and the expression of EMT markers in vitro. Furthermore, overexpression of EBNA1 inhibited the expression of microRNA 200a (miR-200a) and miR-200b and, in turn, up-regulated expression of their target genes, zinc finger E-box binding homeobox 1 ( ZEB1) and ZEB2, which are well known mediators of EMT. In addition, EBNA1-regulated miR-200a and miR-200b expression was mediated by transforming growth factor-β1. The current findings provided novel insight into the vital role of EBNA1 in manipulating a molecular switch of EMT in EBV-positive NPC cells. © 2013 American Cancer Society.

  12. Nucleolin is important for Epstein–Barr virus nuclear antigen 1-mediated episome binding, maintenance, and transcription

    PubMed Central

    Chen, Ya-Lin; Liu, Cheng-Der; Cheng, Chi-Ping; Zhao, Bo; Hsu, Hao-Jen; Shen, Chih-Long; Chiu, Shu-Jun; Kieff, Elliott; Peng, Chih-wen

    2014-01-01

    Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for EBV episome maintenance, replication, and transcription. These effects are mediated by EBNA1 binding to cognate oriP DNA, which comprise 20 imperfect copies of a 30-bp dyad symmetry enhancer and an origin for DNA replication. To identify cell proteins essential for these EBNA1 functions, EBNA1 associated cell proteins were immune precipitated and analyzed by liquid chromatography-tandem mass spectrometry. Nucleolin (NCL) was identified to be EBNA1 associated. EBNA1's N-terminal 100 aa and NCL's RNA-binding domains were critical for EBNA1/NCL interaction. Lentivirus shRNA-mediated NCL depletion substantially reduced EBNA1 recruitment to oriP DNA, EBNA1-dependent transcription of an EBV oriP luciferase reporter, and EBV genome maintenance in lymphoblastoid cell lines. NCL RNA-binding domain K429 was critical for ATP and EBNA1 binding. NCL overexpression increased EBNA1 binding to oriP and transcription, whereas NCL K429A was deficient. Moreover, NCL silencing impaired lymphoblastoid cell line growth. These experiments reveal a surprisingly critical role for NCL K429 in EBNA1 episome maintenance and transcription, which may be a target for therapeutic intervention. PMID:24344309

  13. The Epstein–Barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group A proteins

    PubMed Central

    Coppotelli, Giuseppe; Mughal, Nouman; Callegari, Simone; Sompallae, Ramakrishna; Caja, Laia; Luijsterburg, Martijn S.; Dantuma, Nico P.; Moustakas, Aristidis; Masucci, Maria G.

    2013-01-01

    Viral proteins reprogram their host cells by hijacking regulatory components of protein networks. Here we describe a novel property of the Epstein–Barr virus (EBV) nuclear antigen-1 (EBNA1) that may underlie the capacity of the virus to promote a global remodeling of chromatin architecture and cellular transcription. We found that the expression of EBNA1 in transfected human and mouse cells is associated with decreased prevalence of heterochromatin foci, enhanced accessibility of cellular DNA to micrococcal nuclease digestion and decreased average length of nucleosome repeats, suggesting de-protection of the nucleosome linker regions. This is a direct effect of EBNA1 because targeting the viral protein to heterochromatin promotes large-scale chromatin decondensation with slow kinetics and independent of the recruitment of adenosine triphosphate–dependent chromatin remodelers. The remodeling function is mediated by a bipartite Gly-Arg rich domain of EBNA1 that resembles the AT-hook of High Mobility Group A (HMGA) architectural transcription factors. Similar to HMGAs, EBNA1 is highly mobile in interphase nuclei and promotes the mobility of linker histone H1, which counteracts chromatin condensation and alters the transcription of numerous cellular genes. Thus, by regulating chromatin compaction, EBNA1 may reset cellular transcription during infection and prime the infected cells for malignant transformation. PMID:23358825

  14. Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen.

    PubMed Central

    Jónsson, Z O; Hindges, R; Hübscher, U

    1998-01-01

    The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis. PMID:9545252

  15. PSL, a nuclear cell-cycle associated antigen is increased during retinoic acid-induced differentiation of HL-60 cells.

    PubMed

    Barque, J P; Lagaye, S; Ladoux, A; Della Valle, V; Abita, J P; Larsen, C J

    1987-09-30

    PSL(p55) is a nuclear 55kD antigen present in various mammalian cell systems, which has been first identified by use of human autoimmune antibodies (Barque et al. 1983, EMBO J. 2, 743). It has been shown to be associated with interphase chromatine and to be synthesized in during the S phase of the cell cycle. In this work, we have analysed the status of PSL in promyelocytic HL-60 human cells in exponential or stationary growth, or undergoing granulocytic differentiation in presence of Retinoic acid. By use of 2-dimensional electrophoresis, PSL was found to be composed of two acidic proteins designated p55A and p55B. Unexpectedly, estimated 10-20 fold higher amounts of each species were found in cells treated for 5 days with 10(-6)M Retinoic acid, than in asynchronously growing cells or resting cells. Moreover, the p55A protein was phosphorylated during the process. On the basis of these results, PSL appears to be involved in some steps of the granulocytic differentiation process.

  16. Proliferating cell nuclear antigen expression in brain tumors, and its prognostic role in ependymomas: an immunohistochemical study.

    PubMed

    Schiffer, D; Chiò, A; Giordana, M T; Pezzulo, T; Vigliani, M C

    1993-01-01

    Proliferating cell nuclear antigen (PCNA)/cyclin is currently often investigated immunohistochemically in tumors as a marker of cell proliferation, but many problems remain open concerning its reliability as a prognostic factor. PCNA has been studied in a series of 123 brain tumors using the monoclonal antibody PC10. A clear intra- and inter-tumor variability of PCNA-positive nuclei has been found, but taking into account the tumor areas with the highest number of positive nuclei, a positive correlation between this number and the histological malignancy of tumors has been demonstrated. The staining intensity of nuclei was variable; very-intensely positive nuclei, counted separately, are hypothesized to represent nuclei in S-phase of the cell cycle. In ependymomas the investigation included a quantitative statistical analysis. The number of PCNA-positive nuclei correlated with cell density and mitotic index, but only very intensely positive nuclei showed a significant statistical correlation with survival. In spite of the many possibilities of wrong interpretation of PCNA expression, the most important of which is its deregulation, the method is useful in the practice for prognostic purposes. Its important advantages are the possibility of a retrospective application and a visual analysis of the proliferation potential of tumors.

  17. Proteasome activator subunit PA28 alpha and related Ki antigen (PA28 gamma) are absent from the nuclear fraction purified by sucrose gradient centrifugation.

    PubMed

    Wójcik, C

    1999-02-01

    The aim of the present work was to attempt to partially purify PA28 (REG) alpha and gamma (Ki antigen) in the nuclear fraction from NT2/D1 cells. Nuclei were isolated by the hypertonic sucrose gradient centrifugation method and fractionated into membrane/nucleoplasmic and chromatin/nucleolar fractions. Western blotting with anti-histone and anti-beta-tubulin monoclonal antibodies confirmed the accuracy of the procedure. Proteasomes were present mainly in the cytoplasm but also in the nuclei. Disruption of the nuclear envelope released the proteasomes implying a loose or no binding with the chromatin. PA28 alpha and gamma were detected mainly in the cytosol and to a lesser extent in the crude nuclear pellet, however the purified nuclei were devoid of PA28 alpha and gamma. This indicates, that only a small fraction of the PA28 activator is present in the nuclei as detected by immunofluorescence or/and it is easily removed during nuclear purification.

  18. Cancer Specific Proliferating Cell Nuclear Antigen as a Novel Diagnostic Marker for the Detection of Breast Cancer

    DTIC Science & Technology

    2003-10-01

    CELLS(BIOLOGY), *ANTIGENS, *MAMMARY GLANDS, *BREAST CANCER, TISSUES(BIOLOGY), DETECTION, PEPTIDES, ENZYMES, PROTEINS , DIAGNOSIS(MEDICINE), PATIENTS, BLOOD SERUM, IMMUNOASSAY, GELS, ELECTROPHORESIS, SENSE ORGANS, ESTROGENS.

  19. Immunohistochemical study of p53 and proliferating cell nuclear antigen expression in odontogenic keratocyst and periapical cyst.

    PubMed

    Sajeevan, Thara Purath; Saraswathi, Tillai Rajasekaran; Ranganathan, Kannan; Joshua, Elizabeth; Rao, Uma Devi K

    2014-07-01

    p53 protein is a product of p53 gene, which is now classified as a tumor suppressor gene. The gene is a frequent target for mutation, being seen as a common step in the pathogenesis of many human cancers. Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and plays a critical role in initiation of cell proliferation. The aim of this study is to assess and compare the expression of p53 and PCNA in lining epithelium of odontogenic keratocyst (OKC) and periapical cyst (PA). A total of 20 cases comprising 10 OKC and 10 PA were included in retrospective study. Three paraffin section of 4 μm were cut, one was used for routine hematoxylin and eosin stain, while the other two were used for immunohistochemistry. Statistical analysis was performed using Chi-square test. The level of staining and intensity were assessed in all these cases. OKC showed PCNA expression in all cases (100%), whereas in perapical cyst only 60% of cases exhibited PCNA staining. (1) OKC showed p53 expression in 6 cases (60%) whereas in PA only 10% of the cases exhibited p53 staining. Chi-square test showed PCNA staining intensity was more significant than p53 in OKC. (2) The staining intensity of PA using p53, PCNA revealed that PCNA stating intensity was more significant than p53. OKC shows significant proliferative activity than PA using PCNA and p53. PCNA staining was more intense when compared with p53 in both OKC and PA.

  20. Epstein-Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1.

    PubMed

    Bazot, Quentin; Deschamps, Thibaut; Tafforeau, Lionel; Siouda, Maha; Leblanc, Pascal; Harth-Hertle, Marie L; Rabourdin-Combe, Chantal; Lotteau, Vincent; Kempkes, Bettina; Tommasino, Massimo; Gruffat, Henri; Manet, Evelyne

    2014-09-01

    The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. p53, c-myc p62 and proliferating cell nuclear antigen (PCNA) expression in non-Hodgkin's lymphomas.

    PubMed Central

    Korkolopoulou, P; Oates, J; Kittas, C; Crocker, J

    1994-01-01

    AIMS--To investigate the immunohistochemical expression of p53 protein in non-Hodgkin's lymphomas (NHL) and its relation to that of c-myc p62 oncoprotein and proliferating cell nuclear antigen (PCNA). METHODS--Paraffin wax embedded tissue from 90 non-Hodgkin's lymphomas (72 B cell and 18 T cell) was stained immunohistochemically for p53 protein, c-myc p62 oncoprotein, and PCNA using the monoclonal antibodies DO7, c-myc 1-9 E10, and PC-10, respectively. RESULTS--Of the non-Hodgkin's lymphomas studied, 55 (61%) stained positively for p53 protein. The proportion of positive cases increased from low grade non-Hodgkin's lymphoma and was higher in tumours of T cell origin. The percentage of positive cells (labelling index or LI) was significantly lower in low grade non-Hodgkin's lymphoma, but no difference was established between intermediate and high grade non-Hodgkin's lymphoma. In a large proportion of low grade non-Hodgkin's lymphoma the LI was below 1%. c-myc p62 immunoreactivity was identified in all cases. A significant positive correlation was established between p53 LI and c-myc p62 LI (rs = 0.453) as well as between p53 LI and PCNA LI (rs = 0.338). CONCLUSIONS--p53 immunoreactivity was present in about half the cases of non-Hodgkin's lymphoma and was related to the grade of malignancy and possibly to the B or T cell origin of the tumour. It was also associated with the proliferation state as expressed by PCNA LI and c-myc p62 expression, indicating that the expression of these three cell cycle-related genes might be interrelated. Images PMID:7907610

  2. Effects of the myeloid cell nuclear differentiation antigen on the proliferation, apoptosis and migration of osteosarcoma cells.

    PubMed

    Sun, Chengliang; Liu, Chuanju; Dong, Jun; Li, Dong; Li, Wei

    2014-03-01

    Despite improvements over the past two decades, the outcome for patients with advanced osteosarcoma remains poor. Targeted therapies have emerged as promising treatment options for various malignancies. However, effective targeted cancer therapies require the identification of key molecules in the pathogenesis of cancer. The aim of this study was to evaluate the value of the myeloid cell nuclear differentiation antigen (MNDA), a member of the interferon-inducible p200 (IFI-200) family, as a therapeutic target for osteosarcoma by analyzing the baseline expression of MNDA in human osteosarcoma cells and determining the effect of MNDA overexpression on the proliferation and apoptosis profiles and migration/invasion ability in osteosarcoma cells. To this end, MNDA mRNA abundance in wild-type sarcoma osteogenic (Saos-2) cells was analyzed using reverse transcription-polymerase chain reaction, proliferation/apoptosis profiles and migration/invasion capacity in Saos-2 cells overexpressing a green fluorescence protein (GFP)-human MNDA fusion protein. Saos-2 cells found to be overexpressing GFP alone were assessed by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric analysis and Matrigel Transwell migration assay. The results demonstrated that MNDA mRNA was significantly less abundant in wild-type Saos-2 cells compared with human monocyte-like U-937 cells and MNDA overexpression effectively inhibited proliferation, induced apoptosis and reduced migration/invasiveness in Saos-2 cells compared with GFP overexpression alone. Preliminary observations suggested that MNDA potentially serves as a novel therapeutic target for osteosarcoma.

  3. Variations of Epstein-Barr virus nuclear antigen 1 gene in gastric carcinomas and nasopharyngeal carcinomas from Northern China.

    PubMed

    Wang, Yun; Liu, Xia; Xing, Xiaoming; Cui, Ying; Zhao, Chengquan; Luo, Bing

    2010-02-01

    The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1), the only viral protein consistently expressed in all EBV-associated tumors, is classified into five distinct subtypes: P-ala, P-thr, V-leu, V-val and V-pro based on the signature changes at amino acid residue 487. By now, whether the EBNA1 subtypes preferentially associate with particular malignancies or represent geographical polymorphism remains controversial. In China, most studies of the EBNA1 variations focused on nasopharyngeal carcinoma (NPC) in endemic area, among which some suggested the V-val subtype is preferentially associated with NPC. To characterize the variations of EBNA1 in NPC non-endemic area in China and to explore the association of EBNA1 variations with EBV-associated gastric carcinoma (EBVaGC) and NPC, the C-terminal sequences of EBNA1 were analyzed for 41 EBVaGC, 41 NPC biopsies and 55 throat washing (TW) samples from healthy donors in Northern China. Three major patterns of the EBNA1 variations, V-val, P-thrV and V-leuV, were observed, and V-val was the most common subtype in all the three groups, followed by P-thrV and V-leuV. The distribution of the EBNA1 subtypes among EBVaGC, NPC and healthy donors was not significantly different (P>0.05). In addition, preferential linkages between EBNA1 subtypes and EBNA3C variants were found to exist. There was no evidence that particular EBNA1 subtypes are preferentially associated with EBVaGC or NPC in Northern China, suggesting that EBNA1 gene variations are geographically restricted rather than tumor-specific polymorphisms. (c) 2009 Elsevier B.V. All rights reserved.

  4. The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation.

    PubMed

    Winter, Jody A; Christofi, Panayiotis; Morroll, Shaun; Bunting, Karen A

    2009-08-22

    The high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA) to a resolution of 2.0 A. The overall architecture of HvPCNA is very similar to other known PCNAs, which are highly structurally conserved. Three commonly observed adaptations in halophilic proteins are higher surface acidity, bound ions and increased numbers of intermolecular ion pairs (in oligomeric proteins). HvPCNA possesses the former two adaptations but not the latter, despite functioning as a homotrimer. Strikingly, the positive surface charge considered key to PCNA's role as a sliding clamp is dramatically reduced in the halophilic protein. Instead, bound cations within the solvation shell of HvPCNA may permit sliding along negatively charged DNA by reducing electrostatic repulsion effects. The extent to which individual proteins adapt to halophilic conditions varies, presumably due to their diverse characteristics and roles within the cell. The number of ion pairs observed in the HvPCNA monomer-monomer interface was unexpectedly low. This may reflect the fact that the trimer is intrinsically stable over a wide range of salt concentrations and therefore additional modifications for trimer maintenance in high salt conditions are not required. Halophilic proteins frequently bind anions and cations and in HvPCNA cation binding may compensate for the remarkable reduction in positive charge in the pore region, to facilitate functional interactions with DNA. In this way, HvPCNA may harness its environment as opposed to simply surviving in extreme halophilic conditions.

  5. Proliferative cell nuclear antigen (PCNA) expression in the intestine of Salmo trutta trutta naturally infected with an acanthocephalan

    PubMed Central

    2012-01-01

    Background Changes in the production of proliferating cell nuclear antigen (PCNA), a 36 kd protein involved in protein synthesis, within intestinal epithelia can provide an early indication of deviations to normal functioning. Inhibition or stimulation of cell proliferation and PCNA can be determined through immunohistochemical staining of intestinal tissue. Changes in the expression of PCNA act as an early warning system of changes to the gut and this application has not been applied to the fields of aquatic parasitology and fish health. The current study set out to determine whether a population of wild brown trout, Salmo trutta trutta (L.) harbouring an infection of the acanthocephalan Dentitruncus truttae Sinzar, 1955 collected from Lake Piediluco in Central Italy also effected changes in the expression of PCNA. Methods A total of 29 brown trout were investigated, 19 of which (i.e. 65.5%) were found to harbour acanthocephalans (5–320 worms fish-1). Histological sections of both uninfected and infected intestinal material were immunostained for PCNA. Results The expression of PCNA was observed in the epithelial cells in the intestinal crypts and within the mast cells and fibroblasts in the submucosa layer which is consistent with its role in cell proliferation and DNA synthesis. The number of PCNA-positive cells in both the intestinal epithelium and the submucosa layer in regions close to the point of parasite attachment were significantly higher than the number observed in uninfected individuals and in infected individuals in zones at least 0.7 cm from the point of parasite attachment (ANOVA, p < 0.05). Conclusions An infection of the acanthocephalan D. truttae within the intestinal tract of S. t. trutta effected a significant increase in the number of PCNA positive cells (mast cells and fibroblasts) at the site of parasite attachment when compared to the number of positive cells found in uninfected conspecifics and in tissue zones away from the point

  6. Expression of apoptosis and proliferating cell nuclear antigen (PCNA) in the cardiac conduction system of crib death (SIDS).

    PubMed

    Matturri, L; Ottaviani, G; Lavezzi, A M; Turconi, P; Cazzullo, A; Rossi, L

    2001-07-01

    Aim of this study is to determine the expression of apoptosis and Proliferating Cell Nuclear Antigen (PCNA) in the cardiac conduction system in crib death and explained death (ED) cases. Postnatal morphogenesis of the conducting tissue is an important part of its normal development. In the atrio-ventricular node (AVN) and His bundle (HB) it consists of degeneration, cell death and replacing in an orderly programmed way. However, its nature and its relation to crib death is not yet fully explained. Apoptosis and PCNA were investigated in 8 heart conduction systems of infants dying of crib death and in 3 conduction systems of infants dying of ED as controls. The cardiac conduction system was removed in two blocks: the first included the sino-atrial node (SAN) and the crista terminalis, the second contained the atrio-ventricular node (AVN), His bundle (HB), bifurcation, and bundle branches. In the conduction systems as well as in the common myocardium the PCNA Labeling Index (PCNA-LI) was found to be negative in all cases. The apoptotic indices (AI) in SIDS and in ED were found to have no statistically significant differences (p>0.05). The SAN, in both groups, showed an AI similar to the one detected in common myocardium. In almost all cases, TUNEL labeling was detected in peripheral region of the AVN, close to the atrial myocardium. The AI was higher in the AVN, HB and the initial tract of bundle branches than in the common myocardium (p<0.05; Student's t test).

  7. Combined use of epithelial membrane antigen and nuclear matrix protein 52 as sensitive biomarkers for detection of bladder cancer.

    PubMed

    Attallah, Abdelfattah M; El-Far, Mohamed; Abdallah, Sanaa O; El-Waseef, Ahmed M; Omran, Mohamed M; Abdelrazek, Mohamed A; Attallah, Ahmed A; Saadh, Mohamed J; Radwan, Mohamed; El-waffaey, Kholoud A; Abol-Enei, Hassan

    2015-11-11

    The advent of noninvasive urine-based markers as well as other novel modalities has yielded improved diagnostic accuracy. However, the new markers failed to reach higher sensitivity and specificity. We therefore evaluated the potential role of epithelial membrane antigen (EMA) and nuclear matrix protein 52 (NMP-52) singly and combined as noninvasive biomarkers for the detection of bladder cancer (BC). A total of 160 individuals including 66 patients with BC, 54 patients with benign urologic disorders and 40 healthy volunteers were investigated. Urinary EMA at 130 kDa and NMP at 52 kDa were identified, purified and quantified by Western blot, electroelution and enzyme-linked immunosorbent assay (ELISA). The diagnostic performance of each biomarker and their combination were compared using area under receiver operating characteristic curves (AUC). Mean urinary EMA, 2.42 µg/mL, and NMP-52, 17.85 µg/mL, were significantly elevated in patients with BC compared to controls, 1.18 and 3.44 µg/mL, respectively (p<0.0001). The combined use of these markers yielded values which were increased 4.4- and 13.7-fold in the benign and malignant disease groups, respectively, with respect to the normal group. The values of EMA and NMP-52 were significantly higher in patients with higher-grade tumors than those with lower-grade tumors (p<0.0001). Moreover, this combination could predict all BC stages and grades with 0.91 AUC, 94% sensitivity and 80% specificity. EMA and NMP-52 in combination could be promising noninvasive biomarkers for BC detection.

  8. Proliferating Cell Nuclear Antigen Has an Association with Prognosis and Risks Factors of Cancer Patients: a Systematic Review.

    PubMed

    Lv, Qiongying; Zhang, Juan; Yi, Yuexiong; Huang, Yue; Wang, Yong; Wang, Yijun; Zhang, Wei

    2016-11-01

    Proliferating cell nuclear antigen (PCNA) is reported as a famous marker in various tumors. A couple of articles have been published about the clinical function of PCNA on cancer progression; however, these results are conflicting in some degree. Thus, it is crucial to perform a systematic review and meta-analysis to identify their real actions. Here, we took cervical cancer and glioma as example and then pooled hazard ratios (HRs) or odds ratios (ORs) with 95 % confidence intervals (95 % CIs). In the present study, the PCNA expression in cervical cancer and gliomas patients was both correlated with 5-year-overall survival (OS) (HR = 4.41, 95 % CI 2.71-7.17, p = 0.000; HR = 4.40, 95 % CI 3.00-6.47, p = 0.000; respectively). In addition, a fixed effect model revealed a significant association between PCNA and FIGO stage (OR = 4.48, 95 % CI 3.48-5.77, p = 0.000) or WHO grade (OR = 5.64, 95 % CI 4.15-7.68, p = 0.000), rather than age (OR = 1.01, 95 % CI 0.71-1.43, p = 0.957; OR = 1.00, 95 % CI 0.80-1.24, p = 0.989; respectively). No heterogeneity was observed across all studies. According to funnel plot, no publication bias was reported. In conclusion, our systematic review suggests that PCNA expression is significantly associated with poor 5-year survival, advanced stage or higher WHO grade, which might be suggested as a useful prognostic and diagnostic biomarker, or an effective therapy target in cervical cancer, gliomas, or even more cancers.

  9. Proliferating Cell Nuclear Antigen (PCNA) Regulates Primordial Follicle Assembly by Promoting Apoptosis of Oocytes in Fetal and Neonatal Mouse Ovaries

    PubMed Central

    Zhang, Yuanwei; Jiang, Xiaohua; Zhang, Huan; Ma, Tieliang; Zheng, Wei; Sun, Rui; Shen, Wei; Sha, Jiahao; Cooke, Howard J.; Shi, Qinghua

    2011-01-01

    Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer guanulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries. PMID:21253613

  10. Low power laser irradiation stimulates cell proliferation via proliferating cell nuclear antigen and Ki-67 expression during tissue repair

    NASA Astrophysics Data System (ADS)

    Prabhu, Vijendra; Rao, Bola Sadashiva Satish; Mahato, Krishna Kishore

    2015-03-01

    Low power laser irradiation (LPLI) is becoming an increasingly popular and fast growing therapeutic modality in dermatology to treat various ailments without any reported side effects. In the present study an attempt was made to investigate the proliferative potential of red laser light during tissue repair in Swiss albino mice. To this end, full thickness excisional wounds of diameter 15 mm created on mice were exposed to single dose of Helium-Neon laser (632.8 nm; 7 mW; 4.02 mWcm-2; Linear polarization) at 2 Jcm-2 and 10 Jcm-2 along with un-illuminated controls. The granulation tissues from all the respective experimental groups were harvested on day 10 post-wounding following euthanization. Subsequently, tissue regeneration potential of these laser doses under study were evaluated by monitoring proliferating cell nuclear antigen and Ki-67 following the laser treatment and comparing it with the un-illuminated controls. The percentages of Ki-67 or PCNA positive cells were determined by counting positive nuclei (Ki-67/PCNA) and total nuclei in five random fields per tissue sections. Animal wounds treated with single exposure of the 2 Jcm-2 indicated significant elevation in PCNA (P<0.01) and Ki-67 (P<0.05 compared to un-illuminated control and P<0.01 compared to 10 Jcm-2) expression as compared to other tested experimental groups as evidenced by the microscopy results in the study. In summary, the findings of the present study have clearly demonstrated the regulation of cell proliferation by LPLI via PCNA and Ki-67 expression during tissue regeneration.

  11. Sequence Variation Analysis of Epstein-Barr Virus Nuclear Antigen 1 Gene in the Virus Associated Lymphomas of Northern China.

    PubMed

    Sun, Lingling; Zhao, Zhenzhen; Liu, Song; Liu, Xia; Sun, Zhifu; Luo, Bing

    2015-01-01

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein expressed in all EBV-positive tumors as it is essential for the maintenance, replication and transcription of the virus genome. According to the polymorphism of residue 487 in EBNA1 gene, EBV isolates can be classified into five subtypes: P-ala, P-thr, V-val, V-leu and V-pro. Whether these EBNA1 subtypes contribute to different tissue tropism of EBV and are consequently associated with certain malignancies remain to be determined. To elucidate the relationship, one hundred and ten EBV-positive lymphoma tissues of different types from Northern China, a non-NPC endemic area, were tested for the five subtypes by nested-PCR and DNA sequencing. In addition, EBV type 1 and type 2 classification was typed by using standard PCR assays across type-specific regions of the EBNA3C genes. Four EBNA1 subtypes were identified: V-val (68.2%, 75/110), P-thrV (15.5%, 17/110), V-leuV (3.6%, 4/110) and P-ala (10.9%, 12/110). The distribution of the EBNA1 subtypes in the four lymphoma groups was not significantly different (p = 0.075), neither was that of the EBV type 1/type 2 (p = 0.089). Compared with the previous data of gastric carcinoma (GC), nasopharyngeal carcinoma (NPC) and throat washing (TW) from healthy donors, the distribution of EBNA1 subtypes in lymphoma differed significantly (p = 0.016), with a little higher frequency of P-ala subtype. The EBV type distribution between lymphoma and the other three groups was significantly different (p = 0.000, p = 0.000, p = 0.001, respectively). The proportion of type 1 and type 2 mixed infections was higher in lymphoma than that in GC, NPC and TW. In lymphomas, the distribution of EBNA1 subtypes in the three EBV types was not significantly different (p = 0.546). These data suggested that the variation patterns of EBNA1 gene may be geographic-associated rather than tumor-specific and the role of EBNA1 gene variations in tumorigenesis needs more extensive and

  12. Comparison of cellular localization of thallium-201, proliferating cell nuclear antigen and Ki-67 in C6 gliomas

    SciTech Connect

    Krishna, L.; Katsetos, C.D.; Vender, J.

    1996-05-01

    In order to substantiate the use of thallium-201 scintigraphy as a tool to evaluate the proliferative capacity of a glioma, we compared the patterns of cellular localization of thallium-201 (Tl-201) with established proliferation markers - proliferating cell nuclear antigen (PCNA) and Ki-67 in C6 gliomas. Six Sprague-Dawley rats were stereotactically implanted with C6 glioma cells intracerebrally. On day 7 post-implantation, 50uCi of Tl-201 chloride were injected intravenously to each animal. The animals were sacrificed 60 minutes post-injection and the brain was immediately removed and frozen in dry ice to preserve cellular integrity. Ten um sections of the C6 glioma were mounted on gelatin coated slides. Consecutive slides were used to perform microautoradiographic localization of Tl-201, as well as immunohistochemical localization of PCNA and Ki-67. Localization of all markers were measured by counting and comparing either silver grain density (for Tl-201), or immunostained cells (for PCNA and Ki-67) in at least 1000 cells in glioma vs normal brain. All three markers localized primarily in the glioma as opposed to normal brain at statistically significant levels at p<0.05. Mean indices for glioma vs non-glioma regions were (i) Tl-201: 142 grains/cm{sup 2} vs 11 grains/cm; (ii) PCNA: 92% vs 4%; (iii) Ki-67: 74% vs 3%. The significant and selective localization of the proliferation markers PCNA and Ki-67 as well as Tl-201 in the glioma cells provides validation at a cellular level, that Tl-201 can be used as a proliferation marker. Existing technology ie. Tl-201 scintigraphy, can be used in the management of biopsy-proven gliomas, to measure the proliferative capacity of the tumor. The advantages of using a non-invasive, relatively inexpensive proliferation marker such as Tl-201 scintigraphy include the capacity to evaluate the proliferation potential of the entire glioma, thereby decreasing the sampling errors inherent in evaluating biopsy specimens.

  13. Sequence Variation Analysis of Epstein-Barr Virus Nuclear Antigen 1 Gene in the Virus Associated Lymphomas of Northern China

    PubMed Central

    Sun, Lingling; Zhao, Zhenzhen; Liu, Song; Liu, Xia; Sun, Zhifu; Luo, Bing

    2015-01-01

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein expressed in all EBV-positive tumors as it is essential for the maintenance, replication and transcription of the virus genome. According to the polymorphism of residue 487 in EBNA1 gene, EBV isolates can be classified into five subtypes: P-ala, P-thr, V-val, V-leu and V-pro. Whether these EBNA1 subtypes contribute to different tissue tropism of EBV and are consequently associated with certain malignancies remain to be determined. To elucidate the relationship, one hundred and ten EBV-positive lymphoma tissues of different types from Northern China, a non-NPC endemic area, were tested for the five subtypes by nested-PCR and DNA sequencing. In addition, EBV type 1 and type 2 classification was typed by using standard PCR assays across type-specific regions of the EBNA3C genes. Four EBNA1 subtypes were identified: V-val (68.2%, 75/110), P-thrV (15.5%, 17/110), V-leuV (3.6%, 4/110) and P-ala (10.9%, 12/110). The distribution of the EBNA1 subtypes in the four lymphoma groups was not significantly different (p = 0.075), neither was that of the EBV type 1/type 2 (p = 0.089). Compared with the previous data of gastric carcinoma (GC), nasopharyngeal carcinoma (NPC) and throat washing (TW) from healthy donors, the distribution of EBNA1 subtypes in lymphoma differed significantly (p = 0.016), with a little higher frequency of P-ala subtype. The EBV type distribution between lymphoma and the other three groups was significantly different (p = 0.000, p = 0.000, p = 0.001, respectively). The proportion of type 1 and type 2 mixed infections was higher in lymphoma than that in GC, NPC and TW. In lymphomas, the distribution of EBNA1 subtypes in the three EBV types was not significantly different (p = 0.546). These data suggested that the variation patterns of EBNA1 gene may be geographic-associated rather than tumor-specific and the role of EBNA1 gene variations in tumorigenesis needs more extensive and

  14. Expression of cloned herpesvirus genes. I. Detection of nuclear antigens from herpes simplex virus type 2 inverted repeat regions in transfected mouse cells.

    PubMed Central

    Middleton, M H; Reyes, G R; Ciufo, D M; Buchan, A; Macnab, J C; Hayward, G S

    1982-01-01

    Three different recombinant plasmids containing the entire 15-kilobase L and S inverted repeat sequence of herpes simplex virus type 2 DNA have been introduced into cultured Ltk- or BSC cells by both the calcium and DEAE-dextran transfection procedures. In each case, after 24 h approximately 1% of the cells gave strongly positive nuclear staining when assayed by immunofluorescence with hyperimmune antisera made against early and immediate-early infected-cell polypeptides. The nuclear fluorescence pattern and intensity mimicked that observed within 2 to 3 h after infection of Ltk- cells with either herpes simplex virus type 1 or type 2 wild-type virus. Herpes simplex virus type 1 (KOStsB2)-infected Ltk- cells under nonpermissive conditions did not express these antigens in the nucleus. Therefore, we conclude that either one or both of the 185,000- and 110,000-molecular-weight immediate early proteins, or some other as yet unknown gene product encoded entirely within the inverted repeats, can be transiently expressed in large amounts in transfected cells in the absence of other viral genes or accompanying virion components. Permanent mouse cell lines derived from transfection with these plasmids by using the thymidine kinase coselection procedure did not express sufficient nuclear antigen to be detectable by immunofluorescence. Images PMID:6292452

  15. Molecular recognition of the Thomsen-Friedenreich antigen-threonine conjugate by adhesion/growth regulatory galectin-3: nuclear magnetic resonance studies and molecular dynamics simulations.

    PubMed

    Yongye, Austin B; Calle, Luis; Ardá, Ana; Jiménez-Barbero, Jesús; André, Sabine; Gabius, Hans-Joachim; Martínez-Mayorga, Karina; Cudic, Mare

    2012-09-18

    Nuclear magnetic resonance (NMR) spectroscopy and molecular modeling methods have been strategically combined to elucidate the molecular recognition features of the binding of threonine O-linked Thomsen-Friedenreich (TF) antigen to chimera-type avian galectin-3 (CG-3). Saturation transfer difference (STD) NMR experiments revealed the highest intensities for the H4 protons of both the β-D-Galp and α-D-GalpNAc moieties, with 100 and 71% of relative STD, respectively. The methyl protons of the threonine residue exhibited a small STD effect, <15%, indicating that the interaction of the amino acid with the protein is rather transient. Two-dimensional transferred nuclear Overhauser effect spectroscopy NMR experiments and molecular modeling suggested some differences in conformer populations between the free and bound states. A dynamic binding mode for the TF antigen-CG-3 complex consisting of two poses has been deduced. In one pose, intermolecular interactions were formed between the terminal threonine residue and the receptor. In the second pose, intermolecular interactions involved the internal GalpNAc. The difference in the trend of some shifts in the heteronuclear single-quantum coherence titration spectra indicates some disparities in the binding interactions of CG-3 with lactose and TF antigen. The results obtained from this model of the avian orthologue of human galectin-3 will allow detailed interspecies comparison to give sequence deviations in phylogeny a structural and functional meaning. Moreover, the results indicate that the peptide scaffold presenting TF antigen could be relevant for binding and thus provides a possible route for the design of galectin-3 inhibitors with improved affinity and selectivity.

  16. Simultaneous cytoplasmic and nuclear protein expression of melanoma antigen-A family and NY-ESO-1 cancer-testis antigens represents an independent marker for poor survival in head and neck cancer.

    PubMed

    Laban, Simon; Atanackovic, Djordje; Luetkens, Tim; Knecht, Rainald; Busch, Chia-Jung; Freytag, Marcus; Spagnoli, Giulio; Ritter, Gerd; Hoffmann, Thomas K; Knuth, Alexander; Sauter, Guido; Wilczak, Waldemar; Blessmann, Marco; Borgmann, Kerstin; Muenscher, Adrian; Clauditz, Till S

    2014-09-01

    The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p < 0.0001), 46.6 versus 50.0 versus 109.0 for MAGE-A3/A4 (p = 0.0074) and 13.3 versus 50.0 versus 100.2 months for NY-ESO-1 (p = 0.0019). By multivariate analysis, these factors were confirmed as independent markers for poor survival. HNSCC patients showing protein expression of MAGE-A family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients. © 2014 UICC.

  17. [Effect of fibroblast growth factor 1 on the proliferating cell nuclear antigen expression in submandibular gland of diabetic mice].

    PubMed

    Zhou, Z H; Shi, L; Lang, M J; Chen, Z L; Wang, Y L; He, S

    2017-05-09

    Objective: To examine the proliferating cell nuclear antigen (PCNA) expression in submandibular gland of diabetic mice and to investigate the influence of fibroblast growth factor 1 (FGF-1) on PCNA expression and its possible mechanism. Methods: Sixteen db/db diabetic male mice were randomly divided into diabetic group and diabetic-FGF-1 group (n=8). Eight age-matched db/m mice served as a control group. After FGF-1 was administered intraperitoneally to diabetic-FGF-1 group continuously for 16 weeks, blood glucose and body weight of each mouse in the three groups were detected at 0, 4, 8, 12, 16 weeks. Then the flow rate of saliva in three groups was compared at 0, 8, 16 weeks. At 16 week, bilateral submandibular glands were resected. Then HE staining was performed to observe the histological morphology of submandibular gland and PCNA expression was examined by immunohistochemical staining. Results: Four weeks after administration, the blood glucose in diabetic-FGF-1 group decreased markedly, close to the control group (P>0.05). Weight loss in diabetic-FGF-1 group was noticeable at 8 weeks after administration, but still higher than that in the control group (P<0.05). The flow rate of saliva in diabetic-FGF-1 group increased gradually after administration, which was higher at 8, 16 weeks ([260.1±43.3], [308.5±34.0] mg·min(-1)·kg(-1)) respectively than that in the diabetic group at the same time point ([181.8±37.5], [194.9±49.8] mg·min(-1)·kg(-1)) (P<0.05). Compared with the control group, submandibular glands in diabetic group significantly atrophied and the glandular atrophy in diabetic-FGF-1 group was alleviated. The submandibular gland index in the control group, diabetic group and diabetic-FGF-1 group were (7.45±0.63), (2.23±0.26), (3.97±0.15) mg/g, respectively (P<0.05). HE staining showed that the histological morphology of submandibular gland in diabetic-FGF-1 group was clearer, and acinar and ductal atrophy were less significant than diabetic

  18. Generalized Latent Trait Models.

    ERIC Educational Resources Information Center

    Moustaki, Irini; Knott, Martin

    2000-01-01

    Discusses a general model framework within which manifest variables with different distributions in the exponential family can be analyzed with a latent trait model. Presents a unified maximum likelihood method for estimating the parameters of the generalized latent trait model and discusses the scoring of individuals on the latent dimensions.…

  19. RFHVMn ORF73 is structurally related to the KSHV ORF73 latency-associated nuclear antigen (LANA) and is expressed in retroperitoneal fibromatosis (RF) tumor cells

    SciTech Connect

    Burnside, Kellie L.; Ryan, Jonathan T.; Bielefeldt-Ohmann, Helle; Gregory Bruce, A.; Thouless, Margaret E.; Tsai, Che-Chung; Rose, Timothy M. . E-mail: trose@u.washington.edu

    2006-10-10

    Retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), was first identified in retroperitoneal fibromatosis (RF) tumor lesions of macaques with simian AIDS. We cloned and sequenced the ORF73 latency-associated nuclear antigen (LANA) of RFHVMn from the pig-tailed macaque. RFHVMn LANA is structurally analogous to KSHV ORF73 LANA and contains an N-terminal serine-proline-rich region, a large internal glutamic acidic-rich repeat region and a conserved C-terminal domain. RFHVMn LANA reacts with monoclonal antibodies specific for a glutamic acid-proline dipeptide motif and a glutamic acid-glutamine-rich motif in the KSHV LANA repeat region. Immunohistochemical and immunofluorescence analysis revealed that RFHVMn LANA is a nuclear antigen which is highly expressed in RF spindloid tumor cells. These data suggest that RFHV LANA is an ortholog of KSHV LANA and will function similarly to maintain viral latency and play a role in tumorigenicity in macaques.

  20. Immunohistochemical determination of nuclear antigens by colour image analysis: application for labelling index, estrogen and progesterone receptor status in breast cancer.

    PubMed

    Rostagno, P; Birtwisle, I; Ettore, F; Courdi, A; Gioanni, J; Namer, M; Caldani, C

    1994-12-01

    The immunohistochemical evaluation of 5-bromo-2'-deoxyuridine (BrdU) labelling index, estrogen (ER) and progesterone receptor (PR) status was carried out on the automated computer-assisted image analysis station BIOCOM 500. Special software has been developed to measure nuclear antigens using the immunoperoxidase method with the Harris hematoxylin counterstain. The analysis was based on the different light adsorption spectra of the chromogen diaminobenzidine and Harris's hematoxylin coloration when exposed to light of differing wavelengths. The results obtained by image analysis were compared to previously validated methods. The thymidine labelling index performed by manual procedures and BrdU incorporation performed by image analysis were comparable (linear correlation coefficient r = 0.76, P < 0.001). Comparison of image analysis and dextran coated charcoal assay for ER and PR content revealed excellent sensitivities and specificities (linear correlation coefficient r = 0.87, P < 0.001 for ER and r = 0.93, P < 0.001 for PR). These data suggest that automated image analysis offers a reliable and reproducible procedure for measuring nuclear antigens.

  1. Myeloid cell nuclear differentiation antigen is expressed in a subset of marginal zone lymphomas and is useful in the differential diagnosis with follicular lymphoma.

    PubMed

    Metcalf, Ryan A; Monabati, Ahmad; Vyas, Monika; Roncador, Giovanna; Gualco, Gabriela; Bacchi, Carlos E; Younes, Sheren F; Natkunam, Yasodha; Freud, Aharon G

    2014-08-01

    The diagnosis of marginal zone lymphomas (MZL) is challenged by the lack of specific markers that distinguish them from other low-grade non-Hodgkin B-cell lymphomas. Myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein that labels myelomonocytic cells as well as B lymphocytes that localize to the marginal zone areas of splenic white pulp. We evaluated MNDA expression in a large series of B-cell lymphomas to assess the sensitivity and specificity of this antigen for the characterization of MZL. A total of 440 tissue sections containing extramedullary B-cell lymphomas and 216 bone marrow biopsies containing atypical or neoplastic lymphoid infiltrates were stained for MNDA by immunohistochemistry. Among the extramedullary lymphoma cases, approximately 67% of nodal MZL, 61% of extranodal MZL, and 24% of splenic MZL expressed MNDA. MNDA was also infrequently expressed in other B-cell neoplasms including mantle cell lymphoma (6%), chronic lymphocytic leukemia/small lymphocytic lymphoma (13%), follicular lymphoma (FL) (4%), lymphoplasmacytic lymphoma (25%), and diffuse large B-cell lymphoma (3%). In contrast, MNDA was only expressed in 2.3% of all bone marrow biopsies involved by lymphoid infiltrates, including 2 cases of FL and one case of MZL. Collectively, these data support the inclusion of MNDA in the diagnostic evaluation of extramedullary B-cell lymphomas, particularly those in which the differential diagnosis is between low-grade FL and MZL.

  2. Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mimics Epstein-Barr Virus EBNA1 Immune Evasion through Central Repeat Domain Effects on Protein Processing▿

    PubMed Central

    Kwun, Hyun Jin; da Silva, Suzane Ramos; Shah, Ishita M.; Blake, Neil; Moore, Patrick S.; Chang, Yuan

    2007-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5′ or 3′ end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses. PMID:17522213

  3. Inhibition of nuclear factor-kappa B enhances the capacity of immature dendritic cells to induce antigen-specific tolerance in experimental autoimmune encephalomyelitis.

    PubMed

    Iruretagoyena, Mirentxu I; Sepúlveda, Sofía E; Lezana, J Pablo; Hermoso, Marcela; Bronfman, Miguel; Gutiérrez, Miguel A; Jacobelli, Sergio H; Kalergis, Alexis M

    2006-07-01

    Autoimmune disorders develop as a result of deregulated immune responses that target self-antigens and cause destruction of healthy host tissues. Because dendritic cells (DCs) play an important role in the maintenance of peripheral immune tolerance, we are interested in identifying means of enhancing their therapeutic potential in autoimmune diseases. It is thought that during steady state, DCs are able to anergize potentially harmful T cells bearing T cell receptors that recognize self-peptide-major histocompatibility complexes. The tolerogenic capacity of DCs requires an immature phenotype, which is characterized by a reduced expression of costimulatory molecules. On the contrary, activation of antigen-specific naive T cells is enhanced by DC maturation, a process that involves expression of genes controlled by the transcription factor nuclear factor (NF)-kappaB. We evaluated the capacity of drugs that inhibit NF-kappaB to enhance the tolerogenic properties of immature DCs in the experimental autoimmune encephalomyelitis (EAE) model. We show that andrographolide, a bicyclic diterpenoid lactone, and rosiglitazone, a peroxisome proliferator-activated receptor gamma agonist, were able to interfere with NF-kappaB activation in murine DCs. As a result, treated DCs showed impaired maturation and a reduced capacity to activate antigen-specific T cells. Furthermore, NF-kappaB-blocked DCs had an enhanced tolerogenic capacity and were able to prevent EAE development in mice. The tolerogenic feature was specific for myelin antigens and involved the expansion of regulatory T cells. These data suggest that NF-kappaB blockade is a potential pharmacological approach that can be used to enhance the tolerogenic ability of immature DCs to prevent detrimental autoimmune responses.

  4. Increased expression of CD54, CD18, MHC class II molecules, and proliferating cell nuclear antigen in acute puromycin aminonucleoside nephrosis.

    PubMed

    Fernandez, Lucas; Romero, Maritza; Rincón, Jaimar; Mosquera, Jesús

    2003-01-01

    Cellular infiltration to renal tissues is an important feature during acute puromycin aminonucleoside nephrosis (PAN) in rats. The mechanisms responsible for this infiltration are poorly understood. To elucidate the participation of adhesion molecules in PAN, nephrosis was induced in rats by intraperitoneal puromycin aminonucleoside injection. Controls represent animals injected with a 0.9% saline solution. ICAM-1 (intercellular adhesion molecule 1), CD18 (beta chain of lymphocyte-function-associated antigen), LCA (leukocyte common antigen), ED1 (monocyte/macrophage marker), and proliferating cell nuclear antigen expressions were evaluated in renal tissues 1, 2, and 7 weeks after injection. Frozen sections from PAN rat kidneys showed increased expressions of ICAM-1 and its ligand, and these findings were associated with increased levels of LCA+ and ED1+ cells in glomerulus and interstitium. The kinetics of leukocyte infiltration was similar to the kinetics of ICAM-1 expression: high values at week 2 which returned to normal values at week 7. Increased glomerular and interstitial proliferative activities (proliferating cell nulear antigen positive cells) were also found at week 2 of nephrosis. There was a correlation between ICAM-1 expression and numbers of LCA+ and ED1+ cells and between numbers of LCA+ cells and proliferating cells in glomerulus and interstitium. Correlations between glomerular and tubular ICAM-1 expression, interstitial leukocyte infiltration, and glomerular, interstitial, and tubular proliferative activities with the proteinuria were also observed during the nephrotic phase. In addition, increased lymphocyte binding to PAN renal tissues was observed, and this binding was diminished by anti-LFA-1beta monoclonal antibody pretreatment of lymphocytes. A similar result was found with anti-ICAM-1 monoclonal antibody pretreatment of renal tissues. Our results suggest that increased expression of ICAM-1 and proliferative activity could be important

  5. EGCG debilitates the persistence of EBV latency by reducing the DNA binding potency of nuclear antigen 1

    SciTech Connect

    Chen, Ya-Lin; Tsai, Hsing-Lyn; Peng, Chih-Wen

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Two cell-based reporter platforms were established for screening of EBNA1 inhibitors. Black-Right-Pointing-Pointer EGCG acts as an inhibitor to block EBNA1 binding with the cognate oriP sequence. Black-Right-Pointing-Pointer EGCG debilitates EBNA1-dependent transcription enhancement and episome maintenance. Black-Right-Pointing-Pointer EGCG impairs persistence of EBV latency. Black-Right-Pointing-Pointer EGCG is a potent anti-EBV agent for targeting the latent cascade of EBV. -- Abstract: Because the expression of EBNA1 is prevalent in all EBV-associated tumors, it has become one of the most attractive drug targets for the discovery of anti-EBV compounds. In a cell-based reporter system, EBNA1 consistently upregulated the transcription of an oriP-Luc mini-EBV episome by 6- to 8-fold. The treatment of cells with 50 {mu}M EGCG effectively blocked the binding of EBNA1 to oriP-DNA both in vivo and in vitro, which led to the abrogation of EBNA1-dependent episome maintenance and transcriptional enhancement. Importantly, the anti-EBNA1 effects caused by EGCG ultimately impaired the persistence of EBV latent infection. Our data suggest that the inhibition of EBNA1 activity by EGCG could be a promising starting point for the development of new protocols for anti-EBV therapy.

  6. The ubiquitin-specific protease USP7 modulates the replication of Kaposi's sarcoma-associated herpesvirus latent episomal DNA.

    PubMed

    Jäger, Wiebke; Santag, Susann; Weidner-Glunde, Magdalena; Gellermann, Eva; Kati, Semra; Pietrek, Marcel; Viejo-Borbolla, Abel; Schulz, Thomas F

    2012-06-01

    Kaposi's sarcoma herpesvirus (KSHV) belongs to the gamma-2 Herpesviridae and is associated with three neoplastic disorders: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). The viral latency-associated nuclear antigen 1 (LANA) is expressed in all latently KSHV-infected cells and is involved in viral latent replication and maintenance of the viral genome. We show that LANA interacts with the ubiquitin-specific protease USP7 through its N-terminal TRAF (tumor necrosis factor [TNF] receptor-associated factor) domain. This interaction involves a short sequence (amino acids [aa] 971 to 986) within the C-terminal domain of LANA with strong similarities to the USP7 binding site of the Epstein-Barr virus (EBV) EBNA-1 protein. A LANA mutant with a deletion of the identified USP7 binding site showed an enhanced ability to replicate a plasmid containing the KSHV latent origin of replication but was comparable to the wild-type LANA (LANA WT) with regard to the regulation of viral and cellular promoters. Furthermore, the LANA homologues of two other gamma-2 herpesviruses, MHV68 and RRV, also recruit USP7. Our findings suggest that recruitment of USP7 to LANA could play a role in the regulation of viral latent replication. The recruitment of USP7, and its role in herpesvirus latent replication, previously described for the latent EBNA-1 protein of the gamma-1 herpesvirus (lymphocryptovirus) EBV (M. N. Holowaty et al., J. Biol. Chem. 278:29987-29994, 2003), may thereby be a conserved feature among gammaherpesvirus latent origin binding proteins.

  7. The Ubiquitin-Specific Protease USP7 Modulates the Replication of Kaposi's Sarcoma-Associated Herpesvirus Latent Episomal DNA

    PubMed Central

    Jäger, Wiebke; Santag, Susann; Weidner-Glunde, Magdalena; Gellermann, Eva; Kati, Semra; Pietrek, Marcel; Viejo-Borbolla, Abel

    2012-01-01

    Kaposi's sarcoma herpesvirus (KSHV) belongs to the gamma-2 Herpesviridae and is associated with three neoplastic disorders: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). The viral latency-associated nuclear antigen 1 (LANA) is expressed in all latently KSHV-infected cells and is involved in viral latent replication and maintenance of the viral genome. We show that LANA interacts with the ubiquitin-specific protease USP7 through its N-terminal TRAF (tumor necrosis factor [TNF] receptor-associated factor) domain. This interaction involves a short sequence (amino acids [aa] 971 to 986) within the C-terminal domain of LANA with strong similarities to the USP7 binding site of the Epstein-Barr virus (EBV) EBNA-1 protein. A LANA mutant with a deletion of the identified USP7 binding site showed an enhanced ability to replicate a plasmid containing the KSHV latent origin of replication but was comparable to the wild-type LANA (LANA WT) with regard to the regulation of viral and cellular promoters. Furthermore, the LANA homologues of two other gamma-2 herpesviruses, MHV68 and RRV, also recruit USP7. Our findings suggest that recruitment of USP7 to LANA could play a role in the regulation of viral latent replication. The recruitment of USP7, and its role in herpesvirus latent replication, previously described for the latent EBNA-1 protein of the gamma-1 herpesvirus (lymphocryptovirus) EBV (M. N. Holowaty et al., J. Biol. Chem. 278:29987–29994, 2003), may thereby be a conserved feature among gammaherpesvirus latent origin binding proteins. PMID:22514345

  8. Measurement of Phenotype and Absolute Number of Circulating Heparin-Binding Hemagglutinin, ESAT-6 and CFP-10, and Purified Protein Derivative Antigen-Specific CD4 T Cells Can Discriminate Active from Latent Tuberculosis Infection

    PubMed Central

    Barkham, Timothy M. S.; Tang, Wenying; Kemeny, David M.; Chee, Cynthia Bin-Eng; Wang, Yee T.

    2014-01-01

    The tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are used as adjunctive tests for the evaluation of suspected cases of active tuberculosis (TB). However, a positive test does not differentiate latent from active TB. We investigated whether flow cytometric measurement of novel combinations of intracellular cytokines and surface makers on CD4 T cells could differentiate between active and latent TB after stimulation with Mycobacterium tuberculosis-specific proteins. Blood samples from 60 patients referred to the Singapore Tuberculosis Control Unit for evaluation for active TB or as TB contacts were stimulated with purified protein derivative (PPD), ESAT-6 and CFP-10, or heparin-binding hemagglutinin (HBHA). The CD4 T cell cytokine response (IFN-γ, interleukin-2 [IL-2], interleukin-17A [IL-17A], interleukin-22 [IL-22], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor alpha [TNF-α]) and surface marker expression (CD27, CXCR3, and CD154) were then measured. We found that the proportion of PPD-specific CD4 T cells, defined as CD154+ TNF-α+ cells that were negative for CD27 and positive for GM-CSF, gave the strongest discrimination between subjects with latent and those with active TB (area under the receiver operator characteristic [ROC] curve of 0.9277; P < 0.0001). Also, the proportions and absolute numbers of HBHA-specific CD4 T cells were significantly higher in those with latent TB infection, particularly CD154+ TNF-α+ IFN-γ+ IL-2+ and CD154+ TNF-α+ CXCR3+. Finally, we found that the ratio of ESAT-6- and CFP-10-responding to HBHA-responding CD4 T cells was significantly different between the two study populations. In conclusion, we found novel markers of M. tuberculosis-specific CD4 cells which differentiate between active and latent TB. PMID:25520147

  9. Identifying Patient-Specific Epstein-Barr Nuclear Antigen-1 Genetic Variation and Potential Autoreactive Targets Relevant to Multiple Sclerosis Pathogenesis

    PubMed Central

    Tschochner, Monika; Leary, Shay; Cooper, Don; Strautins, Kaija; Chopra, Abha; Clark, Hayley; Choo, Linda; Dunn, David; James, Ian; Carroll, William M.; Kermode, Allan G.; Nolan, David

    2016-01-01

    Background Epstein-Barr virus (EBV) infection represents a major environmental risk factor for multiple sclerosis (MS), with evidence of selective expansion of Epstein-Barr Nuclear Antigen-1 (EBNA1)-specific CD4+ T cells that cross-recognize MS-associated myelin antigens in MS patients. HLA-DRB1*15-restricted antigen presentation also appears to determine susceptibility given its role as a dominant risk allele. In this study, we have utilised standard and next-generation sequencing techniques to investigate EBNA-1 sequence variation and its relationship to HLA-DR15 binding affinity, as well as examining potential cross-reactive immune targets within the central nervous system proteome. Methods Sanger sequencing was performed on DNA isolated from peripheral blood samples from 73 Western Australian MS cases, without requirement for primary culture, with additional FLX 454 Roche sequencing in 23 samples to identify low-frequency variants. Patient-derived viral sequences were used to predict HLA-DRB1*1501 epitopes (NetMHCII, NetMHCIIpan) and candidates were evaluated for cross recognition with human brain proteins. Results EBNA-1 sequence variation was limited, with no evidence of multiple viral strains and only low levels of variation identified by FLX technology (8.3% nucleotide positions at a 1% cut-off). In silico epitope mapping revealed two known HLA-DRB1*1501-restricted epitopes (‘AEG’: aa 481–496 and ‘MVF’: aa 562–577), and two putative epitopes between positions 502–543. We identified potential cross-reactive targets involving a number of major myelin antigens including experimentally confirmed HLA-DRB1*15-restricted epitopes as well as novel candidate antigens within myelin and paranodal assembly proteins that may be relevant to MS pathogenesis. Conclusions This study demonstrates the feasibility of obtaining autologous EBNA-1 sequences directly from buffy coat samples, and confirms divergence of these sequences from standard laboratory strains

  10. The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins

    PubMed Central

    1993-01-01

    The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length cDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated cDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen cDNA. The central part of the Ki-67 antigen cDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycle-associated nuclear nonhistone proteins. PMID:8227122

  11. Characterization of Epstein-Barr virus type 1 nuclear antigen 3C sequence patterns of nasopharyngeal and gastric carcinomas in northern China.

    PubMed

    Wu, Guocai; Wang, Yun; Chao, Yan; Jia, Yuping; Zhao, Chengquan; Luo, Bing

    2012-05-01

    Epstein-Barr virus nuclear antigen protein 3C (EBNA3C) is a 992-amino-acid protein that has been shown to play a complex regulatory role in the transcription of viral and cellular genes. In this study, we successfully amplified 26 Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs), 50 nasopharyngeal carcinomas (NPCs) and 27 throat washing (TW) samples from healthy donors. Based on a phylogenetic tree, the samples could be divided into three patterns. 3C-6 was the predominant subtype in northern China, and the variations between the strains sequenced in our study and those from southern China and Japan were similar, but differences were also identified. The distribution of EBNA3C subtypes among EBVaGCs, NPCs and healthy donors was not significantly different. These data suggest that EBNA3C gene variations are geographically restricted rather than tumor-specific polymorphisms.

  12. Application of linear discriminant analysis in performance evaluation of extractable nuclear antigen immunoassay systems in the screening and diagnosis of systemic autoimmune rheumatic diseases.

    PubMed

    Pi, David; de Badyn, Monika Hudoba; Nimmo, Mike; White, Rick; Pal, Jason; Wong, Patrick; Phoon, Carmen; O'Connor, Deidre; Pi, Steven; Shojania, Kam

    2012-10-01

    This study applied a linear discriminant analysis model to evaluate the performance of 2 types of commercially available extractable nuclear antigen (ENA) immunoassays for the screening and diagnosis of systemic autoimmune rheumatic diseases (SARDs) in a large tertiary hospital reference laboratory: (1) an enzyme-linked immunosorbent assay (ELISA) and (2) a multiplex bead-based immunoassay (MPBI). The results of the study showed both ENA immunoassays had comparable sensitivity for the detection of SARDs compared with the antinuclear antigen immunofluorescence (ANA-IF) method (ANA-IF: 85.6%, ENA-ELISA: 91.5%, ENA-MPBI: 83.1%, pairwise comparisons with ANA-IF: P > .05). However, both ENA immunoassays offered improved specificity compared with the ANA-IF (ANA-IF: 24.2%; ENA-ELISA: 39.8%; ENA-MPBI: 53.1%; pairwise comparison with ANA-IF: P < .001). The use of a more specific screening immunoassay with comparable sensitivity to ANA-IF is important in a tertiary hospital with high prevalence of non-SARD immune diseases. Diagnostic performance of the ENA/dsDNA components by the MPBI and ELISA methods did not differ significantly (area under the curve [AUC], 81.0% vs 83.0%, respectively, P > .05), but the key ENA/dsDNA variables contributing to the discriminating power of the assays for the diagnosis of specific SARDs were reagent/method dependent.

  13. Systemic lupus erythematosus murine monoclonal DNA-binding antibodies recognize cytoplasmic and nuclear phosphorylated antigens that display cell cycle redistribution in HEp-2 cells.

    PubMed Central

    Bronze-da-Rocha, E; Machado, C; Staines, N A; Sunkel, C E

    1992-01-01

    The immunological basis for the production of autoantibodies characteristic of systemic lupus erythematosus (SLE) against a wide range of antigens remains obscure. The specificity of (NZB x NZW)F1 (BWF1) or MRL/Mp-lpr/lpr (MRL/lpr) mouse monoclonal antibodies (mAb) was examined by immunofluorescence, immunoblotting and immunoprecipitation techniques. Using non-synchronized HEp-2 cells as substrate, the murine mAb were classified by indirect immunofluorescence into five groups on the basis of their staining patterns of subcellular components in interphase and mitotic stages of the cell cycle. The nature of the antigens recognized by the murine lupus was assessed by immunoblotting experiments in total, cytoplasmic and nuclear cell extracts from HEp-2 cells. The six antibodies used recognized in total cell extracts a range of polypeptides with apparent molecular weights from 25,000 to 210,000. Three polypeptides of 130,000, 110,000 and 45,000 MW were recognized by all six antibodies in both nuclear and cytoplasmic extracts. Immunoprecipitation of total cellular extracts labelled with [35S]methionine showed almost the same pattern as obtained in the immunoblotting assay. The labelling in vivo of HEp-2 cells with [32P], followed by the immunoprecipitation of the [32P]cell lysate showed that these mAb recognized phosphorylated proteins. The progressive decrease in reactivity of these mAb following treatment with higher concentrations of alkaline phosphatase in both [32P]cell lysate or nitrocellulose membranes indicates that these mAb recognize phosphorylated epitopes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:1283600

  14. Comparative analysis of oncogenic properties and nuclear factor-kappaB activity of latent membrane protein 1 natural variants from Hodgkin's lymphoma's Reed-Sternberg cells and normal B-lymphocytes.

    PubMed

    Faumont, Nathalie; Chanut, Aurélie; Benard, Alan; Cogne, Nadine; Delsol, Georges; Feuillard, Jean; Meggetto, Fabienne

    2009-03-01

    In Epstein-Barr virus-associated Hodgkin's lymphomas, neoplastic Reed-Sternberg cells and surrounding non-tumor B-cells contain different variants of the LMP1-BNLF1 oncogene. In this study, we raised the question of functional properties of latent membrane protein 1 (LMP1) natural variants from both Reed-Sternberg and non-tumor B-cells. Twelve LMP1 natural variants from Reed-Sternberg cells, non-tumor B-cells of Hodgkin's lymphomas and from B-cells of benign reactive lymph nodes were cloned, sequenced and stably transfected in murine recombinant interleukin-3-dependent Ba/F3 cells to search for relationships between LMP1 cellular origin and oncogenic properties as well as nuclear factor-kappaB activation, and apoptosis protection. LMP1 variants of Reed-Sternberg cell origin were often associated with increased mutation rate and with recurrent genetic events, such as del15bp associated with S to N replacement at codon 309, and four substitutions I85L, F106Y, I122L, and M129I. Oncogenic potential (growth factor-independence plus clonogenicity) was consistently associated with LMP1 variants from Reed-Sternberg cells, but inconstantly for LMP1-variants from non-tumor B-cells. Analysis of LMP1 variants from both normal B-cells and Reed-Sternberg cells indicates that protection against apoptosis through activation of nuclear factor-kappaB - whatever the cellular origin of LMP1 - was maintained intact, regardless of the mutational pattern. Taken together, our results demonstrate that preserved nuclear factor-kappaB activity and protection against apoptosis would be the minimal prerequisites for all LMP1 natural variants from both normal and tumor cells in Hodgkin's lymphomas, and that oncogenic potential would constitute an additional feature for LMP1 natural variants in Reed-Sternberg cells.

  15. Latent Period of Relaxation.

    PubMed

    Kobayashi, M; Irisawa, H

    1961-10-27

    The latent period of relaxation of molluscan myocardium due to anodal current is much longer than that of contraction. Although the rate and the grade of relaxation are intimately related to both the stimulus condition and the muscle tension, the latent period of relaxation remains constant, except when the temperature of the bathing fluid is changed.

  16. Freshwater acclimation induces stress responses and expression of branchial Na+/K(+)-ATPase and proliferating cell nuclear antigen in Takifugu niphobles.

    PubMed

    Tang, Cheng-Hao; Lee, Tsung-Han

    2013-08-01

    Almost the whole life cycle of the grass puffer (Takifugu niphobles) occurs in seawater (SW), but it is also sometimes found in fresh water (FW) rivers. This study aims to evaluate the effects of FW exposure on the stress, osmoregulatory, and physiological responses of the grass puffer. The grass puffers were captured from a local wetland and acclimated to SW (35‰) or FW in the laboratory. In the stress responses, plasma glucose concentrations and the abundances of hepatic and branchial heat shock proteins were higher in the FW group than in the SW group. FW acclimation led to a significant increase in the protein abundance and the specific activity of branchial Na(+)/K(+)-ATPase (NKA). Immunochemical staining showed that the NKA immunoreactive (NKIR) cells of the FW and SW puffer were distributed mainly in gill filaments. Although the number of NKIR cells was similar in the two groups, the protein levels of proliferating cell nuclear antigen (PCNA) of nuclear fractions were elevated in the gills of the FW puffer. The induction of gill PCNA might contribute to cell proliferation which would maintain the amount of NKIR cells or repair DNA when exposed to FW, an osmotically stressful environment. Hence, activation of stress responses would provide the osmoprotection associated with FW adaptation of the grass puffer. Changes of branchial NKA expression and activity for osmoregulatory adjustment were required for stable blood osmolality and muscle water content. Based on our findings, the grass puffer was suggested to be a euryhaline teleost with SW preference.

  17. Complex alternative cytoplasmic protein isoforms of the Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 generated through noncanonical translation initiation.

    PubMed

    Toptan, Tuna; Fonseca, Lidia; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S

    2013-03-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) latency associated-nuclear antigen 1 (LANA1) protein is constitutively expressed in all KSHV-infected cells, as well as in all forms of KSHV-associated malignancies. LANA1 is a multifunctional KSHV oncoprotein containing multiple repeat sequences that is important for viral episome maintenance and the regulation of cellular and viral gene expression. We characterize here multiple LANA1 isoforms and show that ∼50% of LANA1 is naturally generated as N-terminally truncated shoulder proteins that are detected on SDS-PAGE as faster-migrating shoulder bands designated LANA1(S). Higher-molecular-weight LANA1(S) isoforms initiate downstream at noncanonical sites within the N-terminal region, whereas lower-molecular-weight LANA1(S) isoforms initiate downstream within the central repeat 1 domain. LANA1(S) proteins lack an N-terminal nuclear localization signal motif, and some isoforms differ from full-length, canonical LANA1 by localizing to perinuclear and cytoplasmic sites. Although LANA1 has until now been assumed to be solely active in the nucleus, this finding indicates that this major KSHV oncoprotein may have cytoplasmic activities as well. KSHV overcomes its limited genetic coding capacity by generating alternatively initiated protein isoforms that may have distinct biological functions.

  18. Association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope and smoking status in Brazilian patients with rheumatoid arthritis.

    PubMed

    Yazbek, Michel Alexandre; Barros-Mazon, Silvia de; Rossi, Cláudio Lúcio; Londe, Ana Carolina; Costallat, Lilian Tereza Lavras; Bertolo, Manoel Barros

    2011-01-01

    Epstein-Barr virus exposure appears to be an environmental trigger for rheumatoid arthritis that interacts with other risk factors. Relationships among anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status have been observed in patients with rheumatoid arthritis from different populations. To perform an association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status in Brazilian patients with rheumatoid arthritis. In a case-control study, 140 rheumatoid arthritis patients and 143 healthy volunteers who were matched for age, sex, and ethnicity were recruited. Anti-Epstein-Barr nuclear antigen-1 antibodies and anti-cyclic citrullinated peptide antibodies were examined using an enzyme-linked immunosorbent assay, and shared epitope alleles were identified by genotyping. Smoking information was collected from all subjects. A comparative analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status was performed in the patient group. Logistic regression analysis models were used to analyze the risk of rheumatoid arthritis. Anti-Epstein-Barr nuclear antigen-1 antibodies were not associated with anti-cyclic citrullinated peptide antibodies, shared epitope alleles, or smoking status. Anti-cyclic citrullinated peptide antibody positivity was significantly higher in smoking patients with shared epitope alleles (OR = 3.82). In a multivariate logistic regression analysis using stepwise selection, only anti-cyclic citrullinated peptide antibodies were found to be independently associated with rheumatoid arthritis (OR = 247.9). Anti-Epstein-Barr nuclear antigen-1 antibodies did not increase the risk of rheumatoid arthritis and were not associated with the rheumatoid arthritis risk factors studied. Smoking and shared epitope alleles were correlated with anti

  19. Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) interacts with Regulator of Chromosome Condensation (RCC1) dynamically throughout the cell cycle.

    PubMed

    Deschamps, Thibaut; Quentin, Bazot; Leske, Derek M; MacLeod, Ruth; Mompelat, Dimitri; Tafforeau, Lionel; Lotteau, Vincent; Maréchal, Vincent; Baillie, George S; Gruffat, Henri; Wilson, Joanna B; Manet, Evelyne

    2016-12-12

    The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a sequence-specific DNA binding protein which plays an essential role in viral episome replication and segregation, by recruiting the cellular complex of DNA replication onto the origin (oriP) and by tethering the viral DNA onto the mitotic chromosomes. Whereas the mechanisms of viral DNA replication are well documented, those involved in tethering EBNA1 to the cellular chromatin are far from being understood. Here, we have identified Regulator of Chromosome Condensation 1 (RCC1) as a novel cellular partner for EBNA1. RCC1 is the major nuclear guanine nucleotide exchange factor (RanGEF) for the small GTPase Ran enzyme. RCC1, associated with chromatin, is involved in the formation of RanGTP gradients critical for nucleo-cytoplasmic transport, mitotic spindle formation, and nuclear envelope reassembly following mitosis. Using several approaches, we have demonstrated a direct interaction between these two proteins and found that the EBNA1 domains responsible for EBNA1 tethering to the mitotic chromosomes are also involved in the interaction with RCC1. The use of an EBNA1 peptide array confirmed the interaction of RCC1 with these regions and also the importance of the N-terminal region of RCC1 in this interaction. Finally, using confocal microscopy and FRET analysis to follow the dynamics of interaction between the two proteins throughout the cell cycle, we have demonstrated that EBNA1 and RCC1 closely associate on the chromosomes during metaphase, suggesting an essential role for the interaction during this phase, perhaps in tethering EBNA1 to mitotic chromosomes.

  20. Human serum antibodies to a major defined epitope of human herpesvirus 8 small viral capsid antigen.

    PubMed

    Tedeschi, R; De Paoli, P; Schulz, T F; Dillner, J

    1999-04-01

    The major antibody-reactive epitope of the small viral capsid antigen (sVCA) of human herpesvirus 8 (HHV-8) was defined by use of overlapping peptides. Strong IgG reactivity was found among approximately 50% of 44 human immunodeficiency virus-positive or -negative patients with Kaposi's sarcoma and 13 subjects who were seropositive by immunofluorescence assay (IFA) for the latent HHV-8 nuclear antigen. Only 1 of 106 subjects seronegative for both lytic and latent HHV-8 antigens and 10 of 81 subjects IFA-seropositive only for the lytic HHV-8 antigen had strong IgG reactivity to this epitope. Among 534 healthy Swedish women, only 1.3% were strongly seropositive. Comparison of the peptide-based and purified sVCA protein-based ELISAs found 55% sensitivity and 98% specificity. However, only 1 of 452 serum samples from healthy women was positive in both tests. In conclusion, the defined sVCA epitope was a specific, but not very sensitive, serologic marker of active HHV-8 infection. Such infection appears to be rare among Swedish women, even with sexual risk-taking behavior.

  1. Latent fingerprint matching.

    PubMed

    Jain, Anil K; Feng, Jianjiang

    2011-01-01

    Latent fingerprint identification is of critical importance to law enforcement agencies in identifying suspects: Latent fingerprints are inadvertent impressions left by fingers on surfaces of objects. While tremendous progress has been made in plain and rolled fingerprint matching, latent fingerprint matching continues to be a difficult problem. Poor quality of ridge impressions, small finger area, and large nonlinear distortion are the main difficulties in latent fingerprint matching compared to plain or rolled fingerprint matching. We propose a system for matching latent fingerprints found at crime scenes to rolled fingerprints enrolled in law enforcement databases. In addition to minutiae, we also use extended features, including singularity, ridge quality map, ridge flow map, ridge wavelength map, and skeleton. We tested our system by matching 258 latents in the NIST SD27 database against a background database of 29,257 rolled fingerprints obtained by combining the NIST SD4, SD14, and SD27 databases. The minutiae-based baseline rank-1 identification rate of 34.9 percent was improved to 74 percent when extended features were used. In order to evaluate the relative importance of each extended feature, these features were incrementally used in the order of their cost in marking by latent experts. The experimental results indicate that singularity, ridge quality map, and ridge flow map are the most effective features in improving the matching accuracy.

  2. Expression of VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes of Epstein-Barr virus in Pichia pastoris and application of the products in a screening test for patients with nasopharyngeal carcinoma.

    PubMed

    Hu, Bo; Hong, Guoqiang; Li, Zhaoxia; Xu, Jue; Zhu, Zhenyu; Li, Lin

    2007-05-01

    EBV (Epstein-Barr virus) serological tests have been used for many years as accessory diagnostic predictors of NPC (nasopharyngeal carcinoma). To date, IF (indirect immunofluorescence) assays still serve as the 'gold standard' for EBV serodiagnosis. However, IF assays are time-consuming, unsuitable for automatic handling and difficult to standardize. This makes their application in mass screening of populations inconvenient. Some of the technical difficulties associated with IF have been overcome by the development of specific ELISAs, but, at present, high sensitivity and specificity cannot be achieved simultaneously by using recombinant protein-based ELISAs, as the diagnostic value of different fragments of EBV in NPC is different. In an attempt to determine a suitable recombinant EBV protein for diagnostic purposes, fragments of EBV VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes were expressed in the methylotrophic yeast Pichia pastoris, and a novel ELISA was established using P. pastoris-expressed VCA-BALF4 [aa (amino acids) 287-623; the BALF4 gene encodes the EBV glycoprotein gp125], EBNA1 (aa 390-641) and VCA-BFRF3 (the gene BFRF3 encodes a viral structural capsid protein or tegument protein VCA p18) proteins. Serum samples were collected from patients with NPC and healthy controls and were tested using this ELISA. The sensitivity of VCA-BFRF3, VCA-BALF4 and EBNA1 tests in the NPC sera were 65.0 (195/300), 76.3 (229/300) and 81.4% (244/300) respectively, whereas the specificity of normal individuals were 92 (460/500), 96 (480/500) and 95.8% (479/500). The optimum combination is VCA-BALF4 plus EBNA1, which identified 90.3% (271/300) of the NPC patients and had a specificity of 92.8% (464/500) for normal individuals. The results obtained from the evaluation of three antibodies to EBV as markers for detecting NPC suggests that a combination of EBNA1 (aa 390-641) and VCA-BALF4 (aa 287-623) assays would give better results

  3. Latent palmprint matching.

    PubMed

    Jain, Anil K; Feng, Jianjiang

    2009-06-01

    The evidential value of palmprints in forensic applications is clear as about 30 percent of the latents recovered from crime scenes are from palms. While biometric systems for palmprint-based personal authentication in access control type of applications have been developed, they mostly deal with low-resolution (about 100 ppi) palmprints and only perform full-to-full palmprint matching. We propose a latent-to-full palmprint matching system that is needed in forensic applications. Our system deals with palmprints captured at 500 ppi (the current standard in forensic applications) or higher resolution and uses minutiae as features to be compatible with the methodology used by latent experts. Latent palmprint matching is a challenging problem because latent prints lifted at crime scenes are of poor image quality, cover only a small area of the palm, and have a complex background. Other difficulties include a large number of minutiae in full prints (about 10 times as many as fingerprints), and the presence of many creases in latents and full prints. A robust algorithm to reliably estimate the local ridge direction and frequency in palmprints is developed. This facilitates the extraction of ridge and minutiae features even in poor quality palmprints. A fixed-length minutia descriptor, MinutiaCode, is utilized to capture distinctive information around each minutia and an alignment-based minutiae matching algorithm is used to match two palmprints. Two sets of partial palmprints (150 live-scan partial palmprints and 100 latent palmprints) are matched to a background database of 10,200 full palmprints to test the proposed system. Despite the inherent difficulty of latent-to-full palmprint matching, rank-1 recognition rates of 78.7 and 69 percent, respectively, were achieved in searching live-scan partial palmprints and latent palmprints against the background database.

  4. Multiple HLA A11-restricted cytotoxic T-lymphocyte epitopes of different immunogenicities in the Epstein-Barr virus-encoded nuclear antigen 4.

    PubMed

    Gavioli, R; Kurilla, M G; de Campos-Lima, P O; Wallace, L E; Dolcetti, R; Murray, R J; Rickinson, A B; Masucci, M G

    1993-03-01

    Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotoxic T-lymphocyte (CTL) responses. Analyses of target antigen choice have shown that the very strong CTL responses which are often observed through the HLA A11 allele map are due almost entirely to a single transformation-associated EBV protein, the nuclear antigen EBNA4. Here, we sought to determine the number and relative immunogenicities of HLA A11-restricted epitopes within this 938-amino-acid protein. An initial screening with a series of recombinant vaccinia virus vectors encoding progressively truncated forms of EBNA4 was followed by peptide sensitization experiments using overlapping 14- or 15-mers from the entire sequence. These two approaches allowed the identification of five epitope regions located between residues 101 and 115, 416 and 429, 396 and 410, 481 and 495, and 551 and 564 of the EBNA4 molecule. CTL preparations from all seven HLA A11-positive donors tested had demonstrable reactivities against the 416-to-429 peptide, whereas reactivities against the other epitopes either tended to be lost on serial passage or, for some of the donors, were never detected. The immunodominance of the 416-to-429 epitope was further supported by peptide dilution assays using polyclonal effectors and by CTL cloning experiments. Analysis of the 416-to-429 region identified the nanomer 416-424 (IVTDFSVIK) as the cognate peptide. This peptide was able to sensitize targets to lysis by A11-restricted CTL clones at concentrations as low as 5 x 10(-14) M.

  5. A Genome-Wide Integrative Genomic Study Localizes Genetic Factors Influencing Antibodies against Epstein-Barr Virus Nuclear Antigen 1 (EBNA-1)

    PubMed Central

    Rubicz, Rohina; Yolken, Robert; Drigalenko, Eugene; Carless, Melanie A.; Dyer, Thomas D.; Bauman, Lara; Melton, Phillip E.; Kent, Jack W.; Harley, John B.; Curran, Joanne E.; Johnson, Matthew P.; Cole, Shelley A.; Almasy, Laura; Moses, Eric K.; Dhurandhar, Nikhil V.; Kraig, Ellen; Blangero, John; Leach, Charles T.; Göring, Harald H. H.

    2013-01-01

    Infection with Epstein-Barr virus (EBV) is highly prevalent worldwide, and it has been associated with infectious mononucleosis and severe diseases including Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal lymphoma, and lymphoproliferative disorders. Although EBV has been the focus of extensive research, much still remains unknown concerning what makes some individuals more sensitive to infection and to adverse outcomes as a result of infection. Here we use an integrative genomics approach in order to localize genetic factors influencing levels of Epstein Barr virus (EBV) nuclear antigen-1 (EBNA-1) IgG antibodies, as a measure of history of infection with this pathogen, in large Mexican American families. Genome-wide evidence of both significant linkage and association was obtained on chromosome 6 in the human leukocyte antigen (HLA) region and replicated in an independent Mexican American sample of large families (minimum p-value in combined analysis of both datasets is 1.4×10−15 for SNPs rs477515 and rs2516049). Conditional association analyses indicate the presence of at least two separate loci within MHC class II, and along with lymphocyte expression data suggest genes HLA-DRB1 and HLA-DQB1 as the best candidates. The association signals are specific to EBV and are not found with IgG antibodies to 12 other pathogens examined, and therefore do not simply reveal a general HLA effect. We investigated whether SNPs significantly associated with diseases in which EBV is known or suspected to play a role (namely nasopharyngeal lymphoma, Hodgkin lymphoma, systemic lupus erythematosus, and multiple sclerosis) also show evidence of associated with EBNA-1 antibody levels, finding an overlap only for the HLA locus, but none elsewhere in the genome. The significance of this work is that a major locus related to EBV infection has been identified, which may ultimately reveal the underlying mechanisms by which the immune system regulates infection with this pathogen

  6. The rheumatoid arthritis synovial fluid citrullinome reveals novel citrullinated epitopes in apolipoprotein E, myeloid nuclear differentiation antigen, and β-actin.

    PubMed

    van Beers, Joyce J B C; Schwarte, Carla M; Stammen-Vogelzangs, Judith; Oosterink, Els; Božič, Borut; Pruijn, Ger J M

    2013-01-01

    To generate a catalog of citrullinated proteins that are present in the synovia of patients with rheumatoid arthritis (RA) and to elucidate their relevance for the anti-citrullinated protein antibody response in RA. Polypeptides isolated from the synovial fluid of patients with RA were identified by mass spectrometry. Three proteins (apolipoprotein E [Apo E], myeloid nuclear differentiation antigen [MNDA], and β-actin) were studied in more detail, using immunoprecipitation and Western blotting. The presence of autoantibodies to synthetic peptides derived from these proteins in sera from patients with RA, sera from patients with other diseases, and sera from healthy control subjects was studied by enzyme-linked immunosorbent assay (ELISA). RA synovial fluid samples displayed several distinct patterns of citrullinated proteins. Using mass spectrometry, (fragments of) 192 proteins were identified, including 53 citrullinated proteins, some of which contained multiple citrullinated residues. In addition to previously reported citrullinated proteins in RA synovia (e.g., vimentin and fibrinogen), a series of novel citrullinated proteins, including Apo E, MNDA, β-actin, and cyclophilin A, was identified. Immunoprecipitation experiments confirmed the citrullination of Apo E and MNDA. ELISAs demonstrated the presence of autoreactive citrullinated epitopes in Apo E, MNDA, and β-actin. Synovial fluid samples from the inflamed joints of patients with RA contain many citrullinated proteins. Citrullinated Apo E, MNDA, and β-actin are novel antigens identified in RA synovial fluid, and only a limited number of their citrullinated epitopes are targeted by the immune system in RA. Copyright © 2013 by the American College of Rheumatology.

  7. Epstein-Barr Nuclear Antigen 1 (EBNA1)-dependent Recruitment of Origin Recognition Complex (Orc) on oriP of Epstein-Barr Virus with Purified Proteins

    PubMed Central

    Moriyama, Kenji; Yoshizawa-Sugata, Naoko; Obuse, Chikashi; Tsurimoto, Toshiki; Masai, Hisao

    2012-01-01

    Origin recognition complex (Orc) plays an essential role in directing assembly of prereplicative complex at selective sites on chromosomes. However, Orc from vertebrates is reported to bind to DNA in a sequence-nonspecific manner, and it is still unclear how it selects specific genomic loci and how Cdc6, another conserved AAA+ factor known to interact with Orc, participates in this process. Replication from oriP, the latent origin of Epstein-Barr virus, provides an excellent model system for the study of initiation on the host chromosomes because it is known to depend on prereplicative complex factors, including Orc and Mcm. Here, we show that Orc is recruited selectively at the essential dyad symmetry element in nuclear extracts in a manner dependent on EBNA1, which specifically binds to dyad symmetry. With purified proteins, EBNA1 can recruit both Cdc6 and Orc independently on a DNA containing EBNA1 binding sites, and Cdc6 facilitates the Orc recruitment by EBNA1. Purified Cdc6 directly binds to EBNA1, whereas association of Orc with EBNA1 requires the presence of the oriP DNA. Nuclease protection assays suggest that Orc associates with DNA segments on both sides adjacent to the EBNA1 binding sites and that this process is stimulated by the presence of Cdc6. Thus, EBNA1 can direct localized assembly of Orc in a process that is facilitated by Cdc6. The possibility of similar modes of recruitment of Orc/Cdc6 at the human chromosomal origins will be discussed. PMID:22589552

  8. Murine Gammaherpesvirus 68 Expressing Kaposi Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen (LANA) Reveals both Functional Conservation and Divergence in LANA Homologs.

    PubMed

    Gupta, Arundhati; Oldenburg, Darby G; Salinas, Eduardo; White, Douglas W; Forrest, J Craig

    2017-10-01

    Latency-associated nuclear antigen (LANA) is a multifunctional protein encoded by members of the Rhadinovirus genus of gammaherpesviruses. Studies using murine gammaherpesvirus 68 (MHV68) demonstrated that LANA is important for acute replication, latency establishment, and reactivation in vivo Despite structural similarities in their DNA-binding domains (DBDs), LANA homologs from Kaposi sarcoma-associated herpesvirus (KSHV) and MHV68 exhibit considerable sequence divergence. We sought to determine if KSHV and MHV68 LANA homologs are functionally interchangeable. We generated an MHV68 virus that encodes KSHV LANA (kLANA) in place of MHV68 LANA (mLANA) and evaluated the virus's capacity to replicate, establish and maintain latency, and reactivate. kLANA knock-in (KLKI) MHV68 was replication competent in vitro and in vivo but exhibited slower growth kinetics and lower titers than wild-type (WT) MHV68. Following inoculation of mice, KLKI MHV68 established and maintained latency in splenocytes and peritoneal cells but did not reactivate efficiently ex vivo kLANA repressed the MHV68 promoter for ORF50, the gene that encodes the major lytic transactivator protein RTA, while mLANA did not, suggesting a likely mechanism for the KLKI MHV68 phenotypes. Bypassing this repression by providing MHV68 RTA in trans rescued KLKI MHV68 replication in tissue culture and enabled detection of KLKI MHV68 reactivation ex vivo These data demonstrate that kLANA and mLANA are functionally interchangeable for establishment and maintenance of latency and suggest that repression of lytic replication by kLANA, as previously shown with KSHV, is a kLANA-specific function that is transferable to MHV68.IMPORTANCE Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are members of the Rhadinovirus genus of gammaherpesviruses. These viruses establish lifelong infections that place their respective human and murine hosts at risk for cancer. Latency-associated nuclear

  9. Reactivity with A monoclonal antibody to Epstein-Barr virus (EBV) nuclear antigen 1 defines a subset of aggressive breast cancers in the absence of the EBV genome.

    PubMed

    Murray, Paul G; Lissauer, David; Junying, Jia; Davies, Gillian; Moore, Sukhjinder; Bell, Andrew; Timms, Judith; Rowlands, David; McConkey, Christopher; Reynolds, Gary M; Ghataura, Suk; England, David; Caroll, Rebecca; Young, Lawrence S

    2003-05-01

    Previous studies have suggested that common breast cancers are associated with EBV. We used a highly sensitive quantitative real-time PCR method to screen whole tumor sections of breast cancers for the presence of the EBV genome. EBV DNA was detected in 19 of 92 (21%) tumors, but viral load was very low in positive samples (mean = 1.1 copy EBV/1000 cells, maximum = 7.1 copies EBV/1000 cells). Importantly, quantitative real-time PCR failed to detect the EBV genome in microdissected tumor cells from any case. Using a monoclonal antibody (2B4-1) reactive against the EBV nuclear antigen-1, we noted strong staining of tumor nuclei in a proportion of those breast cancers that had tested negative for the presence of the EBV genome. Because nuclear staining with the 2B4-1 antibody was previously observed more frequently in poor prognosis breast cancers, we examined a larger series of breast cancers with complete clinical follow-up. Strong punctate staining of tumor cell nuclei was observed in 47 of 153 (31%) breast cancers; 2B4-1-positive tumors were significantly more likely to be ER-negative (P < 0.0001), to be of higher grade (P = 0.001) and larger (P = 0.03), to involve more regional lymph nodes (P = 0.01), and to have higher Nottingham Prognostic Index scores (P = 0.0003). Conclusions are: (a) EBV can be regularly detected in whole sections of breast cancers but viral copy number is very low; (b) in these cases, tumor cells do not harbor virus; and (c) reactivity with the monoclonal antibody 2B4-1 is detectable in the absence of the EBV genome and is strongly associated with ER-negative breast tumors and with prognostically unfavorable disease. Additional studies should be directed to the identification of this protein and to elucidation of its role in breast cancer.

  10. Ly9 (CD229) Cell-Surface Receptor is Crucial for the Development of Spontaneous Autoantibody Production to Nuclear Antigens

    PubMed Central

    de Salort, Jose; Cuenca, Marta; Terhorst, Cox; Engel, Pablo; Romero, Xavier

    2013-01-01

    The Signaling Lymphocyte Activation Molecule Family (SLAMF) genes, which encode cell-surface receptors that modulate innate and adaptive immune responses, lay within a genomic region of human and mouse chromosome 1 that confers a predisposition for the development of systemic lupus erythematosus (SLE). Herein, we demonstrate that the SLAMF member Ly9 arises as a novel receptor contributing to the reinforcement of tolerance. Specifically, Ly9-deficient mice spontaneously developed features of systemic autoimmunity such as the production of anti-nuclear antibodies (ANA), -dsDNA, and -nucleosome autoantibodies, independently of genetic background [(B6.129) or (BALB/c.129)]. In aged (10- to 12-month-old) Ly9−/− mice key cell subsets implicated in autoimmunity were expanded, e.g., T follicular helper (Tfh) as well as germinal center (GC) B cells. More importantly, in vitro functional experiments showed that Ly9 acts as an inhibitory receptor of IFN-γ producing CD4+ T cells. Taken together, our findings reveal that the Ly9 receptor triggers cell intrinsic safeguarding mechanisms to prevent a breach of tolerance, emerging as a new non-redundant inhibitory cell-surface receptor capable of disabling autoantibody responses. PMID:23914190

  11. Ly9 (CD229) Cell-Surface Receptor is Crucial for the Development of Spontaneous Autoantibody Production to Nuclear Antigens.

    PubMed

    de Salort, Jose; Cuenca, Marta; Terhorst, Cox; Engel, Pablo; Romero, Xavier

    2013-01-01

    The Signaling Lymphocyte Activation Molecule Family (SLAMF) genes, which encode cell-surface receptors that modulate innate and adaptive immune responses, lay within a genomic region of human and mouse chromosome 1 that confers a predisposition for the development of systemic lupus erythematosus (SLE). Herein, we demonstrate that the SLAMF member Ly9 arises as a novel receptor contributing to the reinforcement of tolerance. Specifically, Ly9-deficient mice spontaneously developed features of systemic autoimmunity such as the production of anti-nuclear antibodies (ANA), -dsDNA, and -nucleosome autoantibodies, independently of genetic background [(B6.129) or (BALB/c.129)]. In aged (10- to 12-month-old) Ly9 (-/-) mice key cell subsets implicated in autoimmunity were expanded, e.g., T follicular helper (Tfh) as well as germinal center (GC) B cells. More importantly, in vitro functional experiments showed that Ly9 acts as an inhibitory receptor of IFN-γ producing CD4(+) T cells. Taken together, our findings reveal that the Ly9 receptor triggers cell intrinsic safeguarding mechanisms to prevent a breach of tolerance, emerging as a new non-redundant inhibitory cell-surface receptor capable of disabling autoantibody responses.

  12. Epstein–Barr virus nuclear antigen 3C interact with p73: Interplay between a viral oncoprotein and cellular tumor suppressor

    SciTech Connect

    Sahu, Sushil Kumar; Mohanty, Suchitra; Kumar, Amit; Kundu, Chanakya N.; Verma, Subhash C.; Choudhuri, Tathagata

    2014-01-05

    The p73 protein has structural and functional homology with the tumor suppressor p53, which plays an important role in cell cycle regulation, apoptosis, and DNA repair. The p73 locus encodes both a tumor suppressor (TAp73) and a putative oncogene (ΔNp73). p73 May play a significant role in p53-deficient lymphomas infected with Epstein–Barr virus (EBV). EBV produces an asymptomatic infection in the majority of the global population, but it is associated with several human B-cell malignancies. The EBV-encoded Epstein–Barr virus nuclear antigen 3C (EBNA3C) is thought to disrupt the cell cycle checkpoint by interacting directly with p53 family proteins. Doxorubicin, a commonly used chemotherapeutic agent, induces apoptosis through p53 and p73 signaling such that the lowΔNp73 level promotes the p73-mediated intrinsic pathway of apoptosis. In this report, we investigated the mechanism by which EBV infection counters p73α-induced apoptosis through EBNA3C. - Highlights: • EBV-encoded EBNA3C suppresses doxorubicin-induced apoptosis in B-cell lymphomas. • EBNA3C binds to p73 to suppress its apoptotic effect. • EBNA3C maintains latency by regulating downstream mitochondrial pathways.

  13. DNA polymerases BI and D from the hyperthermophilic archaeon Pyrococcus furiosus both bind to proliferating cell nuclear antigen with their C-terminal PIP-box motifs.

    PubMed

    Tori, Kazuo; Kimizu, Megumi; Ishino, Sonoko; Ishino, Yoshizumi

    2007-08-01

    Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3'-5' exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.

  14. Inhibition of replication and transcription activator and latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus by morpholino oligomers.

    PubMed

    Zhang, Yan-Jin; Wang, Kai-Yu; Stein, David A; Patel, Deendayal; Watkins, Rheba; Moulton, Hong M; Iversen, Patrick L; Matson, David O

    2007-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The KSHV replication and transcription activator (RTA) and latency-associated nuclear antigen (LANA) play key roles in activating KSHV lytic replication and maintaining KSHV latency, respectively. Phosphorodiamidate morpholino oligomers (PMO) are similar to short single-stranded DNA oligomers, but possess a modified backbone that confers highly specific binding and resistance to nucleases. In this study, RTA and LANA mRNA in PEL cells were targeted by antisense peptide-conjugated PMO (P-PMO) in an effort to suppress KSHV replication. Highly efficient P-PMO uptake by PEL cells was observed. Treatment of PEL cells with a RTA P-PMO (RP1) reduced RTA expression in a dose-dependent and sequence-specific manner, and also caused a significant decrease in several KSHV early and late gene products, including vIL-6, vIRF-1, and ORF-K8.1A. KSHV viral DNA levels were reduced both in cells and culture supernatants of RP1 P-PMO-treated cells, indicating that KSHV lytic replication was suppressed. Treatment of BCBL-1 cells with P-PMO against LANA resulted in a reduction of LANA expression. Cell viability assays detected no cytotoxicity from P-PMO alone, within the concentration range used for the experiments in this study. These results suggest that RP1 P-PMO can specifically block KSHV replication, and further study is warranted.

  15. DNA Polymerases BI and D from the Hyperthermophilic Archaeon Pyrococcus furiosus Both Bind to Proliferating Cell Nuclear Antigen with Their C-Terminal PIP-Box Motifs▿

    PubMed Central

    Tori, Kazuo; Kimizu, Megumi; Ishino, Sonoko; Ishino, Yoshizumi

    2007-01-01

    Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3′-5′ exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex. PMID:17496095

  16. Induction of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen by the lytic transactivator RTA: a novel mechanism for establishment of latency.

    PubMed

    Lan, Ke; Kuppers, Daniel A; Verma, Subhash C; Sharma, Nikhil; Murakami, Masanao; Robertson, Erle S

    2005-06-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman desease. Following primary infection, latency is typically established. However, the mechanism by which KSHV establishes latency is not understood. We have reported that the latency-associated nuclear antigen (LANA) can repress RTA (for replication and transcription activator) expression by down-regulating its promoter. In this study, we show that RTA is associated with the virion particle. We also show that RTA can activate the LANA promoter and induce LANA expression in transient reporter assays. Additionally, the transcription of RTA correlates with LANA expression in the early stages of de novo infection of KSHV, and induction of LANA transcription is responsive to induction of RTA with an inducible system. This induction in LANA transcription was dependent on recombination signal sequence binding protein Jkappa (RBP-Jkappa), as a RBP-Jkappa-deficient cell line was significantly delayed and inefficient in LANA transcription with expression of RTA. These studies suggest that RTA contributes to establishment of KSHV latency by activating LANA expression in the early stages of infection by utilizing the major effector of the Notch signaling pathway RBP-Jkappa. This describes a feedback mechanism by which LANA and RTA can regulate each other and is likely to be a key event in the establishment of KSHV latency.

  17. Induction of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen by the Lytic Transactivator RTA: a Novel Mechanism for Establishment of Latency

    PubMed Central

    Lan, Ke; Kuppers, Daniel A.; Verma, Subhash C.; Sharma, Nikhil; Murakami, Masanao; Robertson, Erle S.

    2005-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman desease. Following primary infection, latency is typically established. However, the mechanism by which KSHV establishes latency is not understood. We have reported that the latency-associated nuclear antigen (LANA) can repress RTA (for replication and transcription activator) expression by down-regulating its promoter. In this study, we show that RTA is associated with the virion particle. We also show that RTA can activate the LANA promoter and induce LANA expression in transient reporter assays. Additionally, the transcription of RTA correlates with LANA expression in the early stages of de novo infection of KSHV, and induction of LANA transcription is responsive to induction of RTA with an inducible system. This induction in LANA transcription was dependent on recombination signal sequence binding protein Jκ (RBP-Jκ), as a RBP-Jκ-deficient cell line was significantly delayed and inefficient in LANA transcription with expression of RTA. These studies suggest that RTA contributes to establishment of KSHV latency by activating LANA expression in the early stages of infection by utilizing the major effector of the Notch signaling pathway RBP-Jκ. This describes a feedback mechanism by which LANA and RTA can regulate each other and is likely to be a key event in the establishment of KSHV latency. PMID:15919901

  18. Epstein-Barr virus nuclear antigen 3C interact with p73: Interplay between a viral oncoprotein and cellular tumor suppressor.

    PubMed

    Sahu, Sushil Kumar; Mohanty, Suchitra; Kumar, Amit; Kundu, Chanakya N; Verma, Subhash C; Choudhuri, Tathagata

    2014-01-05

    The p73 protein has structural and functional homology with the tumor suppressor p53, which plays an important role in cell cycle regulation, apoptosis, and DNA repair. The p73 locus encodes both a tumor suppressor (TAp73) and a putative oncogene (ΔNp73). p73 May play a significant role in p53-deficient lymphomas infected with Epstein-Barr virus (EBV). EBV produces an asymptomatic infection in the majority of the global population, but it is associated with several human B-cell malignancies. The EBV-encoded Epstein-Barr virus nuclear antigen 3C (EBNA3C) is thought to disrupt the cell cycle checkpoint by interacting directly with p53 family proteins. Doxorubicin, a commonly used chemotherapeutic agent, induces apoptosis through p53 and p73 signaling such that the lowΔNp73 level promotes the p73-mediated intrinsic pathway of apoptosis. In this report, we investigated the mechanism by which EBV infection counters p73α-induced apoptosis through EBNA3C.

  19. Heat shock factor 1 upregulates transcription of Epstein-Barr Virus nuclear antigen 1 by binding to a heat shock element within the BamHI-Q promoter

    SciTech Connect

    Wang, Feng-Wei; Wu, Xian-Rui; Liu, Wen-Ju; Liao, Yi-Ji; Lin, Sheng; Zong, Yong-Sheng; Zeng, Mu-Sheng; Zeng, Yi-Xin; Mai, Shi-Juan; Xie, Dan

    2011-12-20

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for maintenance of the episome and establishment of latency. In this study, we observed that heat treatment effectively induced EBNA1 transcription in EBV-transformed B95-8 and human LCL cell lines. Although Cp is considered as the sole promoter used for the expression of EBNA1 transcripts in the lymphoblastoid cell lines, the RT-PCR results showed that the EBNA1 transcripts induced by heat treatment arise from Qp-initiated transcripts. Using bioinformatics, a high affinity and functional heat shock factor 1 (HSF1)-binding element within the - 17/+4 oligonucleotide of the Qp was found, and was determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Moreover, heat shock and exogenous HSF1 expression induced Qp activity in reporter assays. Further, RNA interference-mediated HSF1 gene silencing attenuated heat-induced EBNA1 expression in B95-8 cells. These results provide evidence that EBNA1 is a new target for the transcription factor HSF1.

  20. Deviating the level of proliferating cell nuclear antigen in Trypanosoma brucei elicits distinct mechanisms for inhibiting proliferation and cell cycle progression.

    PubMed

    Valenciano, Ana L; Ramsey, Aaron C; Mackey, Zachary B

    2015-01-01

    The DNA replication machinery is spatially and temporally coordinated in all cells to reproduce a single exact copy of the genome per division, but its regulation in the protozoan parasite Trypanosoma brucei is not well characterized. We characterized the effects of altering the levels of proliferating cell nuclear antigen, a key component of the DNA replication machinery, in bloodstream form T. brucei. This study demonstrated that tight regulation of TbPCNA levels was critical for normal proliferation and DNA replication in the parasite. Depleting TbPCNA mRNA reduced proliferation, severely diminished DNA replication, arrested the synthesis of new DNA and caused the parasites to accumulated in G2/M. Attenuating the parasite by downregulating TbPCNA caused it to become hypersensitive to hydroxyurea. Overexpressing TbPCNA in T. brucei arrested proliferation, inhibited DNA replication and prevented the parasite from exiting G2/M. These results indicate that distinct mechanisms of cell cycle arrest are associated with upregulating or downregulating TbPCNA. The findings of this study validate deregulating intra-parasite levels of TbPCNA as a potential strategy for therapeutically exploiting this target in bloodstream form T. brucei.

  1. Drosophila proliferating cell nuclear antigen (cyclin) gene: structure, expression during development, and specific binding of homeodomain proteins to its 5'-flanking region.

    PubMed Central

    Yamaguchi, M; Nishida, Y; Moriuchi, T; Hirose, F; Hui, C C; Suzuki, Y; Matsukage, A

    1990-01-01

    The genomic and cDNA clones for a Drosophila melanogaster proliferating cell nuclear antigen (PCNA) (cyclin) were isolated and sequenced. The coding sequence for a 260-amino-acid residue polypeptide was interrupted by a single short intron of 60 base pairs (bp), and about 70% of the deduced amino acid sequence of the Drosophila PCNA was identical to the rat and human PCNA polypeptides, with conserved unique repeats of leucine in the C-terminal region. Genomic Southern blot hybridization analysis indicates the presence of a single gene for PCNA per genome. The PCNA mRNA was detected at a high level in adult ovaries, unfertilized eggs, and early embryos and at low levels in the other developmental stages. The major transcription initiation site (cap site) was localized at 89 bp upstream from the ATG codon. Neither a TATA box nor a CAAT box was found within the 600-bp region upstream of the cap site. Clusters of 10 bp of sequence similar to the binding sites for Drosophila proteins containing homeodomains were found in the region from -127 to -413. DNase I footprint analysis revealed that the Drosophila homeodomain proteins coded by even-skipped and zerknüllt genes can specifically bind to these sites. These results suggest that the expression of the PCNA gene is under the control of genes coding for homeodomain proteins. Images PMID:1968224

  2. Gene variations of Epstein-Barr virus nuclear antigen 3A in nasopharyngeal carcinomas, gastric carcinomas and healthy carriers in northern China.

    PubMed

    Wang, Xiaofeng; Wu, Guocai; Wang, Yun; Sun, Zhifu; Luo, Bing

    2013-10-01

    The Epstein-Barr virus (EBV) nuclear antigen protein 3A (EBNA-3A), a protein of 944 amino acids, is one of five EBNAs (EBNA-1, -2, -LP, -3A and -3C) essential for conversion of primary B lymphocytes to lymphoblastoid cell lines. To characterize the variations of the EBNA-3A gene and explore the association between EBNA-3A gene variations and EBV-associated diseases, we sequenced the key regions of EBNA-3A in the isolates of 30 EBV-associated gastric carcinomas (EBVaGCs), 44 nasopharyngeal carcinomas (NPCs) and 48 samples from healthy donors in northern China. We found that EBNA-3A shares a common evolutionary origin with isolates from southern China and Japan but has the character of a geographical variant. Based on a phylogenetic tree, all of the samples can be subdivided into three patterns, named 3A-8, 3A-5 and B95-8-like. The distribution of EBNA-3A subtypes among EBVaGC, NPC and healthy donors is not significantly different. The subtype 3A-8 is predominant not only in northern China but also in southern China; it is a geographically associated polymorphism in China.

  3. Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA.

    PubMed

    Hedglin, Mark; Aitha, Mahesh; Benkovic, Stephen J

    2017-07-11

    In humans, proliferating cell nuclear antigen (PCNA) sliding clamps encircling DNA coordinate various aspects of DNA metabolism throughout the cell cycle. A critical aspect of this is restricting PCNA to the vicinity of its DNA target site. For example, PCNA must be maintained at or near primer/template (P/T) junctions during DNA synthesis. With a diverse array of cellular factors implicated, many of which interact with PCNA, DNA, or both, it is unknown how this critical feat is achieved. Furthermore, current biochemical assays that examine the retention of PCNA near P/T junctions are inefficient, discontinuous, and qualitative and significantly deviate from physiologically relevant conditions. To overcome these challenges and limitations, we recently developed a novel and convenient Förster resonance energy transfer (FRET) assay that directly and continuously monitors the retention of human PCNA at a P/T junction. Here we describe in detail the design, methodology, interpretation, and limitations of this quantitative FRET assay using the single-stranded DNA-binding protein, SSB, from Escherichia coli as an example. This powerful tool is broadly applicable to any single-stranded DNA-binding protein and may be utilized and/or expanded upon to dissect DNA metabolic pathways that are dependent upon PCNA.

  4. Pro-recombination Role of Srs2 Protein Requires SUMO (Small Ubiquitin-like Modifier) but Is Independent of PCNA (Proliferating Cell Nuclear Antigen) Interaction.

    PubMed

    Kolesar, Peter; Altmannova, Veronika; Silva, Sonia; Lisby, Michael; Krejci, Lumir

    2016-04-01

    Srs2 plays many roles in DNA repair, the proper regulation and coordination of which is essential. Post-translational modification by small ubiquitin-like modifier (SUMO) is one such possible mechanism. Here, we investigate the role of SUMO in Srs2 regulation and show that the SUMO-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 of SIM in asrs2ΔPIMstrain leads to a decrease in recombination, indicating a pro-recombination role of SUMO. Thus SIM has an ambivalent function in Srs2 regulation; it not only mediates interaction with SUMO-PCNA to promote the anti-recombination function but it also plays a PCNA-independent pro-recombination role, probably by stimulating the formation of recombination complexes. The fact that deletion of PIM suppresses the phenotypes of Srs2 lacking SIM suggests that proper balance between the anti-recombination PCNA-bound and pro-recombination pools of Srs2 is crucial. Notably, sumoylation of Srs2 itself specifically stimulates recombination at the rDNA locus.

  5. Expression of insulin-like growth factor-1 and proliferating cell nuclear antigen in human pulp cells of teeth with complete and incomplete root development.

    PubMed

    Caviedes-Bucheli, J; Canales-Sánchez, P; Castrillón-Sarria, N; Jovel-Garcia, J; Alvarez-Vásquez, J; Rivero, C; Azuero-Holguín, M M; Diaz, E; Munoz, H R

    2009-08-01

    To quantify the expression of insulin-like growth factor-1 (IGF-1) and proliferating cell nuclear antigen (PCNA) in human pulp cells of teeth with complete or incomplete root development, to support the specific role of IGF-1 in cell proliferation during tooth development and pulp reparative processes. Twenty six pulp samples were obtained from freshly extracted human third molars, equally divided in two groups according to root development stage (complete or incomplete root development). All samples were processed and immunostained to determine the expression of IGF-1 and PCNA in pulp cells. Sections were observed with a light microscope at 80x and morphometric analyses were performed to calculate the area of PCNA and IGF-1 immunostaining using digital image software. Mann-Whitney's test was used to determine statistically significant differences between groups (P < 0.05) for each peptide and the co-expression of both. Expression of IGF-1 and PCNA was observed in all human pulp samples with a statistically significant higher expression in cells of pulps having complete root development (P = 0.0009). Insulin-like growth factor-1 and PCNA are expressed in human pulp cells, with a significant greater expression in pulp cells of teeth having complete root development.

  6. Expression of epstein-barr virus nuclear antigen 1 is associated with enhanced expression of CD25 in the Hodgkin cell line L428.

    PubMed

    Kube, D; Vockerodt, M; Weber, O; Hell, K; Wolf, J; Haier, B; Grässer, F A; Müller-Lantzsch, N; Kieff, E; Diehl, V; Tesch, H

    1999-02-01

    Epstein-Barr virus is associated with several human malignancies including Burkitt's lymphoma, nasopharyngeal carcinoma, and Hodgkin's disease (HD). To examine the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1) in the pathogenesis of HD, we transfected the gene into the HD cell line L428. EBNA-1 expression was associated with significantly enhanced CD25 expression (interleukin 2 [IL-2]-receptor alpha chain) in transient and stably transfected L428 cells but did not affect the expression of IL-2 receptor beta and gamma chains. There was no up-regulation of the B-cell activation molecules CD23, CD30, CD39, CD40, CD44, CD71, and CD54 (intercellular adhesion molecule 1) or enhanced production of IL-6, IL-10, lymphotoxin alpha, and the soluble form of CD25. Stable EBNA-1-expressing L428 cells were nontumorigenic in SCID mice but showed enhanced lymphoma development in nonobese diabetic-SCID mice compared to mock-transfected cells.

  7. Specific testing for "isolated" anti-52 kDa SSA/Ro antibodies during standard anti-extractable nuclear antigen testing is of limited clinical value.

    PubMed

    Langguth, Daman M; Morris, Samantha; Clifford, Lynette; Wilson, Robert J; Neil, John; Hogan, Patrick G; Wong, Richard C W

    2007-06-01

    To ascertain whether specific testing for "isolated" anti-52 kDa SSA/Ro antibodies (a-SSA/Ro52) during standard anti-extractable nuclear antigen (ENA) testing is clinically useful. 1438 consecutive sera submitted for anti-ENA testing over 1 year were evaluated for a-SSA/Ro52 using various assays. 7 of 1438 (0.48%) patients were found to have a-SSA/Ro52 without SSA/Ro60 antibodies. Subsequent testing detected a further five patients. Clinical follow-up was possible in 10/12 patients. 2 of these 10 patients had evidence of primary Sjögren's syndrome (SS) and one had systemic lupus erythematosus (SLE), with sicca symptoms and abnormal Schirmer's tests. Five other patients had sicca symptoms, of which four had abnormal Schirmer's tests. "Isolated" anti-52 kDa SSA/Ro antibodies were detected in approximately 0.5% of standard anti-ENA requests, in which their presence was generally not associated with underlying SS or SLE. In view of the increased testing complexity and costs in detecting and confirming these antibodies, specific testing for isolated a-SSA Ro52 antibodies during standard anti-ENA testing seems to be of limited clinical value in a non-obstetric population.

  8. Identification of Small Molecule Proliferating Cell Nuclear Antigen (PCNA) Inhibitor That Disrupts Interactions with PIP-box Proteins and Inhibits DNA Replication*

    PubMed Central

    Punchihewa, Chandanamali; Inoue, Akira; Hishiki, Asami; Fujikawa, Yoshihiro; Connelly, Michele; Evison, Benjamin; Shao, Youming; Heath, Richard; Kuraoka, Isao; Rodrigues, Patrick; Hashimoto, Hiroshi; Kawanishi, Masanobu; Sato, Mamoru; Yagi, Takashi; Fujii, Naoaki

    2012-01-01

    We have discovered that 3,3′,5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC50 of ∼1 μm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser139)histone H2AX induction and cell growth inhibition was observed. PMID:22383522

  9. Replication factor C is a more effective proliferating cell nuclear antigen (PCNA) opener than the checkpoint clamp loader, Rad24-RFC.

    PubMed

    Thompson, Jennifer A; Marzahn, Melissa R; O'Donnell, Mike; Bloom, Linda B

    2012-01-13

    Clamp loaders from all domains of life load clamps onto DNA. The clamp tethers DNA polymerases to DNA to increase the processivity of synthesis as well as the efficiency of replication. Here, we investigated proliferating cell nuclear antigen (PCNA) binding and opening by the Saccharomyces cerevisiae clamp loader, replication factor C (RFC), and the DNA damage checkpoint clamp loader, Rad24-RFC, using two separate fluorescence intensity-based assays. Analysis of PCNA opening by RFC revealed a two-step reaction in which RFC binds PCNA before opening PCNA rather than capturing clamps that have transiently and spontaneously opened in solution. The affinity of RFC for PCNA is about an order of magnitude lower in the absence of ATP than in its presence. The affinity of Rad24-RFC for PCNA in the presence of ATP is about an order magnitude weaker than that of RFC for PCNA, similar to the RFC-PCNA interaction in the absence of ATP. Importantly, fewer open clamp loader-clamp complexes are formed when PCNA is bound by Rad24-RFC than when bound by RFC.

  10. Trigonella foenum (Fenugreek) Induced Apoptosis in Hepatocellular Carcinoma Cell Line, HepG2, Mediated by Upregulation of p53 and Proliferating Cell Nuclear Antigen

    PubMed Central

    Khalil, Mahmoud I. M.; Ibrahim, Mohamed M.; El-Gaaly, Gehan A.; Sultan, Ahmed S.

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and most current therapies are of limited efficacy. Trigonella foenum (Fenugreek) is a traditional herbal plant with antitumor activity, although the mechanisms of its activity remain unclear. Herein, a crude methanol extract was prepared from Fenugreek seeds (FCE) and its anticancer mechanism was evaluated, using HepG2 cell line. Growth-inhibitory effect and apoptosis induction of HepG2 cells were evidenced by MTT assay, cell morphology alteration, apoptosis enzyme-linked immunosorbent assay, flow cytometric analysis, caspase-3 activity, and expression of p53, proapoptotic protein, Bax, and proliferating cell nuclear antigen (PCNA) after (100∼500 μg/mL) FCE treatment for 48 h. Furthermore, FCE was analyzed by Chromatography-Mass Spectrometry (GC/MS). Our results revealed that FCE treatment for 48 h showed a cytotoxic effect and apoptosis induction in a dose-dependent manner that was mediated by upregulation of p53, Bax, PCNA, and caspase-3 activation in HepG2 cells. GC-MS analysis of FCE showed the presence of fourteen bioactive compounds such as Terpenoids and Flavonoids, including two main constituents with anticancer activity, Squalene and Naringenin (27.71% and 24.05%), respectively. Our data introduced FCE as a promising nontoxic herbal with therapeutic potential to induce apoptosis in HepG2 cells through p53, Bax, and PCNA upregulation in caspase-3 dependent manner. PMID:26557712

  11. Allo-antigen stimulated CD8+ T-cells suppress NF-κB and Ets-1 DNA binding activity, and inhibit phosphorylated NF-κB p65 nuclear localization in CD4+ T-cells.

    PubMed

    Nagashima, Ryuichi; Kawakami, Fumitaka; Takahashi, Shinichiro; Obata, Fumiya; Kubo, Makoto

    2014-08-01

    CD8+ T-cells of asymptomatic HIV-1 carriers (AC) suppress human immunodeficiency virus type 1 (HIV-1) replication in a class I major histocompatibility complex (MHC-I)-restricted and -unrestricted manner. In order to investigate the mechanism of MHC-I-unrestricted CD8+ T-cell-mediated HIV-1 suppression, we previously established allo-antigen stimulated CD8+T-cells from HIV-1-uninfected donors. These allo-antigen stimulated CD8+ T-cells suppressed HIV-1 replication in acutely infected autologous CD4+ T-cells when directly co-cultured. To elucidate the mechanism of HIV-1 replication suppression, we analyzed DNA-binding activity and phosphorylation of transcriptional factors associated with HIV-1 replication by electrophoresis mobility shift assay and Western blotting. When CD4+ T-cells were cultured with allo-antigen stimulated CD8+ T-cells, the reduction of NF-κB and Ets-1 DNA-binding activity was observed. Nuclear localization of NF-κB p65 and Ets-1 was suppressed in CD4+ T-cells. Although NF-κB p65 and Ets-1 are known to be regulated by protein kinase A (PKA), no difference was observed in the expression and phosphorylation of the PKA catalytic subunit in CD4+ T-cells cultured with PHA-treated CD8+ T-cells or allo-antigen stimulated CD8+ T-cells. Cyclic AMP is also known to enter through gap junctions, but the suppression of HIV-1 replication mediated by allo-antigen stimulated CD8+ T-cells was not affected by the gap junction inhibitor. The nuclear transport of phosphorylated NF-κB p65 (Ser276) was inhibited only in CD4+ T-cells cultured with allo-antigen stimulated CD8+ T-cells. Our results indicate that allo-antigen stimulated CD8+ T-cells suppress the transcriptional activity of NF-κB p65 or Ets-1 in an antigen-nonspecific manner, and inhibit the nuclear transport of phosphorylated NF-κB p65 (Ser276).

  12. Human liver nucleolar antigens.

    PubMed

    Busch, R K; Busch, H

    1981-10-01

    In an extension of previous studies on the antigens in rat liver nucleoli (R. K. Busch, R. C. Reddy, D. H. Henning, and H. Busch, Proc. Soc. Exp. Biol. Med. 160, 185 (1979); R. K. Busch and H. Busch, Tumori 63, 347 (1977); F. M. Davis, R. K. Busch, L. C. Yeoman, and H. Busch, Cancer Res. 38, 1906 (1978), rabbit antibodies were elicited to human liver nucleoli isolated by the sucrose--Mg2+ method (10). Fluorescent nucleoli were found in liver cryostat sections treated with rabbit anti-human liver nucleolar antibodies followed by fluorescein-conjugated goat anti-rabbit antibodies. In HeLa cells, fluorescence was distributed throughout the nucleus and in a nuclear network but was not localized to the nucleolus. In placental cryostat sections, an overall nuclear fluorescence was observed with some localization to nucleoli. Immunodiffusion analysis revealed two immunoprecipitin bands which appeared to be liver specific. Other immunoprecipitin bands were common to liver, placenta, and HeLa nuclear extracts. Rocket immunoelectrophoresis revealed two liver-specific antigens, one migrating to the cathode and the other to the anode Other rockets exhibited identity to antigens of other nuclear extracts. These results demonstrate the presence of human liver nucleolar-specific antigens which were not found in the HeLa and placental cells.

  13. Potentiation of latent inhibition.

    PubMed

    Rodriguez, Gabriel; Hall, Geoffrey

    2008-07-01

    Rats were given exposure either to an odor (almond) or a compound of odor plus taste (almond plus saline), prior to training in which the odor served as the conditioned stimulus. It was found, for both appetitive and aversive procedures, that conditioning was retarded by preexposure (a latent inhibition effect), and the extent of the retardation was greater in rats preexposed to the compound (i.e., latent inhibition to the odor was potentiated by the presence of the taste). In contrast, the presence of the taste during conditioning itself overshadowed learning about the odor. We argue that the presence of the salient taste in compound with the odor enhances the rate of associative learning, producing a rapid loss in the associability of the odor. This loss of associability will generate both overshadowing and the potentiation of latent inhibition that is observed after preexposure to the compound.

  14. Cyclosporin A promotes proliferating cell nuclear antigen expression and migration of human cytotrophoblast cells via the mitgen-activated protein kinase-3/1-mediated nuclear factor-κB signaling pathways.

    PubMed

    Wang, Song-Cun; Yu, Min; Li, Yan-Hong; Piao, Hai-Lan; Tang, Chuan-Lin; Sun, Chan; Zhu, Rui; Li, Ming Qing; Jin, Li-Ping; Li, Da-Jin; Du, Mei-Rong

    2013-01-01

    Our previous studies have demonstrated that cyclosporin A (CsA) promotes the proliferation and migration of human trophoblasts via the mitgen-activated protein kinase-3/1 (MAPK3/1) pathway. In the present study, we further investigated the role of nuclear factor (NF)-κB in the CsA-induced trophoblast proliferating cell nuclear antigen (PCNA) expression and migration, and its relationship to MAPK3/1 signal. Flow cytometry was used to analyze the expression of PCNA in trophoblasts. The migration of human primary trophoblasts was determined by wound-healing assay and transwell migration assay. Western blot analysis was performed to evaluate the activation of NF-κB p65 and NF-κB inhibitory protein I-κB in human trophoblasts. We found that treatment with CsA promotes PCNA expression and migration of human trophoblast in a dose-associated manner. Blocking of the MAPK3/1 signal abrogated the enhanced PCNA expression and migration in trophoblasts by CsA. In addition, CsA increased the phosphorylation of NF-κB p65 and the inhibitor I-κB in human trophoblasts in a time-related manner. Pretreatment with MAPK3/1 inhibitor U0126 abrogated the phosphorylation of NF-κB p65 and I-κB. Accordingly, the CsA-induced enhancement of PCNA expression and migration in trophoblasts was also decreased. This CsA-induced enhancement in the expression and migration of trophoblasts was abolished by pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor. Thus, our results suggest that CsA promotes PCNA expression and migration of human trophoblasts via MAPK-mediated NF-κB activation.

  15. Identification of Viral and Host Proteins That Interact with Murine Gammaherpesvirus 68 Latency-Associated Nuclear Antigen during Lytic Replication: a Role for Hsc70 in Viral Replication

    PubMed Central

    Salinas, Eduardo; Byrum, Stephanie D.; Moreland, Linley E.; Mackintosh, Samuel G.; Tackett, Alan J.

    2015-01-01

    ABSTRACT Latency-associated nuclear antigen (LANA) is a conserved, multifunctional protein encoded by members of the rhadinovirus subfamily of gammaherpesviruses, including Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68). We previously demonstrated that MHV68 LANA (mLANA) is required for efficient lytic replication. However, mechanisms by which mLANA facilitates viral replication, including interactions with cellular and viral proteins, are not known. Thus, we performed a mass spectrometry-based interaction screen that defined an mLANA protein-protein interaction network for lytic viral replication consisting of 15 viral proteins and 191 cellular proteins, including 19 interactions previously reported in KSHV LANA interaction studies. We also employed a stable-isotope labeling technique to illuminate high-priority mLANA-interacting host proteins. Among the top prioritized mLANA-binding proteins was a cellular chaperone, heat shock cognate protein 70 (Hsc70). We independently validated the mLANA-Hsc70 interaction through coimmunoprecipitation and in vitro glutathione S-transferase (GST) pulldown assays. Immunofluorescence and cellular fractionation analyses comparing wild-type (WT) to mLANA-null MHV68 infections demonstrated mLANA-dependent recruitment of Hsc70 to nuclei of productively infected cells. Pharmacologic inhibition and small hairpin RNA (shRNA)-mediated knockdown of Hsc70 impaired MHV68 lytic replication, which functionally correlated with impaired viral protein expression, reduced viral DNA replication, and failure to form viral replication complexes. Replication of mLANA-null MHV68 was less affected than that of WT virus by Hsc70 inhibition, which strongly suggests that Hsc70 function in MHV68 lytic replication is at least partially mediated by its interaction with mLANA. Together these experiments identify proteins engaged by mLANA during the MHV68 lytic replication cycle and define a previously unknown role for Hsc

  16. Prognostic value and clinicopathological significance of proliferating cell nuclear antigen expression in gastric cancer: a systematic review and meta-analysis

    PubMed Central

    Yin, Songcheng; Li, Zhan; Huang, Jinyu; Miao, Zhifeng; Zhang, Junyan; Lu, Chunyang; Xu, Hao; Xu, Huimian

    2017-01-01

    Background The prognostic significance of proliferating cell nuclear antigen (PCNA) expression in gastric cancer has long been assessed, yet results remain controversial. Therefore, we performed a meta-analysis to assess the prognostic value and clinicopathological significance of PCNA in gastric cancer. Methods A systematic literature search of PubMed, EMBASE, and the Cochrane Library databases was conducted. Summary odds ratios (ORs) and hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated to investigate the correlations between PCNA expression and clinicopathological features, overall survival (OS), and disease-free survival (DFS). Results A total of 19 studies involving 2,852 participants were included in our analysis. The pooled HR indicated that high PCNA expression was significantly associated with poor OS (HR 1.66, 95% CI 1.32–2.08) and DFS (HR 1.81, 95% CI 1.40–2.36). Subgroup analysis revealed that the association between PCNA and OS was also significant in Asian and European patients. In addition, the pooled ORs showed that high PCNA expression was significantly associated with deeper tumor invasion (OR 2.37, 95% CI 1.71–3.27), lymph node metastasis (OR 2.49, 95% CI 1.85–3.35), and advanced stage cancer (OR 1.89, 95% CI 1.36–2.63). Conclusion Our meta-analysis indicates that high PCNA expression might be a prognosticator of poor survival and a promising therapeutic target for gastric cancer patients. PMID:28138255

  17. Extracellular-signal regulated kinase 8 of Trypanosoma brucei uniquely phosphorylates its proliferating cell nuclear antigen homolog and reveals exploitable properties

    PubMed Central

    Valenciano, Ana L.; Knudsen, Giselle M.; Mackey, Zachary B.

    2016-01-01

    ABSTRACT The Trypanosoma brucei subspecies T. brucei gambiense and T. brucei rhodesiense are vector-borne pathogens that cause sleeping sickness also known as Human African Trypanosomiasis (HAT), which is fatal if left untreated. The drugs that treat HAT are ineffective and cause toxic side effects. One strategy for identifying safer and more effective HAT drugs is to therapeutically exploit essential gene targets in T. brucei. Genes that make up a basic mitogen-activated protein kinase (MAPK) network are present in T. brucei. Tb927.10.5140 encodes an essential MAPK that is homologous to the human extracellular-signal regulated kinase 8 (HsERK8) which forms a tight complex with the replication factor proliferating cell nuclear antigen (PCNA) to stabilize intracellular PCNA levels. Here we demonstrate that (TbPCNA) is uniquely phos-phorylated on serine (S) and threonine (T) residues in T. brucei and that TbERK8 phosphorylates TbPCNA at each of these residues. The ability of an ERK8 homolog to phosphorylate a PCNA homolog is a novel biochemical property that is first demonstrated here in T. brucei and may be unique to this pathogen. We demonstrate that the potent HsERK8 inhibitor Ro318220, has an IC50 for TbERK8 that is several hundred times higher than its reported IC50 for HsERK8. This indicated that the active sites of TbERK8 and HsERK8 can be selectively inhibited, which provides a rational basis for discovering inhibitors that specifically target this essential parasite MAPK to kill the parasite. PMID:27589575

  18. Amino Acids of Epstein-Barr Virus Nuclear Antigen 3A Essential for Repression of Jκ-Mediated Transcription and Their Evolutionary Conservation

    PubMed Central

    Dalbiès-Tran, Rozenn; Stigger-Rosser, Evelyn; Dotson, Travis; Sample, Clare E.

    2001-01-01

    Epstein-Barr virus (EBV) nuclear antigen 3A (EBNA-3A) is essential for virus-mediated immortalization of B lymphocytes in vitro and is believed to regulate transcription of cellular and/or viral genes. One known mechanism of regulation is through its interaction with the cellular transcription factor Jκ. This interaction downregulates transcription mediated by EBNA-2 and Jκ. To identify the amino acids that play a role in this interaction, we have generated mutant EBNA-3A proteins. A mutant EBNA-3A protein in which alanine residues were substituted for amino acids 199, 200, and 202 no longer downregulated transcription. Surprisingly, this mutant protein remained able to coimmunoprecipitate with Jκ. Using a reporter gene assay based on the recruitment of Jκ by various regions spanning EBNA-3A, we have shown that this mutation abolished binding of Jκ to the N-proximal region (amino acids 125 to 222) and that no other region of EBNA-3A alone was sufficient to mediate an association with Jκ. To determine the biological significance of the interaction of EBNA-3A with Jκ, we have studied its conservation in the simian lymphocryptovirus herpesvirus papio (HVP) by cloning HVP-3A, the homolog of EBNA-3A encoded by this virus. This 903-amino-acid protein exhibited 37% identity with its EBV counterpart, mainly within the amino-terminal half. HVP-3A also interacted with Jκ through a region located between amino acids 127 and 223 and also repressed transcription mediated through EBNA-2 and Jκ. The evolutionary conservation of this function, in proteins that have otherwise significantly diverged, argues strongly for an important biological role in virus-mediated immortalization of B lymphocytes. PMID:11119577

  19. Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus (KSHV) upregulates survivin expression in KSHV-Associated B-lymphoma cells and contributes to their proliferation.

    PubMed

    Lu, Jie; Verma, Subhash C; Murakami, Masanao; Cai, Qiliang; Kumar, Pankaj; Xiao, Bingyi; Robertson, Erle S

    2009-07-01

    Survivin is a master regulator of cell proliferation and cell viability and is highly expressed in most human tumors. The molecular network linked to survivin expression in tumors has not been completely elucidated. In this study, we show that latency-associated nuclear antigen (LANA), a multifunctional protein of Kaposi's sarcoma-associated herpesvirus (KSHV) that is found in Kaposi's sarcoma tumors, upregulates survivin expression and increases the proliferation of KSHV-infected B cells. Analysis of pathway-specific gene arrays showed that survivin expression was highly upregulated in BJAB cells expressing LANA. The mRNA levels of survivin were also upregulated in HEK 293 and BJAB cells expressing LANA. Similarly, protein levels of survivin were significantly higher in LANA-expressing, as well as KSHV-infected, cells. Survivin promoter activity assays identified GC/Sp1 and p53 cis-acting elements within the core promoter region as being important for LANA activity. Gel mobility shift assays revealed that LANA forms a complex with Sp1 or Sp1-like proteins bound to the GC/Sp1 box of the survivin promoter. In addition, a LANA/p53 complex bound to the p53 cis-acting element within the survivin promoter, indicating that upregulation of survivin expression can also occur through suppression of p53 function. Furthermore, immunohistochemistry analyses revealed that survivin expression was upregulated in KSHV-associated Kaposi's sarcoma tissue, suggesting that LANA plays an important role in the upregulation of survivin expression in KSHV-infected endothelial cells. Knockdown of survivin expression by lentivirus-delivered small hairpin RNA resulted in loss of cell proliferation in KSHV-infected cells. Therefore, upregulation of survivin expression in KSHV-associated human cells contributes to their proliferation.

  20. Prognostic impact of DNA ploidy pattern, S-phase fraction (SPF), and proliferating cell nuclear antigen (PCNA) in patients with primary gastric lymphoma.

    PubMed

    Belessi, Chrysoula J; Parasi, Aikaterini S; Manioudaki, Helen S; Laoutaris, Nikolaos P; Legakis, Nikolaos C; Peros, Georgios Th; Androulakis, Georgios A

    2003-04-01

    DNA ploidy, S-phase fraction (SPF), and proliferating cell nuclear antigen (PCNA) are considered to be significant prognostic factors in non-Hodgkin lymphomas. However, reports on their prognostic importance in gastric lymphoma patients are relatively lacking. In the present study, we retrospectively studied the above-mentioned parameters in 29 patients with primary gastric lymphoma; 11/29 had B-low grade mucosa associated lymphoid tissue lymphoma (B-MALT), while 18/29 had diffuse large B-cell lymphoma (DLBCL), according to WHO classification. Proliferative activity was studied by staining against PCNA; in addition, the prognostic significance of DNA ploidy and SPF, as determined by flow cytometry, were investigated and compared to the results of the PCNA stainings. Seven out of 29 patients were found to have aneuploid tumors; DNA index values were >1 for all aneuploid lymphomas. There was no difference in DNA aneuploidy in MALT vs. DLBCL. The mean percentage of SPF was 11.4. SPF was found significantly lower in MALT vs. DLBCL (P < 0.05). The mean percentage of PCNA positive tumor cells was 52.6. PCNA protein expression was significantly lower in MALT vs. DLBCL (P < 0.0001). There was a significant positive correlation between PCNA score and SPF (P < 0.01, by Spearman analysis). DNA ploidy had no impact on survival in the present study. Both SPF and PCNA expression were important prognostic factors in the univariate analysis; however, in the multivariate analysis, the only independent prognostic factor for survival was PCNA expression. These findings indicate that SPF and PCNA are significant prognostic factors in patients with primary gastric lymphomas. However, in the present study, DNA ploidy had no impact on survival in patients with primary gastric lymphomas. Copyright 2003 Wiley-Liss, Inc.

  1. In vivo dynamics of EBNA1-oriP interaction during latent and lytic replication of Epstein-Barr virus.

    PubMed

    Daikoku, Tohru; Kudoh, Ayumi; Fujita, Masatoshi; Sugaya, Yutaka; Isomura, Hiroki; Tsurumi, Tatsuya

    2004-12-24

    The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is required for maintenance of the viral genome DNA during the latent phase of EBV replication but continues to be synthesized after the induction of viral productive replication. An EBV genome-wide chromatin immunoprecipitation assay revealed that EBNA1 constantly binds to oriP of the EBV genome during not only latent but also lytic infection. Although the total levels of EBNA1 proved constant throughout the latter, the levels of the oriP-bound form were increased as lytic infection proceeded. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins are clustered. Confocal laser microscopic analyses revealed that whereas EBNA1 was distributed broadly in nuclei as fine punctate dots during the latent phase of infection, the protein became redistributed to the viral replication compartments and localized as distinct spots within and/or nearby the compartments after the induction of lytic replication. Taking these findings into consideration, oriP regions of the EBV genome might be organized by EBNA1 into replication domains that may set up scaffolding for lytic replication and transcription.

  2. Latent Semantic Analysis.

    ERIC Educational Resources Information Center

    Dumais, Susan T.

    2004-01-01

    Presents a literature review that covers the following topics related to Latent Semantic Analysis (LSA): (1) LSA overview; (2) applications of LSA, including information retrieval (IR), information filtering, cross-language retrieval, and other IR-related LSA applications; (3) modeling human memory, including the relationship of LSA to other…

  3. Latent Semantic Analysis.

    ERIC Educational Resources Information Center

    Dumais, Susan T.

    2004-01-01

    Presents a literature review that covers the following topics related to Latent Semantic Analysis (LSA): (1) LSA overview; (2) applications of LSA, including information retrieval (IR), information filtering, cross-language retrieval, and other IR-related LSA applications; (3) modeling human memory, including the relationship of LSA to other…

  4. Measuring Latent Quantities

    ERIC Educational Resources Information Center

    McDonald, Roderick P.

    2011-01-01

    A distinction is proposed between measures and predictors of latent variables. The discussion addresses the consequences of the distinction for the true-score model, the linear factor model, Structural Equation Models, longitudinal and multilevel models, and item-response models. A distribution-free treatment of calibration and…

  5. Lipid antigens in immunity

    PubMed Central

    Dowds, C. Marie; Kornell, Sabin-Christin

    2014-01-01

    Lipids are not only a central part of human metabolism but also play diverse and critical roles in the immune system. As such, they can act as ligands of lipid-activated nuclear receptors, control inflammatory signaling through bioactive lipids such as prostaglandins, leukotrienes, lipoxins, resolvins, and protectins, and modulate immunity as intracellular phospholipid- or sphingolipid-derived signaling mediators. In addition, lipids can serve as antigens and regulate immunity through the activation of lipid-reactive T cells, which is the topic of this review. We will provide an overview of the mechanisms of lipid antigen presentation, the biology of lipid-reactive T cells, and their contribution to immunity. PMID:23999493

  6. G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV

    PubMed Central

    Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P.; Robertson, Erle S.; Schildkraut, Carl L.; Verma, Subhash C.

    2016-01-01

    Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. PMID:26837574

  7. Latent effects decision analysis

    DOEpatents

    Cooper, J Arlin [Albuquerque, NM; Werner, Paul W [Albuquerque, NM

    2004-08-24

    Latent effects on a system are broken down into components ranging from those far removed in time from the system under study (latent) to those which closely effect changes in the system. Each component is provided with weighted inputs either by a user or from outputs of other components. A non-linear mathematical process known as `soft aggregation` is performed on the inputs to each component to provide information relating to the component. This information is combined in decreasing order of latency to the system to provide a quantifiable measure of an attribute of a system (e.g., safety) or to test hypotheses (e.g., for forensic deduction or decisions about various system design options).

  8. Variations of Epstein-Barr virus nuclear antigen 1 in Epstein-Barr virus-associated gastric carcinomas from Guangzhou, southern China.

    PubMed

    Chen, Jian-ning; Zhang, Na-na; Jiang, Ye; Hui, Da-yang; Wen, Zi-jin; Li, Hai-gang; Ding, Yun-gang; Du, Hong; Shao, Chun-kui

    2012-01-01

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated malignancies, and play a critical role in the onset, progression, and/or maintenance of these tumors. Based on the signature changes at amino acid residue 487, EBNA1 is classified into five distinct subtypes: P-ala, P-thr, V-leu, V-val and V-pro. In the present study, the sequence variations of EBNA1 in EBV-associated gastric carcinoma (EBVaGC) and throat washing (TW) samples of healthy EBV carriers in Guangzhou, southern China, where nasopharyngeal carcinoma (NPC) is endemic, were analyzed by PCR and DNA sequencing. V-val subtype was the most predominant (53.6%, 15/28) in EBVaGC, followed by P-ala (42.9%, 12/28) and V-leu (32.1%, 9/28) subtypes. In TWs of healthy EBV carriers, V-val subtype was also predominant (85.7%, 18/21). The sequence variations of EBNA1 in EBVaGC were similar to those in TW of healthy EBV carriers (p>0.05), suggesting that the EBV strains in EBVaGC might originate from the viral strains prevalent within the background population. The predominance of V-val subtype in EBVaGC in Guangzhou was similar to that in EBVaGC in northern China and Japan, but was different from that in EBVaGC in America, suggesting that the variations of EBNA1 in EBVaGC represent geographic-associated polymorphisms rather than tumor-specific mutations. In addition, the EBNA1 variations in EBVaGC in gastric remnant carcinoma were also determined. V-leu subtype was detected in all 4 (100%) cases, although 2 cases occurred as mixed infection with P-ala subtype. This is different from the predominant V-val subtype in EBVaGC in conventional gastric carcinoma, suggesting that V-leu might be a subtype that adapts particularly well to the microenvironment within the gastric stump and enters the remnant gastric mucosa epithelia easily. This, to our best knowledge, is the first investigation of EBNA1 polymorphisms in EBVaGC from endemic area of NPC.

  9. Peach latent mosaic viroid: not so latent.

    PubMed

    Flores, Ricardo; Delgado, Sonia; Rodio, María-Elena; Ambrós, Silvia; Hernández, Carmen; Serio, Francesco D I

    2006-07-01

    SUMMARY Taxonomy: Peach latent mosaic viroid (PLMVd) is the type species of the genus Pelamoviroid within the family Avsunviroidae of chloroplastic viroids with hammerhead ribozymes. Physical properties: A small circular RNA of 336-351 nt (differences in size result from the absence or presence of certain insertions) adopting a branched conformation stabilized by a pseudoknot between two kissing loops. This particular conformation is most likely responsible for the insolubility of PLMVd in highly saline conditions (in which other viroids adopting a rod-like conformation are soluble). Both polarity strands are able to form hammerhead structures and to self-cleave during replication as predicted by these ribozymes. Biological properties: Although most infections occur without conspicuous symptoms, certain PLMVd isolates induce leaf mosaics, blotches and in the most extreme cases albinism (peach calico, PC), flower streaking, delays in foliation, flowering and ripening, deformations and decolorations of fruits, which usually present cracked sutures and enlarged roundish stones, bud necrosis, stem pitting and premature ageing of the trees, which also adopt a characteristic growing pattern (open habit). The molecular determinant for PC has been mapped at a 12-14-nt insertion that folds into a hairpin capped by a U-rich loop present only in certain variants. PLMVd is horizontally transmitted by the propagation of infected buds and to a lesser extent by pruning tools and aphids, but not by pollen; the viroid is not vertically transmitted through seed. Interesting features: This provides a suitable system for studying how a minimal non-protein-coding catalytic RNA replicates (subverting a DNA-dependent RNA polymerase to transcribe an RNA template), moves, interferes with the metabolism of its host (inciting specific symptoms and a defensive RNA silencing response) and evolves following a quasi-species model characterized by a complex spectrum of variants.

  10. [PSA variations in persons with latent prostate cancer].

    PubMed

    Stamatiou, K; Danciu, M; Karakos, C; Sofras, F

    2008-01-01

    The introduction and common use of serum PSA (Prostate Specific Antigen) has been demonstrated a useful index on latent prostate cancer diagnostic but in the same time has increased surgical intervention on histological forms with no eventual future evolution. Benign comportment of latent carcinomas being well known in advance, we correlated in vitro serum PSA from latent tumors, with the samples from a control group (prostates without signs of malignization). Levels of PSA were slightly elevated compared to age norms, mainly in cases with a large coexistent hypertrophy. Our reduced sample does not stand any statistic analysis, but this observation could eventually explain increased diagnostic and hyper-treatment of non-important carcinomas from a clinical point of view.

  11. Learning multimodal latent attributes.

    PubMed

    Fu, Yanwei; Hospedales, Timothy M; Xiang, Tao; Gong, Shaogang

    2014-02-01

    The rapid development of social media sharing has created a huge demand for automatic media classification and annotation techniques. Attribute learning has emerged as a promising paradigm for bridging the semantic gap and addressing data sparsity via transferring attribute knowledge in object recognition and relatively simple action classification. In this paper, we address the task of attribute learning for understanding multimedia data with sparse and incomplete labels. In particular, we focus on videos of social group activities, which are particularly challenging and topical examples of this task because of their multimodal content and complex and unstructured nature relative to the density of annotations. To solve this problem, we 1) introduce a concept of semilatent attribute space, expressing user-defined and latent attributes in a unified framework, and 2) propose a novel scalable probabilistic topic model for learning multimodal semilatent attributes, which dramatically reduces requirements for an exhaustive accurate attribute ontology and expensive annotation effort. We show that our framework is able to exploit latent attributes to outperform contemporary approaches for addressing a variety of realistic multimedia sparse data learning tasks including: multitask learning, learning with label noise, N-shot transfer learning, and importantly zero-shot learning.

  12. Latent semantic analysis.

    PubMed

    Evangelopoulos, Nicholas E

    2013-11-01

    This article reviews latent semantic analysis (LSA), a theory of meaning as well as a method for extracting that meaning from passages of text, based on statistical computations over a collection of documents. LSA as a theory of meaning defines a latent semantic space where documents and individual words are represented as vectors. LSA as a computational technique uses linear algebra to extract dimensions that represent that space. This representation enables the computation of similarity among terms and documents, categorization of terms and documents, and summarization of large collections of documents using automated procedures that mimic the way humans perform similar cognitive tasks. We present some technical details, various illustrative examples, and discuss a number of applications from linguistics, psychology, cognitive science, education, information science, and analysis of textual data in general. WIREs Cogn Sci 2013, 4:683-692. doi: 10.1002/wcs.1254 CONFLICT OF INTEREST: The author has declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website. © 2013 John Wiley & Sons, Ltd.

  13. A Latent Transition Model with Logistic Regression

    ERIC Educational Resources Information Center

    Chung, Hwan; Walls, Theodore A.; Park, Yousung

    2007-01-01

    Latent transition models increasingly include covariates that predict prevalence of latent classes at a given time or transition rates among classes over time. In many situations, the covariate of interest may be latent. This paper describes an approach for handling both manifest and latent covariates in a latent transition model. A Bayesian…

  14. Kaposi Sarcoma Herpesvirus (KSHV) Latency-Associated Nuclear Antigen (LANA) recruits components of the MRN (Mre11-Rad50-NBS1) repair complex to modulate an innate immune signaling pathway and viral latency.

    PubMed

    Mariggiò, Giuseppe; Koch, Sandra; Zhang, Guigen; Weidner-Glunde, Magdalena; Rückert, Jessica; Kati, Semra; Santag, Susann; Schulz, Thomas F

    2017-04-01

    Kaposi Sarcoma Herpesvirus (KSHV), a γ2-herpesvirus and class 1 carcinogen, is responsible for at least three human malignancies: Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and Multicentric Castleman's Disease (MCD). Its major nuclear latency protein, LANA, is indispensable for the maintenance and replication of latent viral DNA in infected cells. Although LANA is mainly a nuclear protein, cytoplasmic isoforms of LANA exist and can act as antagonists of the cytoplasmic DNA sensor, cGAS. Here, we show that cytosolic LANA also recruits members of the MRN (Mre11-Rad50-NBS1) repair complex in the cytosol and thereby inhibits their recently reported role in the sensing of cytoplasmic DNA and activation of the NF-κB pathway. Inhibition of NF-κB activation by cytoplasmic LANA is accompanied by increased lytic replication in KSHV-infected cells, suggesting that MRN-dependent NF-κB activation contributes to KSHV latency. Cytoplasmic LANA may therefore support the activation of KSHV lytic replication in part by counteracting the activation of NF-κB in response to cytoplasmic DNA. This would complement the recently described role of cytoplasmic LANA in blocking an interferon response triggered by cGAS and thereby promoting lytic reactivation. Our findings highlight a second point at which cytoplasmic LANA interferes with the innate immune response, as well as the importance of the recently discovered role of cytoplasmic MRN complex members as innate sensors of cytoplasmic DNA for the control of KSHV replication.

  15. Epstein-Barr Virus nuclear antigen 1 (EBNA1) confers resistance to apoptosis in EBV-positive B-lymphoma cells through up-regulation of survivin

    SciTech Connect

    Lu Jie; Murakami, Masanao; Verma, Subhash C.; Cai Qiliang; Haldar, Sabyasachi; Kaul, Rajeev; Wasik, Mariusz A.; Middeldorp, Jaap; Robertson, Erle S.

    2011-02-05

    Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV-infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV-infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells.

  16. Epstein-Barr Virus nuclear antigen 1 (EBNA1) confers resistance to apoptosis in EBV-positive B-lymphoma cells through up-regulation of survivin.

    PubMed

    Lu, Jie; Murakami, Masanao; Verma, Subhash C; Cai, Qiliang; Haldar, Sabyasachi; Kaul, Rajeev; Wasik, Mariusz A; Middeldorp, Jaap; Robertson, Erle S

    2011-02-05

    Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV-infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV-infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  17. Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) Confers Resistance to Apoptosis in EBV positive B-lymphoma Cells through Up-regulation of Survivin

    PubMed Central

    Lu, Jie; Murakami, Masanao; Verma, Subhash C.; Cai, Qiliang; Haldar, Sabyasachi; Kaul, Rajeev; Wasik, Mariusz A.; Middeldorp, Jaap; Robertson, Erle S.

    2014-01-01

    Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells. PMID:21093004

  18. Vpx-containing Dendritic Cell Vaccine Vectors Induce CTLs and Reactivate Latent HIV-1 in vitro

    PubMed Central

    Norton, Thomas D.; Miller, Elizabeth A.; Bhardwaj, Nina; Landau, Nathaniel R.

    2015-01-01

    Eradication of HIV-1 from an infected individual requires a means of inducing production of virus from latently infected cells and stimulating an immune response against the infected cells. We report the development of lentiviral vectors that transduce dendritic cells (DCs) to both induce production of virus from latently infected cells and stimulate antigen-specific CTLs. The vectors package Vpx, a lentiviral accessory protein that counteracts the SAMHD1-mediated block to DC transduction, allowing for long-term expression of vector-encoded proteins. The vectors encode influenza or HIV-1-derived epitopes fused via a self-cleaving peptide to CD40L that releases the peptide into the endoplasmic reticulum for entry into the antigen presentation pathway. Expression of CD40L caused transduced DCs to mature and produce Th1-skewing cytokines. The DCs presented antigen to CD8 T cells, enhancing antigen-specific CTLs. Coculture of the transduced DCs with latently infected cells induced high level virus production, an effect that was mediated by TNF-α. The ability of a DC vaccine to reactivate latent HIV-1 and stimulate an adaptive immune response provides a means to reduce the size of the latent reservoir in patients. This strategy can also be applied to develop DC vaccines for other diseases. PMID:25567537

  19. The central repeat domain 1 of Kaposi's sarcoma-associated herpesvirus (KSHV) latency associated-nuclear antigen 1 (LANA1) prevents cis MHC class I peptide presentation

    SciTech Connect

    Kwun, Hyun Jin; Ramos da Silva, Suzane; Qin Huilian; Ferris, Robert L.; Tan Rusung; Chang Yuan; Moore, Patrick S.

    2011-04-10

    KSHV LANA1, a latent protein expressed during chronic infection to maintain a viral genome, inhibits major histocompatibility complex class I (MHC I) peptide presentation in cis as a means of immune evasion. Through deletional cloning, we localized this function to the LANA1 central repeat 1 (CR1) subregion. Other CR subregions retard LANA1 translation and proteasomal processing but do not markedly inhibit LANA1 peptide processing by MHC I. Inhibition of proteasomal processing ablates LANA1 peptide presentation. Direct expression of LANA1 within the endoplasmic reticulum (ER) overcomes CR1 inhibition suggesting that CR1 acts prior to translocation of cytoplasmic peptides into the ER. By physically separating CR1 from other subdomains, we show that LANA1 evades MHC I peptide processing by a mechanism distinct from other herpesviruses including Epstein-Barr virus (EBV). Although LANA1 and EBV EBNA1 are functionally similar, they appear to use different mechanisms to evade host cytotoxic T lymphocyte surveillance.

  20. ANTIGENIC MODULATION

    PubMed Central

    Old, Lloyd J.; Stockert, Elisabeth; Boyse, Edward A.; Kim, Jae Ho

    1968-01-01

    Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro. An ascites leukemia, phenotype TL.1,2,3, which modulates rapidly and completely when incubated with TL antiserum in vitro, was selected for further study of the phenomenon. Over a wide range of TL antibody concentrations modulation at 37°C was detectable within 10 min and was complete within approximately 1 hr. The cells were initially sensitized to C' by their contact with antibody, thereafter losing this sensitivity to C' lysis together with their sensitivity to TL antibody and C' in the cytotoxic test. The capacity of the cells to undergo modulation was abolished by actinomycin D and by iodoacetamide, and by reducing the temperature of incubation to 0°C. Thus modulation apparently is an active cellular process. Antigens TL. 1,2, and 3 are all modulated by anti-TL.1,3 serum and by anti-TL.3 serum. This modulation affects all three TL components together, even when antibody to one or two of them is lacking. aAnti-TL.2 serum does not induce modulation and in fact impairs modulation by the other TL antibodies. The influence of the TL phenotype of cells upon the demonstrable content of H-2 (D region) isoantigen, first shown in cells modulated in vivo, has been observed with cells modulated in vitro. Cells undergoing modulation show a progressive increase in H-2 (D region) antigen over a period of 4 hr, with no change in H-2 antigens of the K region. Restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ → TL- modulation and may require several cell divisions. PMID:5636556

  1. LATENT LIFE OF ARTERIES.

    PubMed

    Carrel, A

    1910-07-23

    When a segment of artery, killed by heat, formalin or glycerin is transplanted, it undergoes a rapid degeneration. Its muscle fibers disappear while the tissue of the host reacts by building a new wall of connective tissue. When the transplanted vessel has been preserved in a condition of latent life, no degeneration of the wall occurs, or the wall undergoes only partial degeneration. The muscle fibers can keep their normal appearance, even for a long time after the operation. It is, therefore, demonstrated that arteries can be preserved outside of the body in a condition of unmanifested actual life. The best method of preservation consists of placing the vessels, immersed in vaselin, in an ice box, the temperature of which is slightly above the freezing point. From a surgical standpoint, the transplantation of preserved vessels can be used with some safety. When the arteries were kept in defibrinated blood or vaselin and in cold storage, the proportion of positive results was 75 and 80 per cent., and this can probably be increased.

  2. CD4+ T-cell responses to Epstein-Barr virus nuclear antigen EBNA1 in Chinese populations are highly focused on novel C-terminal domain-derived epitopes.

    PubMed

    Tsang, C W; Lin, X; Gudgeon, N H; Taylor, G S; Jia, H; Hui, E P; Chan, A T C; Lin, C K; Rickinson, A B

    2006-08-01

    Epstein-Barr virus nuclear antigen EBNA1, the one viral protein uniformly expressed in nasopharyngeal carcinoma (NPC), represents a prime target for T-cell-based immunotherapy. However, little is known about the EBNA1 epitopes, particularly CD4 epitopes, presented by HLA alleles in Chinese people, the group at highest risk for NPC. We analyzed the CD4+ T-cell responses to EBNA1 in 78 healthy Chinese donors and found marked focusing on a small number of epitopes in the EBNA1 C-terminal region, including a DP5-restricted epitope that was recognized by almost half of the donors tested and elicited responses able to recognize EBNA1-expressing, DP5-positive target cells.

  3. Understanding Latent Heat of Vaporization.

    ERIC Educational Resources Information Center

    Linz, Ed

    1995-01-01

    Presents a simple exercise for students to do in the kitchen at home to determine the latent heat of vaporization of water using typical household materials. Designed to stress understanding by sacrificing precision for simplicity. (JRH)

  4. Understanding Latent Heat of Vaporization.

    ERIC Educational Resources Information Center

    Linz, Ed

    1995-01-01

    Presents a simple exercise for students to do in the kitchen at home to determine the latent heat of vaporization of water using typical household materials. Designed to stress understanding by sacrificing precision for simplicity. (JRH)

  5. Predicting Latent Class Scores for Subsequent Analysis

    ERIC Educational Resources Information Center

    Petersen, Janne; Bandeen-Roche, Karen; Budtz-Jorgensen, Esben; Larsen, Klaus Groes

    2012-01-01

    Latent class regression models relate covariates and latent constructs such as psychiatric disorders. Though full maximum likelihood estimation is available, estimation is often in three steps: (i) a latent class model is fitted without covariates; (ii) latent class scores are predicted; and (iii) the scores are regressed on covariates. We propose…

  6. Predicting Latent Class Scores for Subsequent Analysis

    ERIC Educational Resources Information Center

    Petersen, Janne; Bandeen-Roche, Karen; Budtz-Jorgensen, Esben; Larsen, Klaus Groes

    2012-01-01

    Latent class regression models relate covariates and latent constructs such as psychiatric disorders. Though full maximum likelihood estimation is available, estimation is often in three steps: (i) a latent class model is fitted without covariates; (ii) latent class scores are predicted; and (iii) the scores are regressed on covariates. We propose…

  7. Immune parameters differentiating active from latent tuberculosis infection in humans.

    PubMed

    Lee, Ji Yeon; Jung, Young Won; Jeong, Ina; Joh, Joon-Sung; Sim, Soo Yeon; Choi, Boram; Jee, Hyeon-Gun; Lim, Dong-Gyun

    2015-12-01

    Tuberculosis remains a highly prevalent infectious disease worldwide. Identification of the immune parameters that differentiate active disease from latent infection will facilitate the development of efficient control measures as well as new diagnostic modalities for tuberculosis. Here, we investigated the cytokine production profiles of monocytes and CD4(+) T lymphocytes upon encountering mycobacterial antigens. In addition, cytokines and lipid mediators with immune-modulating activities were examined in plasma samples ex vivo. Comparison of these parameters in active tuberculosis patients and healthy subjects with latent infection revealed that, active tuberculosis was associated with diminished Th1-type cytokine secretion from CD4(+) T cells and less augmented inflammatory cytokine secretion from monocytes induced by IFN-γ than that in latent tuberculosis infection. In addition, a higher plasma concentration of lipoxin A4 and lower ratio of prostaglandin E2 to lipoxin A4 were observed in active cases than in latent infections. These findings have implications for preparing new therapeutic strategies and for differential diagnosis of the two types of tuberculosis infection.

  8. Extended culture enhances sensitivity of a gamma interferon assay for latent Mycobacterium tuberculosis infection.

    PubMed

    Cehovin, Ana; Cliff, Jacqueline M; Hill, Philip C; Brookes, Roger H; Dockrell, Hazel M

    2007-06-01

    To test the hypothesis that prolonged culture would enhance the sensitivity of latent tuberculosis detection by a gamma interferon release assay, blood samples from 33 household contacts of Gambian tuberculosis patients were stimulated with Mycobacterium tuberculosis-specific antigens. After 24 h of culture, 66% were positive, compared to 93% after 6 days of culture.

  9. [The immunohistochemical determination of p53 and of proliferating cell nuclear antigen in the epithelial nuclei of benign prostatic hyperplasia following the accident at the Chernobyl Atomic Electric Power Station].

    PubMed

    Romanenko, A M; Vozianov, S O; Zabarko, L B

    1999-01-01

    With the purpose of studying into the morphogenesis and proliferous activity of the prostatic epithelium under a long-term exposure to low doses of ionizing radiation there have been conducted comparative histological and immunohistochemical (expression of p53 and proliferous cellular nuclear antigen-PCNA) investigations designed to study benign prostatic hyperplasia in patients living in those Ukraine territories affected by radionuclide contamination (group III), residents of Kiev (group II), and patients having been operated on before the Chernobyl accident, having constituted the control group I. It has been found out that the incidence of prostatic intraepithelial neoplasia (PIN), the level of nuclear expression of proteins p53 (in the PIN epithelium) and PCNA (in the epithelium of both benign prostatic hyperplasia and PIN) of patients in groups II and III are by far higher as compared with those in group I. The stroma of benign prostatic hyperplasia in patients of groups II and III was clearly different from that in the control group in that the former was characterized by apparent phenomena of hyalinosis, sclerosis, fibrosis, and extensive inflammatory infiltration, which changes can be explained by a long-term systematic exposure of prostatic tissue to low doses of ionizing radiation.

  10. Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes

    SciTech Connect

    Igarashi, Aki; Yamagata, Kousuke; Sugai, Tomokazu; Takahashi, Yukari; Sugawara, Emiko; Tamura, Akihiro; Yaegashi, Hajime; Yamagishi, Noriko; Takahashi, Tsubasa; Isogai, Masamichi; Takahashi, Hideki; Yoshikawa, Nobuyuki

    2009-04-10

    Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.

  11. Latent geometry of bipartite networks

    NASA Astrophysics Data System (ADS)

    Kitsak, Maksim; Papadopoulos, Fragkiskos; Krioukov, Dmitri

    2017-03-01

    Despite the abundance of bipartite networked systems, their organizing principles are less studied compared to unipartite networks. Bipartite networks are often analyzed after projecting them onto one of the two sets of nodes. As a result of the projection, nodes of the same set are linked together if they have at least one neighbor in common in the bipartite network. Even though these projections allow one to study bipartite networks using tools developed for unipartite networks, one-mode projections lead to significant loss of information and artificial inflation of the projected network with fully connected subgraphs. Here we pursue a different approach for analyzing bipartite systems that is based on the observation that such systems have a latent metric structure: network nodes are points in a latent metric space, while connections are more likely to form between nodes separated by shorter distances. This approach has been developed for unipartite networks, and relatively little is known about its applicability to bipartite systems. Here, we fully analyze a simple latent-geometric model of bipartite networks and show that this model explains the peculiar structural properties of many real bipartite systems, including the distributions of common neighbors and bipartite clustering. We also analyze the geometric information loss in one-mode projections in this model and propose an efficient method to infer the latent pairwise distances between nodes. Uncovering the latent geometry underlying real bipartite networks can find applications in diverse domains, ranging from constructing efficient recommender systems to understanding cell metabolism.

  12. Latent geometry of bipartite networks.

    PubMed

    Kitsak, Maksim; Papadopoulos, Fragkiskos; Krioukov, Dmitri

    2017-03-01

    Despite the abundance of bipartite networked systems, their organizing principles are less studied compared to unipartite networks. Bipartite networks are often analyzed after projecting them onto one of the two sets of nodes. As a result of the projection, nodes of the same set are linked together if they have at least one neighbor in common in the bipartite network. Even though these projections allow one to study bipartite networks using tools developed for unipartite networks, one-mode projections lead to significant loss of information and artificial inflation of the projected network with fully connected subgraphs. Here we pursue a different approach for analyzing bipartite systems that is based on the observation that such systems have a latent metric structure: network nodes are points in a latent metric space, while connections are more likely to form between nodes separated by shorter distances. This approach has been developed for unipartite networks, and relatively little is known about its applicability to bipartite systems. Here, we fully analyze a simple latent-geometric model of bipartite networks and show that this model explains the peculiar structural properties of many real bipartite systems, including the distributions of common neighbors and bipartite clustering. We also analyze the geometric information loss in one-mode projections in this model and propose an efficient method to infer the latent pairwise distances between nodes. Uncovering the latent geometry underlying real bipartite networks can find applications in diverse domains, ranging from constructing efficient recommender systems to understanding cell metabolism.

  13. The latent class twin method.

    PubMed

    Baker, Stuart G

    2016-09-01

    The twin method refers to the use of data from same-sex identical and fraternal twins to estimate the genetic and environmental contributions to a trait or outcome. The standard twin method is the variance component twin method that estimates heritability, the fraction of variance attributed to additive genetic inheritance. The latent class twin method estimates two quantities that are easier to interpret than heritability: the genetic prevalence, which is the fraction of persons in the genetic susceptibility latent class, and the heritability fraction, which is the fraction of persons in the genetic susceptibility latent class with the trait or outcome. We extend the latent class twin method in three important ways. First, we incorporate an additive genetic model to broaden the sensitivity analysis beyond the original autosomal dominant and recessive genetic models. Second, we specify a separate survival model to simplify computations and improve convergence. Third, we show how to easily adjust for covariates by extending the method of propensity scores from a treatment difference to zygosity. Applying the latent class twin method to data on breast cancer among Nordic twins, we estimated a genetic prevalence of 1%, a result with important implications for breast cancer prevention research. © 2016, The International Biometric Society.

  14. Epstein-Barr virus nuclear antigen 3A promotes cellular proliferation by repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1.

    PubMed

    Tursiella, Melissa L; Bowman, Emily R; Wanzeck, Keith C; Throm, Robert E; Liao, Jason; Zhu, Junjia; Sample, Clare E

    2014-10-01

    Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14(ARF) and p16(INK4a) expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14(ARF) and p16I(NK4a). By contrast, p16(INK4a) was not detectably expressed in Wp-R BL and the low-level expression of p14(ARF) was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21(WAF1/CIP1), a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21(WAF1/CIP1) expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the

  15. Epstein-Barr Virus Nuclear Antigen 3A Promotes Cellular Proliferation by Repression of the Cyclin-Dependent Kinase Inhibitor p21WAF1/CIP1

    PubMed Central

    Tursiella, Melissa L.; Bowman, Emily R.; Wanzeck, Keith C.; Throm, Robert E.; Liao, Jason; Zhu, Junjia; Sample, Clare E.

    2014-01-01

    Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14ARF and p16INK4a expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14ARF and p16INK4a. By contrast, p16INK4a was not detectably expressed in Wp-R BL and the low-level expression of p14ARF was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21WAF1/CIP1, a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21WAF1/CIP1 expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the proliferation of

  16. Stable Phenotypic Changes of the Host T Cells Are Essential to the Long-Term Stability of Latent HIV-1 Infection.

    PubMed

    Seu, Lillian; Sabbaj, Steffanie; Duverger, Alexandra; Wagner, Frederic; Anderson, Joshua C; Davies, Elizabeth; Wolschendorf, Frank; Willey, Christopher D; Saag, Michael S; Goepfert, Paul; Kutsch, Olaf

    2015-07-01

    The extreme stability of the latent HIV-1 reservoir in the CD4(+) memory T cell population prevents viral eradication with current antiretroviral therapy. It has been demonstrated that homeostatic T cell proliferation and clonal expansion of latently infected T cells due to viral integration into specific genes contribute to this extraordinary reservoir stability. Nevertheless, given the constant exposure of the memory T cell population to specific antigen or bystander activation, this reservoir stability seems remarkable, unless it is assumed that latent HIV-1 resides exclusively in memory T cells that recognize rare antigens. Another explanation for the stability of the reservoir could be that the latent HIV-1 reservoir is associated with an unresponsive T cell phenotype. We demonstrate here that host cells of latent HIV-1 infection events were functionally altered in ways that are consistent with the idea of an anergic, unresponsive T cell phenotype. Manipulations that induced or mimicked an anergic T cell state promoted latent HIV-1 infection. Kinome analysis data reflected this altered host cell phenotype at a system-wide level and revealed how the stable kinase activity changes networked to stabilize latent HIV-1 infection. Protein-protein interaction networks generated from kinome data could further be used to guide targeted genetic or pharmacological manipulations that alter the stability of latent HIV-1 infection. In summary, our data demonstrate that stable changes to the signal transduction and transcription factor network of latently HIV-1 infected host cells are essential to the ability of HIV-1 to establish and maintain latent HIV-1 infection status. The extreme stability of the latent HIV-1 reservoir allows the infection to persist for the lifetime of a patient, despite completely suppressive antiretroviral therapy. This extreme reservoir stability is somewhat surprising, since the latently HIV-1 infected CD4(+) memory T cells that form the

  17. Gastric carcinoma: monoclonal epithelial malignant cells expressing Epstein-Barr virus latent infection protein.

    PubMed Central

    Imai, S; Koizumi, S; Sugiura, M; Tokunaga, M; Uemura, Y; Yamamoto, N; Tanaka, S; Sato, E; Osato, T

    1994-01-01

    In 1000 primary gastric carcinomas, 70 (7.0%) contained Epstein-Barr virus (EBV) genomic sequences detected by PCR and Southern blots. The positive tumors comprised 8 of 9 (89%) undifferentiated lymphoepithelioma-like carcinomas, 27 of 476 (5.7%) poorly differentiated adenocarcinomas, and 35 of 515 (6.8%) moderately to well-differentiated adenocarcinomas. In situ EBV-encoded small RNA 1 hybridization and hematoxylin/eosin staining in adjacent sections showed that the EBV was present in every carcinoma cell but was not significantly present in lymphoid stroma and in normal mucosa. Two-color immunofluorescence and hematoxylin/eosin staining in parallel sections revealed that every keratin-positive epithelial malignant cell expressed EBV-determined nuclear antigen 1 (EBNA1) but did not significantly express CD45+ infiltrating leukocytes. A single fused terminal fragment was detected in each of the EBNA1-expressing tumors, thereby suggesting that the EBV-carrying gastric carcinomas represent clonal proliferation of cells infected with EBV. The carcinoma cells had exclusively EBNA1 but not EBNA2, -3A, -3B, and -3C; leader protein; and latent membrane protein 1 because of methylation. The patients with EBV-carrying gastric carcinoma had elevated serum EBV-specific antibodies. The EBV-specific cellular immunity was not significantly reduced; however, the cytotoxic T-cell target antigens were not expressed. These findings strongly suggest a causal relation between a significant proportion of gastric carcinoma and EBV, and the virus-carrying carcinoma cells may evade immune surveillance. Images PMID:8090780

  18. The life span of major histocompatibility complex-peptide complexes influences the efficiency of presentation and immunogenicity of two class I-restricted cytotoxic T lymphocyte epitopes in the Epstein-Barr virus nuclear antigen 4

    PubMed Central

    1996-01-01

    We have investigated the reactivity to two human histocompatibility leukocyte antigen (HLA) A11-restricted cytotoxic T lymphocyte (CTL) epitopes derived from amino acids 416-424 (IVTDFSVIK, designated IVT) and 399-408 (AVFDRKSVAK, designated AVF) of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) 4. A strong predominance of CTL clones specific for the IVT epitope was demonstrated in polyclonal cultures generated by stimulation of lymphocytes from the EBV-seropositive donor BK with the autologous B95.8 virus-transformed lymphoblastoid cell line (LCL). This was not due to intrinsic differences of CTL efficiency since clones specific for the two epitopes lysed equally well A11- positive phytohemagglutinin blasts and LCLs pulsed with the relevant synthetic peptide. Irrespective of the endogenous levels of EBNA4 expression, untreated LCLs were lysed more efficiently by the IVT- specific effectors, suggesting that a higher density of A11-IVT complexes is presented at the cell surface. In accordance, 10-50-fold higher amounts of IVT peptides were found in high-performance liquid chromatography fractions of acid extracts corresponding to an abundance of about 350-12,800 IVT and 8-760 AVF molecules per cell. Peptide- mediated competition of CTL sensitization, transport assays in streptolysin-O permeabilized cells, and induction of A11 expression in the transporter associated with antigen presentation-deficient T2/A11 transfectant demonstrated that the IVT and AVF peptides bind with similar affinities to A11, are translocated with equal efficiency to the endoplasmic reticulum, and form complexes of comparable stability over a wide range of temperature and pH conditions. A rapid surface turnover of A11 molecules containing the AVF peptide was demonstrated in metabolically active T2/A11 cells corresponding to a half-life of approximately 3.5 as compared to approximately 2 h for molecules induced at 26 degrees C in the absence of exogenous peptides and >12 h for IVT

  19. Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity

    SciTech Connect

    Hamada, Fumika N.; Koshiyama, Akiyo; Namekawa, Satoshi H.; Ishii, Satomi; Iwabata, Kazuki; Sugawara, Hiroko; Nara, Takayuki Y.; Sakaguchi, Kengo . E-mail: kengo@rs.noda.tus.ac.jp; Sawado, Tomoyuki

    2007-01-26

    PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg{sup 2+} ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.

  20. Evaluation of a new ELISA for the detection of specific IgG to the Epstein-Barr nuclear antigen 1 (EBNA-1).

    PubMed

    Schenk, Birgit I; Michel, Patrik O; Enders, Gisela; Thilo, Niklas; Radtke, Martina; Oker-Blom, Christian; Franke, Dieter

    2007-01-01

    Diagnosis of acute primary Epstein-Barr Virus (EBV) infection is predominantly performed by serology. Detection of specific antibodies to defined EBV antigens is considered state of the art. Antibodies to EBNA-1 are not produced early in primary infection and a positive EBNA-1 serology is a sign of past infection. Therefore EBNA-1 serology plays a crucial role for EBV routine diagnosis. In the present study the quantitative EBV EBNA-1-IgG-ELISA PKS medac was evaluated regarding its suitability for routine diagnosis. Using clinically and diagnostically defined serum samples (141 from seronegative, 111 from acute infected, and 52 from individuals with past infection) as well as 100 sera from healthy blood donors the diagnostic performance of the assay was investigated. Furthermore, precision, performance of the single-point quantitation (SPQ), and suitability of the assay for automation were evaluated. Compared to the pre-definition of the serum panel a sensitivity of 100% and a specificity of 99.6% was found. The measurement of the blood donor sera resulted in an anti-EBNA-1 IgG prevalence of 93% and an agreement of 99% with the results of a commercial ELISA used as reference. Regarding intra-assay variation, interassay variation (performed manually and automatically), and person-to-person variation a coefficient of variation < 10% was found with reactive samples. A good dilution linearity (r2 = 0.961), an excellent correlation of SPQ vs. the calibration curve (r2 = 0.997), and between the results of manually vs. automatically performed test runs (r2 > 0.995) was found. The evaluation has shown that the assay meets the demands of routine diagnosis very well.

  1. Evaluation of the Architect Epstein-Barr Virus (EBV) viral capsid antigen (VCA) IgG, VCA IgM, and EBV nuclear antigen 1 IgG chemiluminescent immunoassays for detection of EBV antibodies and categorization of EBV infection status using immunofluorescence assays as the reference method.

    PubMed

    Corrales, Isabel; Giménez, Estela; Navarro, David

    2014-05-01

    Commercial immunoassays for detecting IgG and IgM antibodies against Epstein-Barr virus (EBV), viral capsid antigens (VCA), and IgGs toward EBV nuclear antigen-1 (EBNA-1) are routinely used in combination to categorize EBV infection status. In this study, we evaluated the performances of the Architect EBV VCA IgG, VCA IgM, and EBNA-1 IgG chemiluminescent microparticle assays (CMIAs) in EBV serological analyses using indirect immunofluorescence assays and anticomplement immunofluorescence assays as the reference methods for VCA IgG, VCA IgM, and EBNA-1 IgG antibody detection, respectively. A total of 365 serum samples representing different EBV serological profiles were included in this study. The κ values (concordances between the results) obtained in the Architect CMIA and those in the reference assays were 0.905 (P < 0.0001) for VCA IgM, 0.889 (P < 0.0001) for VCA IgG, and 0.961 (P < 0.0001) for EBNA-1 IgG. The sensitivities and specificities were, respectively, 91.08% and 99.48% for VCA IgM, 99.23% and 86.27% for VCA IgG, and 96.77% and 99.16% for EBNA-1 IgG. The sensitivities and specificities of the Architect CMIA panel were, respectively, 99.15% and 98.6% for diagnosing a primary infection, 97.62% and 93.39% for diagnosing a past EBV infection, and 92.42% and 97.82% for diagnosing the absence of an EBV infection. In summary, we demonstrated that the Architect EBV antibody panel performs very well for EBV antibody detection and correctly categorizes clinically relevant EBV infection states.

  2. Epstein-Barr nuclear antigen 1 (EBNA1)-dependent recruitment of origin recognition complex (Orc) on oriP of Epstein-Barr virus with purified proteins: stimulation by Cdc6 through its direct interaction with EBNA1.

    PubMed

    Moriyama, Kenji; Yoshizawa-Sugata, Naoko; Obuse, Chikashi; Tsurimoto, Toshiki; Masai, Hisao

    2012-07-06

    Origin recognition complex (Orc) plays an essential role in directing assembly of prereplicative complex at selective sites on chromosomes. However, Orc from vertebrates is reported to bind to DNA in a sequence-nonspecific manner, and it is still unclear how it selects specific genomic loci and how Cdc6, another conserved AAA(+) factor known to interact with Orc, participates in this process. Replication from oriP, the latent origin of Epstein-Barr virus, provides an excellent model system for the study of initiation on the host chromosomes because it is known to depend on prereplicative complex factors, including Orc and Mcm. Here, we show that Orc is recruited selectively at the essential dyad symmetry element in nuclear extracts in a manner dependent on EBNA1, which specifically binds to dyad symmetry. With purified proteins, EBNA1 can recruit both Cdc6 and Orc independently on a DNA containing EBNA1 binding sites, and Cdc6 facilitates the Orc recruitment by EBNA1. Purified Cdc6 directly binds to EBNA1, whereas association of Orc with EBNA1 requires the presence of the oriP DNA. Nuclease protection assays suggest that Orc associates with DNA segments on both sides adjacent to the EBNA1 binding sites and that this process is stimulated by the presence of Cdc6. Thus, EBNA1 can direct localized assembly of Orc in a process that is facilitated by Cdc6. The possibility of similar modes of recruitment of Orc/Cdc6 at the human chromosomal origins will be discussed.

  3. Estimation in Latent Trait Models.

    ERIC Educational Resources Information Center

    Rigdon, Steven E.; Tsutakawa, Robert K.

    Estimation of ability and item parameters in latent trait models is discussed. When both ability and item parameters are considered fixed but unknown, the method of maximum likelihood for the logistic or probit models is well known. Discussed are techniques for estimating ability and item parameters when the ability parameters or item parameters…

  4. Indexing by Latent Semantic Analysis.

    ERIC Educational Resources Information Center

    Deerwester, Scott; And Others

    1990-01-01

    Describes a new method for automatic indexing and retrieval called latent semantic indexing (LSI). Problems with matching query words with document words in term-based information retrieval systems are discussed, semantic structure is examined, singular value decomposition (SVD) is explained, and the mathematics underlying the SVD model is…

  5. Indexing by Latent Semantic Analysis.

    ERIC Educational Resources Information Center

    Deerwester, Scott; And Others

    1990-01-01

    Describes a new method for automatic indexing and retrieval called latent semantic indexing (LSI). Problems with matching query words with document words in term-based information retrieval systems are discussed, semantic structure is examined, singular value decomposition (SVD) is explained, and the mathematics underlying the SVD model is…

  6. Euphorbia Kansui Reactivates Latent HIV

    PubMed Central

    Cary, Daniele C.; Fujinaga, Koh; Peterlin, B. Matija

    2016-01-01

    While highly active anti-retroviral therapy has greatly improved the lives of HIV infected individuals, these treatments are unable to eradicate the virus. Current approaches to reactivate the virus have been limited by toxicity, lack of an orally available therapy, and limited responses in primary CD4+ T cells and in clinical trials. The PKC agonist ingenol, purified from Euphorbia plants, is a potent T cell activator and reactivates latent HIV. Euphorbia kansui itself has been used for centuries in traditional Chinese medicine to treat ascites, fluid retention, and cancer. We demonstrate that an extract of this plant, Euphorbia kansui, is capable of recapitulating T cell activation induced by the purified ingenol. Indeed, Euphorbia kansui induced expression of the early T cell activation marker CD69 and P-TEFb in a dose-dependent manner. Furthermore, Euphorbia kansui reactivated latent HIV in a CD4+ T cell model of latency and in HIV+ HAART suppressed PBMC. When combined with the other latency reversing agents, the effective dose of Euphorbia kansui required to reactive HIV was reduced 10-fold and resulted in synergistic reactivation of latent HIV. We conclude that Euphorbia Euphorbia kansui reactivates latent HIV and activates CD4+ T cells. When used in combination with a latency reversing agent, the effective dose of Euphorbia kansui is reduced; which suggests its application as a combination strategy to reactivate latent HIV while limiting the toxicity due to global T cell activation. As a natural product, which has been used in traditional medicine for thousands of years, Euphorbia kansui is attractive as a potential treatment strategy, particularly in resource poor countries with limited treatment options. Further clinical testing will be required to determine its safety with current anti-retroviral therapies. PMID:27977742

  7. The non-canonical protein binding site at the monomer-monomer interface of yeast proliferating cell nuclear antigen (PCNA) regulates the Rev1-PCNA interaction and Polζ/Rev1-dependent translesion DNA synthesis.

    PubMed

    Sharma, Neeru M; Kochenova, Olga V; Shcherbakova, Polina V

    2011-09-23

    Rev1 and DNA polymerase ζ (Polζ) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Polζ lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Polζ with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pull-down and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Polζ-dependent TLS pathway. Taken together, the data suggest that Polζ and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.

  8. Host-pathogen interactions in latent Mycobacterium tuberculosis infection: identification of new targets for tuberculosis intervention.

    PubMed

    Lin, May Young; Ottenhoff, Tom H M

    2008-03-01

    Mycobacterium tuberculosis (M. tuberculosis) is one of the worlds' most successful and sophisticated pathogens. It is estimated that over 2 billion people today harbour latent M. tuberculosis infection without any clinical symptoms. Since most new cases of active tuberculosis (TB) arise from this (growing) number of latently infected individuals, urgent measures to control TB reactivation are required, including more effective drugs and new TB vaccines. The currently widely used BCG vaccines, as well as most new generation TB-vaccines that are being developed are designed as prophylactic or as BCG-booster vaccines. Unfortunately, many of these vaccines are unlikely to be effective in individuals already latently infected with M. tuberculosis. Here we argue that detailed analysis of M. tuberculosis genes that are switched on predominantly during the latent stage of infection may lead to the identification of new M. tuberculosis targets for drug and vaccine development. First, we will describe essential host-pathogen interactions in TB with particular emphasis on TB latency and persistent infection. Subsequently, we will focus on a novel group of late-stage specific genes, encoded by the M. tuberculosis dormancy (dosR) regulon, and summarize recent studies describing human T-cell recognition of these dormancy antigens in relation to (latent) M. tuberculosis infection. We will discuss the possible relevance of these new classes of antigens for new TB intervention strategies.

  9. Comparative study of different latent infections of herpes simplex virus type I in a murine model.

    PubMed

    Huang, Wen; Zhao, Ping; Chen, Xiao; Li, Ping; Zhao, Gaonian; Xu, Mingming; Chen, Xiuying; Xie, Peng

    2014-01-01

    This study aims to compare the different latent infections of herpes simplex virus type I in a murine model. One hundred and twenty BALB/c mice were randomly assigned into either of three groups: intravenous inoculation group, ocular abrasion group, and intranasal inoculation group. Six weeks later, the trigeminal ganglia (TG) were removed to detect the expression of HSV-I antigen. HSV DNA in TG was also detected by polymerase chain reaction to confirm latent infection. The rate of HSV DNA in TG detected in the intravenous inoculation group was 18/22 and 22/26 in the ocular abrasion group, both of which were higher than the rate detected in the intranasal inoculation group (18/30). The expression of HSV antigen in TG in these three groups was all negative. Mortality rate in the intravenous inoculation group was 8/30, which was much higher than those of the two other groups. Intranasal virus dripping, cornea abrasion, and intravenous injection can detect latent HSV-I infection in a murine model. Compared to two other groups, the cornea abrasion group showed less severe signs, a quicker recovery rate in acute infection, and higher incidence rate of latent infection. Therefore, it is an ideal method in the presence of latent HSV-I infection.

  10. Mycobacteria-specific cytokine responses as correlates of treatment response in active and latent tuberculosis.

    PubMed

    Clifford, Vanessa; Tebruegge, Marc; Zufferey, Christel; Germano, Susie; Forbes, Ben; Cosentino, Lucy; McBryde, Emma; Eisen, Damon; Robins-Browne, Roy; Street, Alan; Denholm, Justin; Curtis, Nigel

    2017-08-01

    A biomarker indicating successful tuberculosis (TB) therapy would assist in determining appropriate length of treatment. This study aimed to determine changes in mycobacteria-specific antigen-induced cytokine biomarkers in patients receiving therapy for latent or active TB, to identify biomarkers potentially correlating with treatment success. A total of 33 adults with active TB and 36 with latent TB were followed longitudinally over therapy. Whole blood stimulation assays using mycobacteria-specific antigens (CFP-10, ESAT-6, PPD) were done on samples obtained at 0, 1, 3, 6 and 9 months. Cytokine responses (IFN-γ, IL-1ra, IL-2, IL-10, IL-13, IP-10, MIP-1β, and TNF-α) in supernatants were measured by Luminex xMAP immunoassay. In active TB cases, median IL-1ra (with CFP-10 and with PPD stimulation), IP-10 (CFP-10, ESAT-6), MIP-1β (ESAT-6, PPD), and TNF-α (ESAT-6) responses declined significantly over the course of therapy. In latent TB cases, median IL-1ra (CFP-10, ESAT-6, PPD), IL-2 (CFP-10, ESAT-6), and IP-10 (CFP-10, ESAT-6) responses declined significantly. Mycobacteria-specific cytokine responses change significantly over the course of therapy, and their kinetics in active TB differ from those observed in latent TB. In particular, mycobacteria-specific IL-1ra responses are potential correlates of successful therapy in both active and latent TB. Copyright © 2017 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  11. Growth fraction in non-small cell lung cancer estimated by proliferating cell nuclear antigen and comparison with Ki-67 labeling and DNA flow cytometry data.

    PubMed Central

    Fontanini, G.; Pingitore, R.; Bigini, D.; Vignati, S.; Pepe, S.; Ruggiero, A.; Macchiarini, P.

    1992-01-01

    Results generated by the immunohistochemical staining with PC10, a new monoclonal antibody recognizing PCNA (a nuclear protein associated with cell proliferation) in formalin-fixed and paraffin-embedded tissue were compared with those of Ki-67 labeling and DNA flow cytometry in 47 consecutive non-small cell lung cancer (NSCLC). PCNA reactivity was observed in all samples and confined to the nuclei of cancer cells. Its frequency ranged from 0 to 80% (37.7 +/- 23.6) and larger sized, early-staged and DNA aneuploid tumors expressed a significant higher number of PCNA-reactive cells. The PCNA and Ki-67 labeling rates were closely correlated (r = 0.383, P = 0.009). By flow cytometry, we observed a good correlation among PCNA labeling and S-phase fraction (r = 0.422, P = .0093) and G1 phase (r = 0.303, P = .051) of the cell cycle. Results indicate that PCNA labeling with PC10 is a simple method for assessing the proliferative activity in formalin-fixed, paraffin-embedded tissue of NSCLC and correlates well with Ki-67 labeling and S-phase fraction of the cell cycle. Images Figure 2 PMID:1361306

  12. Functional interaction between the Fanconi Anemia D2 protein and proliferating cell nuclear antigen (PCNA) via a conserved putative PCNA interaction motif.

    PubMed

    Howlett, Niall G; Harney, Julie A; Rego, Meghan A; Kolling, Frederick W; Glover, Thomas W

    2009-10-16

    Fanconi Anemia (FA) is a rare recessive disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The FA proteins and the familial breast cancer susceptibility gene products, BRCA1 and FANCD1/BRCA2, function cooperatively in the FA-BRCA pathway to repair damaged DNA and to prevent cellular transformation. Activation of this pathway occurs via the mono-ubiquitination of the FANCD2 protein, targeting it to nuclear foci where it co-localizes with FANCD1/BRCA2, RAD51, and PCNA. The regulation of the mono-ubiquitination of FANCD2, as well as its function in DNA repair remain poorly understood. In this study, we have further characterized the interaction between the FANCD2 and PCNA proteins. We have identified a highly conserved, putative FANCD2 PCNA interaction motif (PIP-box), and demonstrate that mutation of this motif disrupts FANCD2-PCNA binding and precludes the mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant protein fails to correct the mitomycin C hypersensitivity of FA-D2 patient cells. Our results suggest that PCNA may function as a molecular platform to facilitate the mono-ubiquitination of FANCD2 and activation of the FA-BRCA pathway.

  13. Latent Growth Modeling for Logistic Response Functions

    ERIC Educational Resources Information Center

    Choi, Jaehwa; Harring, Jeffrey R.; Hancock, Gregory R.

    2009-01-01

    Throughout much of the social and behavioral sciences, latent growth modeling (latent curve analysis) has become an important tool for understanding individuals' longitudinal change. Although nonlinear variations of latent growth models appear in the methodological and applied literature, a notable exclusion is the treatment of growth following…

  14. A Multicomponent Latent Trait Model for Diagnosis

    ERIC Educational Resources Information Center

    Embretson, Susan E.; Yang, Xiangdong

    2013-01-01

    This paper presents a noncompensatory latent trait model, the multicomponent latent trait model for diagnosis (MLTM-D), for cognitive diagnosis. In MLTM-D, a hierarchical relationship between components and attributes is specified to be applicable to permit diagnosis at two levels. MLTM-D is a generalization of the multicomponent latent trait…

  15. A Latent Class Model for Rating Data.

    ERIC Educational Resources Information Center

    Rost, Jurgen

    1985-01-01

    A latent class model for rating data is presented which provides an alternative to the latent trait approach of analyzing test data. It is the analog of Andrich's binomial Rasch model for Lazarsfeld's latent class analysis (LCA). Response probabilities for rating categories follow a binomial distribution and depend on class-specific item…

  16. Latent Growth Modeling for Logistic Response Functions

    ERIC Educational Resources Information Center

    Choi, Jaehwa; Harring, Jeffrey R.; Hancock, Gregory R.

    2009-01-01

    Throughout much of the social and behavioral sciences, latent growth modeling (latent curve analysis) has become an important tool for understanding individuals' longitudinal change. Although nonlinear variations of latent growth models appear in the methodological and applied literature, a notable exclusion is the treatment of growth following…

  17. The antigens - Volume VII

    SciTech Connect

    Sela, M. )

    1987-01-01

    This book contains four chapters. They are: Ir Genes: Antigen-Specific Genetic Regulation of the Immune Response; Molecular Genetics of Class II (Ia) Antigens; Antigen-Specific T Cell Clones and T Cell Factors; and Infection and Autoimmunity.

  18. Expression of the Epstein-Barr Virus-Encoded Epstein-Barr Virus Nuclear Antigen 1 in Hodgkin’s Lymphoma Cells Mediates Up-Regulation of CCL20 and the Migration of Regulatory T Cells

    PubMed Central

    Baumforth, Karl R.N.; Birgersdotter, Anna; Reynolds, Gary M.; Wei, Wenbin; Kapatai, Georgia; Flavell, Joanne R.; Kalk, Emma; Piper, Karen; Lee, Steve; Machado, Lee; Hadley, Kerry; Sundblad, Anne; Sjoberg, Jan; Bjorkholm, Magnus; Porwit, Anna A.; Yap, Lee-Fah; Teo, Soohwang; Grundy, Richard G.; Young, Lawrence S.; Ernberg, Ingemar; Woodman, Ciaran B.J.; Murray, Paul G.

    2008-01-01

    In ∼50% of patients with Hodgkin’s lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4+ lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4+ T cells that inhibit effector CD4+ and CD8+ T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity. PMID:18502823

  19. Notch1, Notch2, and Epstein-Barr virus-encoded nuclear antigen 2 signaling differentially affects proliferation and survival of Epstein-Barr virus-infected B cells.

    PubMed

    Kohlhof, Hella; Hampel, Franziska; Hoffmann, Reinhard; Burtscher, Helmut; Weidle, Ulrich H; Hölzel, Michael; Eick, Dirk; Zimber-Strobl, Ursula; Strobl, Lothar J

    2009-05-28

    The canonical mode of transcriptional activation by both the Epstein-Barr viral protein, Epstein-Barr virus-encoded nuclear antigen 2 (EBNA2), and an activated Notch receptor (Notch-IC) requires their recruitment to RBPJ, suggesting that EBNA2 uses the Notch pathway to achieve B-cell immortalization. To gain further insight into the biologic equivalence between Notch-IC and EBNA2, we performed a genome-wide expression analysis, revealing that Notch-IC and EBNA2 exhibit profound differences in the regulation of target genes. Whereas Notch-IC is more potent in regulating genes associated with differentiation and development, EBNA2 is more potent in inducing viral and cellular genes involved in proliferation, survival, and chemotaxis. Because both EBNA2 and Notch-IC induced the expression of cell cycle-associated genes, we analyzed whether Notch1-IC or Notch2-IC can replace EBNA2 in B-cell immortalization. Although Notch-IC could drive quiescent B cells into the cell cycle, B-cell immortalization was not maintained, partially due to an increased apoptosis rate in Notch-IC-expressing cells. Expression analysis revealed that both EBNA2 and Notch-IC induced the expression of proapoptotic genes, but only in EBNA2-expressing cells were antiapoptotic genes strongly up-regulated. These findings suggest that Notch signaling in B cells and B-cell lymphomas is only compatible with proliferation if pathways leading to antiapototic signals are active.

  20. Comparison of automated multiplexed bead-based ANA screening assay with ELISA for detecting five common anti-extractable nuclear antigens and anti-dsDNA in systemic rheumatic diseases.

    PubMed

    Kim, Yoonjung; Park, Yongjung; Lee, Eun Young; Kim, Hyon-Suk

    2012-01-18

    A newly developed and totally automated Luminex-based assay, the BioPlex™ 2200 system, is able to detect various autoantibodies simultaneously from a single sample. We compared the BioPlex™ 2200 system with ELISA for the detection of six autoantibodies. A total of 127 serum samples from the patients with systemic rheumatic diseases were collected and assayed with the BioPlex™ 2200 system (Bio-Rad, USA) and conventional ELISA (INOVA Diagnostics, USA) for 5 anti-extractable nuclear antigens. Additionally, relative sensitivity of the BioPlex™ 2200 system for detecting anti-dsDNA was evaluated with 79 specimens from SLE patients, which were positive for anti-dsDNA by ELISA. The concordance rates between ELISA and the BioPlex ranged from 88.1% for anti-RNP to 95.2% for anti-Scl-70, and the kappa coefficients between the results by the two assays were from 0.48 to 0.67. Among the 79 anti-dsDNA positive specimens by ELISA, seventy-eight (98.7%) showed positive results for anti-dsDNA by the BioPlex. The BioPlex™ 2200 system showed comparable results with those by conventional ELISA for detecting autoantibodies, and this automated assay could measure multifarious autoantibodies concurrently in a single sample. It could be effectively used in clinical laboratories for screening autoimmune diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Icariin inhibits oxidized low-density lipoprotein-induced proliferation of vascular smooth muscle cells by suppressing activation of extracellular signal-regulated kinase 1/2 and expression of proliferating cell nuclear antigen.

    PubMed

    Hu, Yanwu; Liu, Kai; Yan, Mengtong; Zhang, Yang; Wang, Yadi; Ren, Liqun

    2016-03-01

    Icariin, a flavonoid isolated from the traditional Chinese herbal medicine Epimedium brevicornum Maxim, has been shown to possess anti-inflammatory, anti‑oxidant and anti-atherosclerotic activities in vivo and in vitro. The aim of the present study was to investigate the effects of icariin on oxidized low‑density lipoprotein (ox-LDL)-induced proliferation of vascular smooth muscle cells (VSMCs) and the possible underlying mechanism. VSMCs were cultured and pre‑treated with various concentrations of icariin (0, 10, 20 or 40 µm) prior to stimulation by ox‑LDL (50 µg/ml). Cell proliferation was evaluated by an MTT assay. Flow cytometry was used to study the influence of icariin on the cell cycle. Proliferating cell nuclear antigen (PCNA) expression and phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2 were detected by western blot analysis. The results indicated that icariin significantly inhibited ox‑LDL‑induced proliferation of VSMCs and phosphorylation of ERK1/2. Furthermore, icariin also blocked the ox‑LDL‑induced cell‑cycle progression at G1/S‑interphase and downregulated the expression of PCNA in VSMCs. In conclusion, the present study indicated for the first time that icariin reduced the amount of ox‑LDL‑induced proliferation of VSMCs through suppression of PCNA expression and inactivation of ERK1/2.

  2. Modulatory Effect of Taurine on 7,12-Dimethylbenz(a)Anthracene-Induced Alterations in Detoxification Enzyme System, Membrane Bound Enzymes, Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue.

    PubMed

    Vanitha, Manickam Kalappan; Baskaran, Kuppusamy; Periyasamy, Kuppusamy; Selvaraj, Sundaramoorthy; Ilakkia, Aruldoss; Saravanan, Dhiravidamani; Venkateswari, Ramachandran; Revathi Mani, Balasundaram; Anandakumar, Pandi; Sakthisekaran, Dhanapal

    2016-08-01

    The modulatory effect of taurine on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na(+) /K(+) ATPase, Ca(2+) ATPase, and Mg(2+) ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione-S-transferase and UDP-glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA-induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA-induced breast cancer.

  3. Amino acid substitution analyses of the DNA contact region, two amphipathic alpha-helices and a recognition-helix-like helix outside the dimeric beta-barrel of Epstein-Barr virus nuclear antigen 1.

    PubMed

    Fujita, T; Ikeda, M; Kusano, S; Yamazaki, M; Ito, S; Obayashi, M; Yanagi, K

    2001-01-01

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1), which is essential for EBV latency, homodimerizes and binds to the EBV replication origin, oriP. We analyzed the dimerization/DNA-binding domain of EBNA-1 by random and site-directed amino acid substitution. Random point mutations that resulted in reduced DNA binding clustered in the DNA contact region (a.a. 461-473) and at or near the termini of alpha-helix II (514-527). Three substitutions of Gly in the DNA contact region each greatly reduced binding to a single binding site oligonucleotide. Substitutions at and near the termini of alpha-helix II diminished DNA binding. A helix-deforming substitution in alpha-helix I (477-489) blocked DNA binding. A helix-deforming substitution in alpha-helix III (568-582) abolished dimerization and DNA binding. Similarities in surface electrostatic properties and conserved amino acids were found between alpha-helix II and recognition helices of papillomavirus E2 proteins. The basic DNA contact region is crucial for the specific interaction of EBNA-1 with a single binding site. Alpha-helix I477 is indispensable for oriP binding, and alpha-helix III568 contributes to the homodimeric structure of EBNA-1. Alpha-helix II514 contributes to oriP binding, perhaps changing its alignment with DNA. Copyright 2001 S. Karger AG, Basel

  4. p53-independent increase in p21WAF1 and reciprocal down-regulation of cyclin A and proliferating cell nuclear antigen in bromodeoxyuridine-mediated growth arrest of human melanoma cells.

    PubMed

    Strasberg Rieber, M; Welch, D R; Miele, M E; Rieber, M

    1996-02-01

    Differentially regulated expression of activators and inhibitors of cyclin-dependent kinases (cdks) modulate cell cycle progression. In normal fibroblasts, these complexes consist of the cdk inhibitor p21WAF1/PCNA/G1 cyclin/cdk. We now show that bromodeoxyuridine (BrdUrd), a thymidine analogue and radiation sensitizer, inhibits growth and activity of cyclin A-cdk2 kinase in metastatic C8161 and nonmetastatic neo 6.3/C8161 human melanoma cells. Inhibition is not due to altered levels of cyclin D or catalytic cdk2 but involves a decrease in cyclin A and proliferating cell nuclear antigen, paralleled by higher levels of p21WAF1 without increases in p53. In contrast to serum starvation, which prevents accumulation of cyclins A and D in normal fibroblasts, such treatment did not down-regulate either cyclin in these melanoma cells, implying an aberrant control for G1 cyclins in these tumor cells. However, cyclin A was decreased by BrdUrd, suggesting that this pyrimidine analogue arrests melanoma cells at a G1 transition point, unlike that of serum starvation. This is the first report indicating that the antitumor therapeutic action of BrdUrd may be mediated by a p53-independent reciprocal effect on activators and inhibitors of cdk kinases.

  5. Identifying sites bound by Epstein-Barr virus nuclear antigen 1 (EBNA1) in the human genome: defining a position-weighted matrix to predict sites bound by EBNA1 in viral genomes.

    PubMed

    Dresang, Lindsay R; Vereide, David T; Sugden, Bill

    2009-04-01

    We identified binding sites for Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) in the human genome using chromatin immunoprecipitation and microarrays. The sequences for these newly identified sites were used to generate a position-weighted matrix (PWM) for EBNA1's DNA-binding sites. This PWM helped identify additional DNA-binding sites for EBNA1 in the genomes of EBV, Kaposi's sarcoma-associated herpesvirus, and cercopithecine herpesvirus 15 (CeHV-15) (also called herpesvirus papio 15). In particular, a homologue of the Rep* locus in EBV was predicted in the genome of CeHV-15, which is notable because Rep* of EBV was not predicted by the previously developed consensus sequence for EBNA1's binding DNA. The Rep* of CeHV-15 functions as an origin of DNA synthesis in the EBV-positive cell line Raji; this finding thus builds on a set of DNA-binding sites for EBNA1 predicted in silico.

  6. Impaired Cytokine but Enhanced Cytotoxic Marker Expression in Mycobacterium tuberculosis-Induced CD8+ T Cells in Individuals With Type 2 Diabetes and Latent Mycobacterium tuberculosis Infection.

    PubMed

    Kumar, Nathella Pavan; Moideen, Kadar; George, Parakkal Jovvian; Dolla, Chandrakumar; Kumaran, Paul; Babu, Subash

    2016-03-01

    Type 2 diabetes mellitus (DM) is a risk factor for tuberculosis among individuals with latent Mycobacterium tuberculosis infection. To explore the influence of DM on CD8(+) T-cell responses during latent M. tuberculosis infection, we estimated the cytokine and cytotoxic marker expression pattern in individuals with latent M. tuberculosis infection with DM and those with latent M. tuberculosis infection without DM. Among individuals with latent M. tuberculosis infection, those with DM had diminished frequencies of CD8(+) T-helper type 1 (Th1), Th2, and Th17 cells following stimulation by M. tuberculosis antigen and enhanced frequencies of CD8(+) T cells expressing cytotoxic markers, compared with those without DM. Thus, our results suggest that coincident DM modulates CD8(+) T-cell function during latent M. tuberculosis infection.

  7. Latent period in clinical radiation myelopathy

    SciTech Connect

    Schultheiss, T.E.; Higgins, E.M.; El-Mahdi, A.M.

    1984-07-01

    Seventy-seven papers containing data on more than 300 cases of radiation myelopathy have been analyzed. The data suggest that the latent periods are similar in the cervical and thoracic levels of the spinal cord and are bimodally distributed. Myelopathy of lumbar cord apparently has a shorter latent period. As in controlled animal experiments, the latent period decreases with increasing dose. Furthermore, the variation in latent periods also decreases with dose. It is also seen that retreated patients and pediatric or adolescent patients have greatly reduced latent periods. The implications of these findings as they compare with the animal data are discussed.

  8. Recognition of distinct HLA-DQA1 promoter elements by a single nuclear factor containing Jun and Fos or antigenically related proteins.

    PubMed Central

    Neve Ombra, M; Autiero, M; DeLerma Barbaro, A; Barretta, R; Del Pozzo, G; Guardiola, J

    1993-01-01

    The activity of MHC class II promoters depends upon conserved regulatory signals one of which, the extended X-box, contains in its X2 subregion a sequence related to the cAMP response element, CRE and to the TPA response element, TRE. Accordingly, X2 is recognized by the AP-1 factor and by other c-Jun or c-Fos containing heterodimers. We report that the X-box dependent promoter activity of the HLA-DQA1 gene is down-modulated by an array of DNA elements each of which represented twice either in an invertedly or directly repeated orientation. In this frame, we describe a nuclear binding factor, namely DBF, promiscuously interacting with two of these additional signals, delta and sigma, and with a portion of the X-box, namely the X-core, devoid of X2. The presence of a single factor recognizing divergent DNA sequences was indicated by the finding that these activities were co-eluted from a heparin-Sepharose column and from DNA affinity columns carrying different DNA binding sites as ligands. Competition experiments made with oligonucleotides representing wild type and mutant DNA elements showed that each DNA element specifically inhibited the binding of the others, supporting the contention that DBF is involved in recognition of different targets. Furthermore, we found that DBF also exhibits CRE/TRE binding activity and that this activity can be competed out by addition of an excess of sigma, delta and X-core oligonucleotides. Anti-Jun peptide and anti-Fos peptide antibodies blocked not only the binding activity of DBF, but also its X-core and sigma binding; this blockade was removed by the addition of the Jun or Fos peptides against which the antibodies had been raised. In vitro synthesized Jun/Fos was able to bind to all these boxes, albeit with seemingly different affinities. The cooperativity of DBF interactions may explain the modulation of the X-box dependent promoter activity mediated by the accessory DNA elements described here. Images PMID:8493100

  9. Incidence of persistent viraemia and latent feline leukaemia virus infection in cats with lymphoma.

    PubMed

    Stützer, Bianca; Simon, Karin; Lutz, Hans; Majzoub, Monir; Hermanns, Walter; Hirschberger, Johannes; Sauter-Louis, Carola; Hartmann, Katrin

    2011-02-01

    In the past, feline leukaemia virus (FeLV) infection, and also latent FeLV infection, were commonly associated with lymphoma and leukaemia. In this study, the prevalence of FeLV provirus in tumour tissue and bone marrow in FeLV antigen-negative cats with these tumours was assessed. Seventy-seven diseased cats were surveyed (61 antigen-negative, 16 antigen-positive). Blood, bone marrow, and tumour samples were investigated by two polymerase chain reaction (PCR) assays detecting deoxyribonucleic acid (DNA) sequences of the long terminal repeats (LTR) and the envelope (env) region of the FeLV genome. Immunohistochemistry (IHC) was performed in bone marrow and tumour tissue. None of the antigen-negative cats with lymphoma was detectably infected with latent FeLV. The prevalence of FeLV viraemia in cats with lymphoma was 20.8%. This suggests that causes other than FeLV play a role in tumorigenesis, and that latent FeLV infection is unlikely to be responsible for most feline lymphomas and leukaemias. Copyright © 2010. Published by Elsevier Ltd.

  10. Tuberculosis Infection and Latent Tuberculosis

    PubMed Central

    2016-01-01

    Active tuberculosis (TB) has a greater burden of TB bacilli than latent TB and acts as an infection source for contacts. Latent tuberculosis infection (LTBI) is the state in which humans are infected with Mycobacterium tuberculosis without any clinical symptoms, radiological abnormality, or microbiological evidence. TB is transmissible by respiratory droplet nucleus of 1–5 µm in diameter, containing 1–10 TB bacilli. TB transmission is affected by the strength of the infectious source, infectiousness of TB bacilli, immunoresistance of the host, environmental stresses, and biosocial factors. Infection controls to reduce TB transmission consist of managerial activities, administrative control, engineering control, environmental control, and personal protective equipment provision. However, diagnosis and treatment for LTBI as a national TB control program is an important strategy on the precondition that active TB is not missed. Therefore, more concrete evidences for LTBI management based on clinical and public perspectives are needed. PMID:27790271

  11. Latent heat of vehicular motion

    NASA Astrophysics Data System (ADS)

    Ahmadi, Farzad; Berrier, Austin; Habibi, Mohammad; Boreyko, Jonathan

    2016-11-01

    We have used the thermodynamic concept of latent heat, where a system loses energy due to a solid-to-liquid phase transition, to study the flow of a group of vehicles moving from rest. During traffic flow, drivers keep a large distance from the car in front of them to ensure safe driving. When a group of cars comes to a stop, for example at a red light, drivers voluntarily induce a "phase transition" from this "liquid phase" to a close-packed "solid phase." This phase transition is motivated by the intuition that maximizing displacement before stopping will minimize the overall travel time. To test the effects of latent heat on flow efficiency, a drone captured the dynamics of cars flowing through an intersection on a Smart Road where the initial spacing between cars at the red light was systematically varied. By correlating the experimental results with the Optimal Velocity Model (OVM), we find that the convention of inducing phase transitions at intersections offers no benefit, as the lag time (latent heat) of resumed flow offsets the initial increase in displacement. These findings suggest that in situations where gridlock is not an issue, drivers should not decrease their spacing during stoppages in order to maximize safety with no loss in flow efficiency.

  12. Streamlined, automated protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) using ÄKTAxpress™

    PubMed Central

    Ludwig, Cornelia; Wear, Martin A.; Walkinshaw, Malcolm D.

    2010-01-01

    We developed streamlined, automated purification protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) from Escherichia coli, using the ÄKTAxpress™ chromatography system. The automated 2-step (cation exchange and size exclusion) purification protocol for untagged hCypA results in final purity and yields of ⩾93% and ∼5 mg L−1 of original cell culture, respectively, in under 12 h, including all primary sample processing and column equilibration steps. The novel automated 4-step (anion exchange, desalt, heparin-affinity and size exclusion, in linear sequence) purification protocol for untagged hPCNA results in final purity and yields of ⩾87% and ∼4 mg L−1 of original cell culture, respectively, in under 24 h, including all primary sample processing and column equilibration steps. This saves in excess of four full working days when compared to the traditional protocol, producing protein with similar final yield, purity and activity. Furthermore, it limits a time-dependent protein aggregation, a problem with the traditional protocol that results in a loss of final yield. Both automated protocols were developed to use generic commercially available pre-packed columns and automatically prepared minimal buffers, designed to eliminate user and system variations, maximize run reproducibility, standardize yield and purity between batches, increase throughput and reduce user input to a minimum. Both protocols represent robust generic methods for the automated production of untagged hCypA and hPCNA. PMID:19995609

  13. Mechanistic Control of Carcinoembryonic Antigen-related Cell Adhesion Molecule-1 (CEACAM1) Splice Isoforms by the Heterogeneous Nuclear Ribonuclear Proteins hnRNP L, hnRNP A1, and hnRNP M*

    PubMed Central

    Dery, Kenneth J.; Gaur, Shikha; Gencheva, Marieta; Yen, Yun; Shively, John E.; Gaur, Rajesh K.

    2011-01-01

    Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3′ to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1. PMID:21398516

  14. Effects of resveratrol on ARPE-19 cell proliferation and migration via regulating the expression of proliferating cell nuclear antigen, P21, P27 and p38MAPK/MMP-9

    PubMed Central

    Hao, Xiao-Ning; Wang, Wen-Jie; Chen, Jian; Zhou, Qing; Qu, Yi-Xin; Liu, Xiao-Yong; Xu, Wei

    2016-01-01

    AIM To explore whether resveratrol (Res) can inhibit human retinal pigment epithelial cell (ARPE-19 cell) proliferation and migration, and to research the molecular mechanisms. METHODS ARPE-19 cells were pretreated with various concentrations at 0, 50, 100, 150, 200 and 300 µmol/L of Res, and with 0 µmol/L Res as the control for 24, 48 and 72h. The cell proliferation, apoptosis and migration were measured with cell counting kit-8 (CCK-8), flow cytometry, and wound-healing and Transwell assays, respectively. The expression of proliferating cell nuclear antigen (PCNA), P21 and P27, as well as matrix metalloproteinase-9 (MMP-9) and p38 mitogen-activated protein kinases (p38MAPK) was identified by Western blot. RESULTS Cell proliferation was effectively inhibited by Res (P<0.05). When pretreated with Res, cells arrested in S-phase increased remarkably (P<0.05), but the apoptosis ratios showed no significant difference between the treatment and control groups (P>0.05). Cell migration was suppressed by Res both in wound-healing assay and Transwell migration assay (P<0.05). Decreases of PCNA, MMP-9 and p38MAPK, as well as increases of P21 and P27 were detected by Western blot (P<0.05). CONCLUSION Res can inhibit APRE-19 cell proliferation and migration in a concentration-dependent manner with up-regulation of the expression of P21 and P27, and down-regulation of PCNA, MMP-9 and p38MAPK. PMID:28003970

  15. Fusion of Epstein-Barr virus nuclear antigen-1-derived glycine-alanine repeat to trans-dominant HIV-1 Gag increases inhibitory activities and survival of transduced cells in vivo.

    PubMed

    Hammer, Diana; Wild, Jens; Ludwig, Christine; Asbach, Benedikt; Notka, Frank; Wagner, Ralf

    2008-06-01

    Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.

  16. The C-terminal region of p21SDI1/WAF1/CIP1 is involved in proliferating cell nuclear antigen binding but does not appear to be required for growth inhibition.

    PubMed

    Nakanishi, M; Robetorye, R S; Pereira-Smith, O M; Smith, J R

    1995-07-21

    The cyclin-dependent kinase (Cdk) inhibitor p21SDI1/WAF1/CIP1 has been found to be involved in cell senescence, cell cycle arrest, and differentiation. p21SDI1 inhibits the activity of several Cdks, in contrast to other inhibitors such as p15INK4B and p16INK4A, which act on specific cyclin-Cdk complexes. Of interest were reports that p21SDI1 also bound proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA polymerase delta, and inhibited DNA replication but not DNA repair in vitro. To better understand the function of this interaction in vivo, we first determined the region of p21SDI1 that was needed for PCNA binding. Analysis of deletion mutants of p21SDI1, which covered the majority of the protein, revealed that deletion of either amino acids 142-147 or 149-154 resulted in loss of ability to bind a glutathione S-transferase-PCNA fusion protein. Site-directed mutagenesis in this region led to the identification of the PCNA binding motif RQXXMTXFYXXXR and demonstrated that mutation of either amino acid Met-147 or Phe-150 resulted in almost complete ablation of PCNA binding. Interestingly, when we determined DNA synthesis inhibitory activity of deletion mutants or point mutants that were unable to bind Cdk2 and/or PCNA, we found that loss of binding to PCNA did not affect inhibitory activity, whereas lack of Cdk2 binding greatly reduced the same. This result suggests that the primary mechanism for inhibition of DNA synthesis by p21SDI1 occurs via inhibition of Cdk activity.

  17. Boron neutron capture synovectomy (BNCS) as a potential therapy for rheumatoid arthritis: radiobiological studies at RA-1 Nuclear Reactor in a model of antigen-induced arthritis in rabbits.

    PubMed

    Trivillin, Verónica A; Bruno, Leandro J; Gatti, David A; Stur, Mariela; Garabalino, Marcela A; Hughes, Andrea Monti; Castillo, Jorge; Pozzi, Emiliano C C; Wentzeis, Luis; Scolari, Hugo; Schwint, Amanda E; Feldman, Sara

    2016-11-01

    Rheumatoid arthritis is a chronic autoimmune pathology characterized by the proliferation and inflammation of the synovium. Boron neutron capture synovectomy (BNCS), a binary treatment modality that combines the preferential incorporation of boron carriers to target tissue and neutron irradiation, was proposed to treat the pathological synovium in arthritis. In a previous biodistribution study, we showed the incorporation of therapeutically useful boron concentrations to the pathological synovium in a model of antigen-induced arthritis (AIA) in rabbits, employing two boron compounds approved for their use in humans, i.e., decahydrodecaborate (GB-10) and boronophenylalanine (BPA). The aim of the present study was to perform low-dose BNCS studies at the RA-1 Nuclear Reactor in the same model. Neutron irradiation was performed post intra-articular administration of BPA or GB-10 to deliver 2.4 or 3.9 Gy, respectively, to synovium (BNCS-AIA). AIA and healthy animals (no AIA) were used as controls. The animals were followed clinically for 2 months. At that time, biochemical, magnetic resonance imaging (MRI) and histological studies were performed. BNCS-AIA animals did not show any toxic effects, swelling or pain on palpation. In BNCS-AIA, the post-treatment levels of TNF-α decreased in four of six rabbits and IFN-γ levels decreased in five of six rabbits. In all cases, MRI images of the knee joint in BNCS-AIA resembled those of no AIA, with no necrosis or periarticular effusion. Synovial membranes of BNCS-AIA were histologically similar to no AIA. BPA-BNCS and GB-10-BNCS, even at low doses, would be therapeutically useful for the local treatment of rheumatoid arthritis.

  18. Stable interaction between the human proliferating cell nuclear antigen loader complex Ctf18-replication factor C (RFC) and DNA polymerase {epsilon} is mediated by the cohesion-specific subunits, Ctf18, Dcc1, and Ctf8.

    PubMed

    Murakami, Takeshi; Takano, Ryuji; Takeo, Satoshi; Taniguchi, Rina; Ogawa, Kaori; Ohashi, Eiji; Tsurimoto, Toshiki

    2010-11-05

    One of the proliferating cell nuclear antigen loader complexes, Ctf18-replication factor C (RFC), is involved in sister chromatid cohesion. To examine its relationship with factors involved in DNA replication, we performed a proteomics analysis of Ctf18-interacting proteins. We found that Ctf18 interacts with a replicative DNA polymerase, DNA polymerase ε (pol ε). Co-immunoprecipitation with recombinant Ctf18-RFC and pol ε demonstrated that their binding is direct and mediated by two distinct interactions, one weak and one stable. Three subunits that are specifically required for cohesion in yeast, Ctf18, Dcc1, and Ctf8, formed a trimeric complex (18-1-8) and together enabled stable binding with pol ε. The C-terminal 23-amino acid stretch of Ctf18 was necessary for the trimeric association of 18-1-8 and was required for the stable interaction. The weak interaction was observed with alternative loader complexes including Ctf18-RFC(5), which lacks Dcc1 and Ctf8, suggesting that the common loader structures, including the RFC small subunits (RFC2-5), are responsible for the weak interaction. The two interaction modes, mediated through distinguishable structures of Ctf18-RFC, both occurred through the N-terminal half of pol ε, which includes the catalytic domain. The addition of Ctf18-RFC or Ctf18-RFC(5) to the DNA synthesis reaction caused partial inhibition and stimulation, respectively. Thus, Ctf18-RFC has multiple interactions with pol ε that promote polymorphic modulation of DNA synthesis. We propose that their interaction alters the DNA synthesis mode to enable the replication fork to cooperate with the establishment of cohesion.

  19. mRNA Structural Constraints on EBNA1 Synthesis Impact on In Vivo Antigen Presentation and Early Priming of CD8+ T Cells

    PubMed Central

    Lekieffre, Lea; Bhat, Purnima; Martinez, Michelle; Croft, Nathan P.; Kaplan, Warren; Tellam, Ross L.; Khanna, Rajiv

    2014-01-01

    Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8+ T cell epitopes by CD11c+ dendritic cells in draining lymph nodes and early priming of antigen-specific CD8+ T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8+ T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection. PMID:25299404

  20. mRNA Structural constraints on EBNA1 synthesis impact on in vivo antigen presentation and early priming of CD8+ T cells.

    PubMed

    Tellam, Judy T; Zhong, Jie; Lekieffre, Lea; Bhat, Purnima; Martinez, Michelle; Croft, Nathan P; Kaplan, Warren; Tellam, Ross L; Khanna, Rajiv

    2014-10-01

    Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8(+) T cell epitopes by CD11c(+) dendritic cells in draining lymph nodes and early priming of antigen-specific CD8(+) T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8(+) T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection.

  1. Bayesian variable selection for latent class models.

    PubMed

    Ghosh, Joyee; Herring, Amy H; Siega-Riz, Anna Maria

    2011-09-01

    In this article, we develop a latent class model with class probabilities that depend on subject-specific covariates. One of our major goals is to identify important predictors of latent classes. We consider methodology that allows estimation of latent classes while allowing for variable selection uncertainty. We propose a Bayesian variable selection approach and implement a stochastic search Gibbs sampler for posterior computation to obtain model-averaged estimates of quantities of interest such as marginal inclusion probabilities of predictors. Our methods are illustrated through simulation studies and application to data on weight gain during pregnancy, where it is of interest to identify important predictors of latent weight gain classes.

  2. Latent Heating from TRMM Satellite Measurements

    NASA Astrophysics Data System (ADS)

    Tao, W.; Takayabu, Y. N.; Shige, S.; Lang, S. E.; Olson, W. S.

    2012-12-01

    Rainfall production is a fundamental process within the Earth's hydrological cycle because it represents both a principal forcing term in surface water budgets, and its energetics corollary, latent heating, is the principal source of atmospheric diabatic heating. Latent heat release itself is a consequence of phase changes between the vapor, liquid, and frozen states of water. The properties of the vertical distribution of latent heat release modulate large-scale meridional and zonal circulations within the Tropics - as well as modify the energetic efficiencies of mid-latitude weather systems. This paper highlights the retrieval of latent heat release from satellite measurements generated by the Tropical Rainfall Measuring Mission (TRMM) satellite observatory, which was launched in November 1997 as a joint American-Japanese space endeavor. Since then, TRMM measurements have been providing an accurate four-dimensional account of rainfall over the global Tropics and sub-tropics - information which can be used to estimate the space-time structure of latent heating across the Earth's low latitudes. A set of algorithm methodologies has been developed to estimate latent heating based on rain rate profile retrievals obtained from TRMM measurements. These algorithms are briefly described followed by a discussion of the foremost latent heating products that can be generated from them. The investigation then provides an overview of how TRMM-derived latent heating information is currently being used in conjunction with global weather and climate models, concluding with remarks intended to stimulate further research on latent heating retrieval from satellites.

  3. Estimating and Interpreting Latent Variable Interactions: A Tutorial for Applying the Latent Moderated Structural Equations Method

    ERIC Educational Resources Information Center

    Maslowsky, Julie; Jager, Justin; Hemken, Douglas

    2015-01-01

    Latent variables are common in psychological research. Research questions involving the interaction of two variables are likewise quite common. Methods for estimating and interpreting interactions between latent variables within a structural equation modeling framework have recently become available. The latent moderated structural equations (LMS)…

  4. Information-Theoretic Latent Distribution Modeling: Distinguishing Discrete and Continuous Latent Variable Models

    ERIC Educational Resources Information Center

    Markon, Kristian E.; Krueger, Robert F.

    2006-01-01

    Distinguishing between discrete and continuous latent variable distributions has become increasingly important in numerous domains of behavioral science. Here, the authors explore an information-theoretic approach to latent distribution modeling, in which the ability of latent distribution models to represent statistical information in observed…

  5. Optimization-Based Model Fitting for Latent Class and Latent Profile Analyses

    ERIC Educational Resources Information Center

    Huang, Guan-Hua; Wang, Su-Mei; Hsu, Chung-Chu

    2011-01-01

    Statisticians typically estimate the parameters of latent class and latent profile models using the Expectation-Maximization algorithm. This paper proposes an alternative two-stage approach to model fitting. The first stage uses the modified k-means and hierarchical clustering algorithms to identify the latent classes that best satisfy the…

  6. Estimating and Interpreting Latent Variable Interactions: A Tutorial for Applying the Latent Moderated Structural Equations Method

    ERIC Educational Resources Information Center

    Maslowsky, Julie; Jager, Justin; Hemken, Douglas

    2015-01-01

    Latent variables are common in psychological research. Research questions involving the interaction of two variables are likewise quite common. Methods for estimating and interpreting interactions between latent variables within a structural equation modeling framework have recently become available. The latent moderated structural equations (LMS)…

  7. Optimization-Based Model Fitting for Latent Class and Latent Profile Analyses

    ERIC Educational Resources Information Center

    Huang, Guan-Hua; Wang, Su-Mei; Hsu, Chung-Chu

    2011-01-01

    Statisticians typically estimate the parameters of latent class and latent profile models using the Expectation-Maximization algorithm. This paper proposes an alternative two-stage approach to model fitting. The first stage uses the modified k-means and hierarchical clustering algorithms to identify the latent classes that best satisfy the…

  8. The Latent Structure of Autistic Traits: A Taxometric, Latent Class and Latent Profile Analysis of the Adult Autism Spectrum Quotient

    ERIC Educational Resources Information Center

    James, Richard J.; Dubey, Indu; Smith, Danielle; Ropar, Danielle; Tunney, Richard J.

    2016-01-01

    Autistic traits are widely thought to operate along a continuum. A taxometric analysis of Adult Autism Spectrum Quotient data was conducted to test this assumption, finding little support but identifying a high severity taxon. To understand this further, latent class and latent profile models were estimated that indicated the presence of six…

  9. Semi-Nonparametric Methods for Detecting Latent Non-Normality: A Fusion of Latent Trait and Ordered Latent Class Modeling

    ERIC Educational Resources Information Center

    Schmitt, J. Eric; Mehta, Paras D.; Aggen, Steven H.; Kubarych, Thomas S.; Neale, Michael C.

    2006-01-01

    Ordered latent class analysis (OLCA) can be used to approximate unidimensional latent distributions. The main objective of this study is to evaluate the method of OLCA in detecting non-normality of an unobserved continuous variable (i.e., a common factor) used to explain the covariation between dichotomous item-level responses. Using simulation,…

  10. The Latent Structure of Autistic Traits: A Taxometric, Latent Class and Latent Profile Analysis of the Adult Autism Spectrum Quotient

    ERIC Educational Resources Information Center

    James, Richard J.; Dubey, Indu; Smith, Danielle; Ropar, Danielle; Tunney, Richard J.

    2016-01-01

    Autistic traits are widely thought to operate along a continuum. A taxometric analysis of Adult Autism Spectrum Quotient data was conducted to test this assumption, finding little support but identifying a high severity taxon. To understand this further, latent class and latent profile models were estimated that indicated the presence of six…

  11. Semi-Nonparametric Methods for Detecting Latent Non-Normality: A Fusion of Latent Trait and Ordered Latent Class Modeling

    ERIC Educational Resources Information Center

    Schmitt, J. Eric; Mehta, Paras D.; Aggen, Steven H.; Kubarych, Thomas S.; Neale, Michael C.

    2006-01-01

    Ordered latent class analysis (OLCA) can be used to approximate unidimensional latent distributions. The main objective of this study is to evaluate the method of OLCA in detecting non-normality of an unobserved continuous variable (i.e., a common factor) used to explain the covariation between dichotomous item-level responses. Using simulation,…

  12. Consequences of Fitting Nonidentified Latent Class Models

    ERIC Educational Resources Information Center

    Abar, Beau; Loken, Eric

    2012-01-01

    Latent class models are becoming more popular in behavioral research. When models with a large number of latent classes relative to the number of manifest indicators are estimated, researchers must consider the possibility that the model is not identified. It is not enough to determine that the model has positive degrees of freedom. A well-known…

  13. Latent Memory for Sensitization in "Aplysia"

    ERIC Educational Resources Information Center

    Philips, Gary T.; Tzvetkova, Ekaterina I.; Marinesco, Stephane; Carew, Thomas J.

    2006-01-01

    In the analysis of memory it is commonly observed that, even after a memory is apparently forgotten, its latent presence can still be revealed in a subsequent learning task. Although well established on a behavioral level, the mechanisms underlying latent memory are not well understood. To begin to explore these mechanisms, we have used "Aplysia,"…

  14. Latent Memory for Sensitization in "Aplysia"

    ERIC Educational Resources Information Center

    Philips, Gary T.; Tzvetkova, Ekaterina I.; Marinesco, Stephane; Carew, Thomas J.

    2006-01-01

    In the analysis of memory it is commonly observed that, even after a memory is apparently forgotten, its latent presence can still be revealed in a subsequent learning task. Although well established on a behavioral level, the mechanisms underlying latent memory are not well understood. To begin to explore these mechanisms, we have used "Aplysia,"…

  15. [New tests for diagnosis of latent tubercolosis].

    PubMed

    Palumbo, Emilio

    2007-12-01

    Two new tests (QuantiFERON-TB and T-SPOT.TB) for diagnosis of latent tuberculosis are on the market. They measure the release of interferon-gamma in whole blood in response to stimulation by PDD. They offer a more accurate approach than tuberculin skin test for identification of individuals with latent tuberculosis infection.

  16. A Vernacular for Linear Latent Growth Models

    ERIC Educational Resources Information Center

    Hancock, Gregory R.; Choi, Jaehwa

    2006-01-01

    In its most basic form, latent growth modeling (latent curve analysis) allows an assessment of individuals' change in a measured variable X over time. For simple linear models, as with other growth models, parameter estimates associated with the a construct (amount of X at a chosen temporal reference point) and b construct (growth in X per unit…

  17. Cognitive Diagnosis Using Latent Trait Models.

    ERIC Educational Resources Information Center

    Samejima, Fumiko

    This paper discusses the competency space approach to diagnosing misconceptions, skill, and knowledge acquisition. In some approaches that combine misconceptions, skill, and knowledge acquisition, the latent ability theta is used more or less as an insignificant element, but in the competency space approach, a multidimensional latent space is…

  18. Sampling Weights in Latent Variable Modeling

    ERIC Educational Resources Information Center

    Asparouhov, Tihomir

    2005-01-01

    This article reviews several basic statistical tools needed for modeling data with sampling weights that are implemented in Mplus Version 3. These tools are illustrated in simulation studies for several latent variable models including factor analysis with continuous and categorical indicators, latent class analysis, and growth models. The…

  19. Latent Heating from TRMM Satellite Measurements

    NASA Technical Reports Server (NTRS)

    Tao, W.-K.; Smith, E.; Olson, W.

    2005-01-01

    Rainfall production is a fundamental process within the Earth;s hydrological cycle because it represents both a principal forcing term in surface water budgets, and its energetics corollary, latent heating, is the principal source of atmospheric diabatic heating. Latent heat release itself is a consequence of phase changes between the vapor, liquid, and frozen states of water. The properties of the vertical distribution of latent heat release modulate large-scale meridional and zonal circulations with the Tropics - as well as modify the energetic efficiencies of mid-latitude weather systems. This paper highlights the retrieval of observatory, which was launched in November 1997 as a joint American-Japanese space endeavor. Since then, TRMM measurements have been providing an accurate four-dimensional amount of rainfall over the global Tropics and sub-tropics - information which can be used to estimate the spacetime structure of latent heating across the Earth's low latitudes. A set of algorithm methodologies has and continues to be developed to estimate latent heating based on rain rate profile retrievals obtained from TRMM measurements. These algorithms are briefly described followed by a discussion of the foremost latent heating products that can be generate from them. The investigation then provides an overview of how TRMM-derived latent heating information is currently being used in conjunction with global weather and climate models, concluding with remarks intended to stimulate further research on latent heating retrieval from satellites.

  20. Introduction to Latent Class Analysis with Applications

    ERIC Educational Resources Information Center

    Porcu, Mariano; Giambona, Francesca

    2017-01-01

    Latent class analysis (LCA) is a statistical method used to group individuals (cases, units) into classes (categories) of an unobserved (latent) variable on the basis of the responses made on a set of nominal, ordinal, or continuous observed variables. In this article, we introduce LCA in order to demonstrate its usefulness to early adolescence…

  1. Introduction to Latent Class Analysis with Applications

    ERIC Educational Resources Information Center

    Porcu, Mariano; Giambona, Francesca

    2017-01-01

    Latent class analysis (LCA) is a statistical method used to group individuals (cases, units) into classes (categories) of an unobserved (latent) variable on the basis of the responses made on a set of nominal, ordinal, or continuous observed variables. In this article, we introduce LCA in order to demonstrate its usefulness to early adolescence…

  2. Identification of a negative regulatory cis-element in the enhancer core region of the prostate-specific antigen promoter: implications for intersection of androgen receptor and nuclear factor-kappaB signalling in prostate cancer cells.

    PubMed Central

    Cinar, Bekir; Yeung, Fan; Konaka, Hiroyuki; Mayo, Marty W; Freeman, Michael R; Zhau, Haiyen E; Chung, Leland W K

    2004-01-01

    The NF-kappaB (nuclear factor-kappaB) transcription factors mediate activation of a large number of gene promoters containing diverse kappaB-site sequences. Here, PSA (prostate-specific antigen) was used as an AR (androgen receptor)-responsive gene to examine the underlying mechanism by which the NF-kappaB p65 transcription factor down-regulates the transcriptional activity of AR in cells. We observed that activation of NF-kappaB by TNFalpha (tumour necrosis factor alpha) inhibited both basal and androgen-stimulated PSA expression, and that this down-regulation occurred at the promoter level, as confirmed by the super-repressor IkappaBalpha (S32A/S36A), a dominant negative inhibitor of NF-kappaB. Using a linker-scanning mutagenesis approach, we identified a cis -element, designated XBE (X-factor-binding element), in the AREc (androgen response element enhancer core) of the PSA promoter, which negatively regulated several AR-responsive promoters, including that of PSA. When three copies of XBE in tandem were juxtaposed to GRE4 (glucocorticoid response element 4), a 4-6-fold reduction of inducible GRE4 activity was detected in three different cell lines, LNCaP, ARCaP-AR and PC3-AR. Bioinformatics and molecular biochemical studies indicated that XBE is a kappaB-like element that binds specifically to the NF-kappaB p65 subunit; consistent with these observations, only NF-kappaB p65, but not the NF-kappaB p50 subunit, was capable of inhibiting AR-mediated PSA promoter transactivation in LNCaP cells. In addition, our data also showed that AR binds to XBE, as well as to the kappaB consensus site, and that the transfection of AR inhibits the kappaB-responsive promoter in transient co-transfection assays. Collectively, these data indicate that cross-modulation between AR and NF-kappaB p65 transcription factors may occur by a novel mechanism involving binding to a common cis -DNA element. PMID:14715080

  3. Variable Assessment in Latent Class Models

    PubMed Central

    Zhang, Q.; Ip, E. H.

    2014-01-01

    The latent class model provides an important platform for jointly modeling mixed-mode data — i.e., discrete and continuous data with various parametric distributions. Multiple mixed-mode variables are used to cluster subjects into latent classes. While the mixed-mode latent class analysis is a powerful tool for statisticians, few studies are focused on assessing the contribution of mixed-mode variables in discriminating latent classes. Novel measures are derived for assessing both absolute and relative impacts of mixed-mode variables in latent class analysis. Specifically, the expected posterior gradient and the Kolmogorov variation of the posterior distribution, as well as related properties are studied. Numerical results are presented to illustrate the measures. PMID:24910486

  4. Latent inhibition in human adults without masking.

    PubMed

    Escobar, Martha; Arcediano, Francisco; Miller, Ralph R

    2003-09-01

    Latent inhibition refers to attenuated responding to Cue X observed when the X-outcome pairings are preceded by X-alone presentations. It has proven difficult to obtain in human adults unless the preexposure (X-alone) presentations are embedded within a masking (i.e., distracting) task. The authors hypothesized that the difficulty in obtaining latent inhibition with unmasked tasks is related to the usual training procedures, in which the preexposure and conditioning experiences are separated by a set of instructions. Experiment 1 reports latent inhibition without masking in a task in which preexposure and conditioning occur without interruption. Experiments 2 and 3 demonstrate that this attenuation in responding to target Cue X does not pass a summation test for conditioned inhibition and is context specific, thereby confirming that it is latent inhibition. Experiments 3 and 4 confirm that introducing instructions between preexposure and conditioning disrupts latent inhibition.

  5. Improving knowledge management systems with latent semantic analysis

    SciTech Connect

    Sebok, A.; Plott, C.; LaVoie, N.

    2006-07-01

    Latent Semantic Analysis (LSA) offers a technique for improving lessons learned and knowledge management systems. These systems are expected to become more widely used in the nuclear industry, as experienced personnel leave and are replaced by younger, less-experienced workers. LSA is a machine learning technology that allows searching of text based on meaning rather than predefined keywords or categories. Users can enter and retrieve data using their own words, rather than relying on constrained language lists or navigating an artificially structured database. LSA-based tools can greatly enhance the usability and usefulness of knowledge management systems and thus provide a valuable tool to assist nuclear industry personnel in gathering and transferring worker expertise. (authors)

  6. Treatment of Latent Tuberculosis Infection.

    PubMed

    Haley, Connie A

    2017-04-01

    There are approximately 56 million people who harbor Mycobacterium tuberculosis that may progress to active tuberculosis (TB) at some point in their lives. Modeling studies suggest that if only 8% of these individuals with latent TB infection (LTBI) were treated annually, overall global incidence would be 14-fold lower by 2050 compared to incidence in 2013, even in the absence of additional TB control measures. This highlights the importance of identifying and treating latently infected individuals, and that this intervention must be scaled up to achieve the goals of the Global End TB Strategy. The efficacy of LTBI treatment is well established, and the most commonly used regimen is 9 months of daily self-administered isoniazid. However, its use has been hindered by limited provider awareness of the benefits, concern about potential side effects such as hepatotoxicity, and low rates of treatment completion. There is increasing evidence that shorter rifamycin-based regimens are as effective, better tolerated, and more likely to be completed compared to isoniazid. Such regimens include four months of daily self-administered rifampin monotherapy, three months of once weekly directly observed isoniazid-rifapentine, and three months of daily self-administered isoniazid-rifampin. The success of LTBI treatment to prevent additional TB disease relies upon choosing an appropriate regimen individualized to the patient, monitoring for potential adverse clinical events, and utilizing strategies to promote adherence. Safer, more cost-effective, and more easily completed regimens are needed and should be combined with interventions to better identify, engage, and retain high-risk individuals across the cascade from diagnosis through treatment completion of LTBI.

  7. Phase I trial of recombinant modified vaccinia ankara encoding Epstein-Barr viral tumor antigens in nasopharyngeal carcinoma patients.

    PubMed

    Hui, Edwin P; Taylor, Graham S; Jia, Hui; Ma, Brigette B Y; Chan, Stephen L; Ho, Rosalie; Wong, Wai-Lap; Wilson, Steven; Johnson, Benjamin F; Edwards, Ceri; Stocken, Deborah D; Rickinson, Alan B; Steven, Neil M; Chan, Anthony T C

    2013-03-15

    Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma, a high incidence tumor in Chinese populations, in which tumor cells express the two EBV antigens EB nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2). Here, we report the phase I trial of a recombinant vaccinia virus, MVA-EL, which encodes an EBNA1/LMP2 fusion protein designed to boost T-cell immunity to these antigens. The vaccine was delivered to Hong Kong patients with nasopharyngeal carcinoma to determine a safe and immunogenic dose. The patients, all in remission more than 12 weeks after primary therapy, received three intradermal MVA-EL vaccinations at three weekly intervals, using five escalating dose levels between 5 × 10(7) and 5 × 10(8) plaque-forming unit (pfu). Blood samples were taken during prescreening, immediately before vaccination, one week afterward and at intervals up to one year later. Immunogenicity was tested by IFN-γ ELIspot assays using complete EBNA1 and LMP2 15-mer peptide mixes and known epitope peptides relevant to patient MHC type. Eighteen patients were treated, three per dose level one to four and six at the highest dose, without dose-limiting toxicity. T-cell responses to one or both vaccine antigens were increased in 15 of 18 patients and, in many cases, were mapped to known CD4 and CD8 epitopes in EBNA1 and/or LMP2. The range of these responses suggested a direct relationship with vaccine dose, with all six patients at the highest dose level giving strong EBNA1/LMP2 responses. We concluded that MVA-EL is both safe and immunogenic, allowing the highest dose to be forwarded to phase II studies examining clinical benefit.

  8. Latent Heating from TRMM Satellite Measurements

    NASA Technical Reports Server (NTRS)

    Tao, Wei-Kuo; Smith, E. A.; Adler, R.; Haddad, Z.; Hou, A.; Iguchi, T.; Kakar, R.; Krishnamurti, T.; Kummerow, C.; Lang, S.

    2004-01-01

    Rainfall production is the fundamental variable within the Earth's hydrological cycle because it is both the principal forcing term in surface water budgets and its energetics corollary, latent heating, is the principal source of atmospheric diabatic heating. Latent heat release itself is a consequence of phase changes between the vapor, liquid, and frozen states of water. The properties of the vertical distribution of latent heat release modulate large-scale meridional and zonal circulations within the tropics - as well as modifying the energetic efficiencies of midlatitude weather systems. This paper focuses on the retrieval of latent heat release from satellite measurements generated by the Tropical Rainfall Measuring Mission (TRMM) satellite observatory, which was launched in November 1997 as a joint American-Japanese space endeavor. Since then, TRMM measurements have been providing an accurate four-dimensional account of rainfall over the global tropics and sub-tropics, information which can be used to estimate the space-time structure of latent heating across the Earth's low latitudes. The paper examines how the observed TRMM distribution of rainfall has advanced an understanding of the global water and energy cycle and its consequent relationship to the atmospheric general circulation and climate via latent heat release. A set of algorithm methodologies that are being used to estimate latent heating based on rain rate retrievals from the TRMM observations are described. The characteristics of these algorithms and the latent heating products that can be generated from them are also described, along with validation analyses of the heating products themselves. Finally, the investigation provides an overview of how TRMM-derived latent heating information is currently being used in conjunction with global weather and climate models, concluding with remarks intended to stimulate further research on latent heating retrieval from satellites.

  9. Analysis of latent tracks for MeV protons in CR-39

    NASA Astrophysics Data System (ADS)

    Kar, S.; Borghesi, M.; Romagnani, L.; Takahashi, S.; Zayats, A.; Malka, V.; Fritzler, S.; Schiavi, A.

    2007-02-01

    For protons of energy up to a few MeV, the temporal evolution of etched latent tracks in CR-39 nuclear track detector has been numerically modeled by assuming that the electronic energy loss of the protons governs the latent track formation. The technique is applied in order to obtain the energy spectrum of high intensity laser driven proton beams, with high accuracy. The precise measurement of the track length and areal track density have been achieved by scanning short etched, highly populated CR-39 employing atomic force microscope.

  10. Orientation field estimation for latent fingerprint enhancement.

    PubMed

    Feng, Jianjiang; Zhou, Jie; Jain, Anil K

    2013-04-01

    Identifying latent fingerprints is of vital importance for law enforcement agencies to apprehend criminals and terrorists. Compared to live-scan and inked fingerprints, the image quality of latent fingerprints is much lower, with complex image background, unclear ridge structure, and even overlapping patterns. A robust orientation field estimation algorithm is indispensable for enhancing and recognizing poor quality latents. However, conventional orientation field estimation algorithms, which can satisfactorily process most live-scan and inked fingerprints, do not provide acceptable results for most latents. We believe that a major limitation of conventional algorithms is that they do not utilize prior knowledge of the ridge structure in fingerprints. Inspired by spelling correction techniques in natural language processing, we propose a novel fingerprint orientation field estimation algorithm based on prior knowledge of fingerprint structure. We represent prior knowledge of fingerprints using a dictionary of reference orientation patches. which is constructed using a set of true orientation fields, and the compatibility constraint between neighboring orientation patches. Orientation field estimation for latents is posed as an energy minimization problem, which is solved by loopy belief propagation. Experimental results on the challenging NIST SD27 latent fingerprint database and an overlapped latent fingerprint database demonstrate the advantages of the proposed orientation field estimation algorithm over conventional algorithms.

  11. Immunogenicity of 60 novel latency-related antigens of Mycobacterium tuberculosis

    PubMed Central

    Serra-Vidal, Mᵃdel Mar; Latorre, Irene; Franken, Kees L. C. M.; Díaz, Jéssica; de Souza-Galvão, Maria Luiza; Casas, Irma; Maldonado, José; Milà, Cèlia; Solsona, Jordi; Jimenez-Fuentes, M. Ángeles; Altet, Neus; Lacoma, Alícia; Ruiz-Manzano, Juan; Ausina, Vicente; Prat, Cristina; Ottenhoff, Tom H. M.; Domínguez, José

    2014-01-01

    The aim of our work here was to evaluate the immunogenicity of 60 mycobacterial antigens, some of which have not been previously assessed, notably a novel series of in vivo-expressed Mycobacterium tuberculosis (IVE-TB) antigens. We enrolled 505 subjects and separated them in individuals with and without latent tuberculosis infection (LTBI) vs. patients with active tuberculosis (TB). Following an overnight and 7 days stimulation of whole blood with purified recombinant M. tuberculosis antigens, interferon-γ (IFN-γ) levels were determined by ELISA. Several antigens could statistically significantly differentiate the groups of individuals. We obtained promising antigens from all studied antigen groups [dormancy survival regulon (DosR regulon) encoded antigens; resuscitation-promoting factors (Rpf) antigens; IVE-TB antigens; reactivation associated antigens]. Rv1733, which is a probable conserved transmembrane protein encoded in DosR regulon, turned out to be very immunogenic and able to discriminate between the three defined TB status, thus considered a candidate biomarker. Rv2389 and Rv2435n, belonging to Rpf family and IVE-TB group of antigens, respectively, also stood out as LTBI biomarkers. Although more studies are needed to support our findings, the combined use of these antigens would be an interesting approach to TB immunodiagnosis candidates. PMID:25339944

  12. Recent advances in testing for latent TB.

    PubMed

    Schluger, Neil W; Burzynski, Joseph

    2010-12-01

    After more than a century of relying on skin testing for the diagnosis of latent TB infection, clinicians now have access to blood-based diagnostics in the form of interferon γ release assays (IGRAs). These tests are generally associated with higher sensitivity and specificity for diagnosis of latent TB infection. This article reviews the indications for testing and treatment of latent TB infection in the overall context of a TB control program and describes how IGRAs might be used in specific clinical settings and populations, including people having close contact with an active case of TB, the foreign born, and health-care workers.

  13. Extraction of latent images from printed media

    NASA Astrophysics Data System (ADS)

    Sergeyev, Vladislav; Fedoseev, Victor

    2015-12-01

    In this paper we propose an automatic technology for extraction of latent images from printed media such as documents, banknotes, financial securities, etc. This technology includes image processing by adaptively constructed Gabor filter bank for obtaining feature images, as well as subsequent stages of feature selection, grouping and multicomponent segmentation. The main advantage of the proposed technique is versatility: it allows to extract latent images made by different texture variations. Experimental results showing performance of the method over another known system for latent image extraction are given.

  14. Nuclear Futures Analysis and Scenario Building

    SciTech Connect

    Arthur, E.D.; Beller, D.; Canavan, G.H.; Krakowski, R.A.; Peterson, P.; Wagner, R.L.

    1999-07-09

    This LDRD project created and used advanced analysis capabilities to postulate scenarios and identify issues, externalities, and technologies associated with future ''things nuclear''. ''Things nuclear'' include areas pertaining to nuclear weapons, nuclear materials, and nuclear energy, examined in the context of future domestic and international environments. Analysis tools development included adaptation and expansion of energy, environmental, and economics (E3) models to incorporate a robust description of the nuclear fuel cycle (both current and future technology pathways), creation of a beginning proliferation risk model (coupled to the (E3) model), and extension of traditional first strike stability models to conditions expected to exist in the future (smaller force sizes, multipolar engagement environments, inclusion of actual and latent nuclear weapons (capability)). Accomplishments include scenario development for regional and global nuclear energy, the creation of a beginning nuclear architecture designed to improve the proliferation resistance and environmental performance of the nuclear fuel cycle, and numerous results for future nuclear weapons scenarios.

  15. Messenger RNA Sequence Rather than Protein Sequence Determines the Level of Self-synthesis and Antigen Presentation of the EBV-encoded Antigen, EBNA1

    PubMed Central

    Tellam, Judy T.; Lekieffre, Lea; Zhong, Jie; Lynn, David J.; Khanna, Rajiv

    2012-01-01

    Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr) encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a novel approach to

  16. Messenger RNA sequence rather than protein sequence determines the level of self-synthesis and antigen presentation of the EBV-encoded antigen, EBNA1.

    PubMed

    Tellam, Judy T; Lekieffre, Lea; Zhong, Jie; Lynn, David J; Khanna, Rajiv

    2012-12-01

    Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr) encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a novel approach to

  17. Retrieved Latent Heating from TRMM

    NASA Technical Reports Server (NTRS)

    Tao, Wei-Kuo; Smith, Eric A.; Houze Jr, Robert

    2008-01-01

    The global hydrological cycle is central to the Earth's climate system, with rainfall and the physics of precipitation formation acting as the key links in the cycle. Two-thirds of global rainfall occurs in the tropics with the associated latent heating (LH) accounting for three-fourths of the total heat energy available to the Earth's atmosphere. In addition, fresh water provided by tropical rainfall and its variability exerts a large impact upon the structure and motions of the upper ocean layer. In the last decade, it has been established that standard products of LH from satellite measurements, particularly TRMM measurements, would be a valuable resource for scientific research and applications. Such products would enable new insights and investigations concerning the complexities of convection system life cycles, the diabatic heating controls and feedbacks related to meso-synoptic circulations and their forecasting, the relationship of tropical patterns of LH to the global circulation and climate, and strategies for improving cloud parameterizations in environmental prediction models. The status of retrieved TRMM LH products, TRMM LH inter-comparison and validation project, current TRMM LH applications and critic issues/action items (based on previous five TRMM LH workshops) is presented in this article.

  18. Reactivation of Latent Viruses in Space

    NASA Technical Reports Server (NTRS)

    Pierson, D. L.; Mehta, S. K.; Tyring, S. K.; Lugg, D. J.

    1999-01-01

    Reactivation of latent viruses is an important health risk for people working and living in physically isolated extreme environments such as Antarctica and space. Preflight quarantine does not significantly reduce the risk associated with latent viruses, however, pharmaceutical countermeasures are available for some viruses. The molecular basis of latency is not fully understood, but physical and psychosocial stresses are known to initiate the reactivation of latent viruses. Presumably, stress induced changes in selected hormones lead to alterations in the cell- mediated immune (CMI) response resulting in increased shedding of latent viruses. Limited access to space makes the use of ground-based analogs essential. The Australian Antarctic stations serve as a good stress model and simulate many aspects of space flight. Closed environmental chambers have been used to simulate space flight since the Skylab missions and have also proven to be a valuable analog of selected aspects of space flight.

  19. Reactivation of Latent Viruses in Space

    NASA Technical Reports Server (NTRS)

    Pierson, D. L.; Mehta, S. K.; Tyring, S. K.; Lugg, D. J.

    1999-01-01

    Reactivation of latent viruses is an important health risk for people working and living in physically isolated extreme environments such as Antarctica and space. Preflight quarantine does not significantly reduce the risk associated with latent viruses, however, pharmaceutical countermeasures are available for some viruses. The molecular basis of latency is not fully understood, but physical and psychosocial stresses are known to initiate the reactivation of latent viruses. Presumably, stress induced changes in selected hormones lead to alterations in the cell- mediated immune (CMI) response resulting in increased shedding of latent viruses. Limited access to space makes the use of ground-based analogs essential. The Australian Antarctic stations serve as a good stress model and simulate many aspects of space flight. Closed environmental chambers have been used to simulate space flight since the Skylab missions and have also proven to be a valuable analog of selected aspects of space flight.

  20. AntigenMap 3D: an online antigenic cartography resource.

    PubMed

    Barnett, J Lamar; Yang, Jialiang; Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2012-05-01

    Antigenic cartography is a useful technique to visualize and minimize errors in immunological data by projecting antigens to 2D or 3D cartography. However, a 2D cartography may not be sufficient to capture the antigenic relationship from high-dimensional immunological data. AntigenMap 3D presents an online, interactive, and robust 3D antigenic cartography construction and visualization resource. AntigenMap 3D can be applied to identify antigenic variants and vaccine strain candidates for pathogens with rapid antigenic variations, such as influenza A virus. http://sysbio.cvm.msstate.edu/AntigenMap3D

  1. Latent phenotypes pervade gene regulatory circuits

    PubMed Central

    2014-01-01

    Background Latent phenotypes are non-adaptive byproducts of adaptive phenotypes. They exist in biological systems as different as promiscuous enzymes and genome-scale metabolic reaction networks, and can give rise to evolutionary adaptations and innovations. We know little about their prevalence in the gene expression phenotypes of regulatory circuits, important sources of evolutionary innovations. Results Here, we study a space of more than sixteen million three-gene model regulatory circuits, where each circuit is represented by a genotype, and has one or more functions embodied in one or more gene expression phenotypes. We find that the majority of circuits with single functions have latent expression phenotypes. Moreover, the set of circuits with a given spectrum of functions has a repertoire of latent phenotypes that is much larger than that of any one circuit. Most of this latent repertoire can be easily accessed through a series of small genetic changes that preserve a circuit’s main functions. Both circuits and gene expression phenotypes that are robust to genetic change are associated with a greater number of latent phenotypes. Conclusions Our observations suggest that latent phenotypes are pervasive in regulatory circuits, and may thus be an important source of evolutionary adaptations and innovations involving gene regulation. PMID:24884746

  2. Latent phenotypes pervade gene regulatory circuits.

    PubMed

    Payne, Joshua L; Wagner, Andreas

    2014-05-30

    Latent phenotypes are non-adaptive byproducts of adaptive phenotypes. They exist in biological systems as different as promiscuous enzymes and genome-scale metabolic reaction networks, and can give rise to evolutionary adaptations and innovations. We know little about their prevalence in the gene expression phenotypes of regulatory circuits, important sources of evolutionary innovations. Here, we study a space of more than sixteen million three-gene model regulatory circuits, where each circuit is represented by a genotype, and has one or more functions embodied in one or more gene expression phenotypes. We find that the majority of circuits with single functions have latent expression phenotypes. Moreover, the set of circuits with a given spectrum of functions has a repertoire of latent phenotypes that is much larger than that of any one circuit. Most of this latent repertoire can be easily accessed through a series of small genetic changes that preserve a circuit's main functions. Both circuits and gene expression phenotypes that are robust to genetic change are associated with a greater number of latent phenotypes. Our observations suggest that latent phenotypes are pervasive in regulatory circuits, and may thus be an important source of evolutionary adaptations and innovations involving gene regulation.

  3. Identification of T cell-signaling pathways that stimulate latent HIV in primary cells

    PubMed Central

    Brooks, David G.; Arlen, Philip A.; Gao, Lianying; Kitchen, Christina M. R.; Zack, Jerome A.

    2003-01-01

    Eradication of HIV infection depends on the elimination of a small, but stable population of latently infected T cells. After the discontinuation of therapy, activation of latent virus can rekindle infection. To purge this reservoir, it is necessary to define cellular signaling pathways that lead to activation of latent HIV. We used the SCID-hu (Thy/Liv) mouse model of HIV latency to analyze a broad array of T cell-signaling pathways and show in primary, quiescent cells that viral induction depends on the activation of two primary intracellular signaling pathways, protein kinase C or nuclear factor of activated T cells (NF-AT). In contrast, inhibition or activation of other important T cell stimulatory pathways (such as mitogen-activated protein kinase, calcium flux, or histone deacetylation) do not significantly induce virus expression. We found that the activation of NF-κB is critical to viral reactivation; however, all pathways that stimulate NF-κBdonot reactivate latent virus. Our studies further show that inhibition of NF-κB does not prevent activation of HIV by NF-AT, indicating that these pathways can function independently to activate the HIV LTR. Thus, we define several molecular pathways that trigger HIV reactivation from latency and provide evidence that latent HIV infection is maintained by the functional lack of particular transcription factors in quiescent cells. PMID:14569007

  4. A potential alpha-helix motif in the amino terminus of LANA encoded by Kaposi's sarcoma-associated herpesvirus is critical for nuclear accumulation of HIF-1alpha in normoxia.

    PubMed

    Cai, Qiliang; Murakami, Masanao; Si, Huaxin; Robertson, Erle S

    2007-10-01

    Hypoxia-inducible factor 1 (HIF-1) is a ubiquitously expressed transcriptional regulator involved in induction of numerous genes associated with angiogenesis and tumor growth. Kaposi's sarcoma, associated with increased angiogenesis, is a highly vascularized, endothelial cell-derived tumor. Previously, we have shown that the latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) targets the HIF-1alpha suppressors von Hippel-Lindau protein and p53 for degradation via its suppressor of cytokine signaling-box motif, which recruits the EC5S ubiquitin complex. Here we further show that HIF-1alpha was aberrantly accumulated in KSHV latently infected primary effusion lymphoma (PEL) cells, as well as HEK293 cells infected with KSHV, and also show that a potential alpha-helical amino-terminal domain of LANA was important for HIF-1alpha nuclear accumulation in normoxic conditions. Moreover, we have now determined that this association was dependent on the residues 46 to 89 of LANA and the oxygen-dependent degradation domain of HIF-1alpha. Introduction of specific small interfering RNA against LANA into PEL cells also resulted in a diminished nuclear accumulation of HIF-1alpha. Therefore, these data show that LANA can function not only as an inhibitor of HIF-1alpha suppressor proteins but can also induce nuclear accumulation of HIF-1alpha during KSHV latent infection.

  5. Not to wake a sleeping giant: new insights into host-pathogen interactions identify new targets for vaccination against latent Mycobacterium tuberculosis infection.

    PubMed

    Lin, May Young; Ottenhoff, Tom H M

    2008-05-01

    Mycobacterium tuberculosis is one of the worlds' most successful and sophisticated pathogens. It is estimated that over 2 billion people today harbour latent M. tuberculosis infection without any clinical symptoms. As most new cases of active tuberculosis (TB) arise from this (growing) number of latently infected individuals, urgent measures to control TB reactivation are required, including post-exposure/therapeutic vaccines. The current bacille Calmette-Guérin (BCG) vaccine and all new generation TB vaccines being developed and tested are essentially designed as prophylactic vaccines. Unfortunately, these vaccines are unlikely to be effective in individuals already latently infected with M. tuberculosis. Here, we argue that detailed analysis of M. tuberculosis genes that are switched on predominantly during latent stage infection may lead to the identification of new antigenic targets for anti-TB strategies. We will describe essential host-pathogen interactions in TB with particular emphasis on TB latency and persistent infection. Subsequently, we will focus on novel groups of late-stage specific genes, encoded amongst others by the M. tuberculosis dormancy (dosR) regulon, and summarise recent studies describing human T-cell recognition of these dormancy antigens in relation to (latent) M. tuberculosis infection. We will discuss the possible relevance of these new classes of antigens for vaccine development against TB.

  6. Habituation, latent inhibition, and extinction.

    PubMed

    Jordan, Wesley P; Todd, Travis P; Bucci, David J; Leaton, Robert N

    2015-06-01

    In two conditioned suppression experiments with a latent inhibition (LI) design, we measured the habituation of rats in preexposure, their LI during conditioning, and then extinction over days. In the first experiment, lick suppression, the preexposed group (PE) showed a significant initial unconditioned response (UR) to the target stimulus and significant long-term habituation (LTH) of that response over days. The significant difference between the PE and nonpreexposed (NPE) groups on the first conditioning trial was due solely to the difference in their URs to the conditioned stimulus (CS)-a habituated response (PE) and an unhabituated response (NPE). In the second experiment, bar-press suppression, little UR to the target stimulus was apparent during preexposure, and no detectable LTH. Thus, there was no difference between the PE and NPE groups on the first conditioning trial. Whether the UR to the CS confounds the interpretation of LI (Exp. 1) or not (Exp. 2) can only be known if the UR is measured. In both experiments, LI was observed in acquisition. Also in both experiments, rats that were preexposed and then conditioned to asymptote were significantly more resistant to extinction than were the rats not preexposed. This result contrasts with the consistently reported finding that preexposure either produces less resistance to extinction or has no effect on extinction. The effect of stimulus preexposure survived conditioning to asymptote and was reflected directly in extinction. These two experiments provide a cautionary procedural note for LI experiments and have shown an unexpected extinction effect that may provide new insights into the interpretation of LI.

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

    PubMed

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

    2017-11-01

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

  8. A Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 ORF50 deletion mutant is defective for reactivation of latent virus and DNA replication.

    PubMed

    Xu, Yiyang; AuCoin, David P; Huete, Alicia Rodriguez; Cei, Sylvia A; Hanson, Lisa J; Pari, Gregory S

    2005-03-01

    Kaposi's sarcoma-associated herpesvirus (also called human herpesvirus type 8 [HHV8]) latently infects a number of cell types. Reactivation of latent virus can occur by treatment with the phorbol ester tetradecanoyl phorbol acetate (TPA) or with the transfection of plasmids expressing the lytic switch activator protein K-Rta, the gene product of ORF50. K-Rta expression is sufficient for the activation of the entire lytic cycle and the transactivation of viral genes necessary for DNA replication. In addition, recent evidence has suggested that K-Rta may participate directly in the initiation of lytic DNA synthesis. We have now generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a large deletion within the ORF50 locus. This BAC, BAC36Delta50, failed to produce infectious virus upon treatment with TPA and was defective for DNA synthesis. Expression of K-Rta in trans in BAC36Delta50-containing cells was able to abolish both defects. Real-time PCR revealed that K-bZIP, ORF40/41, and K8.1 were not expressed when BAC36Delta50-containing cells were induced with TPA. However, the mRNA levels of ORF57 were over fivefold higher in TPA-treated BAC36Delta50-containing cells than those observed in similarly treated wild-type BAC-containing cells. In addition, immunohistochemical analysis showed that while the latency-associated nuclear antigen (LANA) was expressed in the mutant BAC-containing cells, ORF59 and K8.1 expression was not detected in TPA-induced BAC36Delta50-containing cells. These results showed that K-Rta is essential for lytic viral reactivation and transactivation of viral genes contributing to DNA replication.

  9. Transcutaneous antigen delivery system

    PubMed Central

    Lee, Mi-Young; Shin, Meong-Cheol; Yang, Victor C.

    2013-01-01

    Transcutaneous immunization refers to the topical application of antigens onto the epidermis. Transcutaneous immunization targeting the Langerhans cells of the skin has received much attention due to its safe, needle-free, and noninvasive antigen delivery. The skin has important immunological functions with unique roles for antigen-presenting cells such as epidermal Langerhans cells and dermal dendritic cells. In recent years, novel vaccine delivery strategies have continually been developed; however, transcutaneous immunization has not yet been fully exploited due to the penetration barrier represented by the stratum corneum, which inhibits the transport of antigens and adjuvants. Herein we review recent achievements in transcutaneous immunization, focusing on the various strategies for the enhancement of antigen delivery and vaccination efficacy. [BMB Reports 2013; 46(1): 17-24] PMID:23351379

  10. Latent Curve Models and Latent Change Score Models Estimated in R

    ERIC Educational Resources Information Center

    Ghisletta, Paolo; McArdle, John J.

    2012-01-01

    In recent years the use of the latent curve model (LCM) among researchers in social sciences has increased noticeably, probably thanks to contemporary software developments and the availability of specialized literature. Extensions of the LCM, like the the latent change score model (LCSM), have also increased in popularity. At the same time, the R…

  11. Latent Curve Models and Latent Change Score Models Estimated in R

    ERIC Educational Resources Information Center

    Ghisletta, Paolo; McArdle, John J.

    2012-01-01

    In recent years the use of the latent curve model (LCM) among researchers in social sciences has increased noticeably, probably thanks to contemporary software developments and the availability of specialized literature. Extensions of the LCM, like the the latent change score model (LCSM), have also increased in popularity. At the same time, the R…

  12. Latent-Trait Latent-Class Analysis of Self-Disclosure in the Work Environment

    ERIC Educational Resources Information Center

    Maij-de Meij, Annette M.; Kelderman, Henk; van der Flier, Henk

    2005-01-01

    Based on the literature about self-disclosure, it was hypothesized that different groups of subjects differ in their pattern of self-disclosure with respect to different areas of social interaction. An extended latent-trait latent-class model was proposed to describe these general patterns of self-disclosure. The model was used to analyze the data…

  13. Detection of interleukin-2 in addition to interferon-gamma discriminates active tuberculosis patients, latently infected individuals, and controls.

    PubMed

    Biselli, R; Mariotti, S; Sargentini, V; Sauzullo, I; Lastilla, M; Mengoni, F; Vanini, V; Girardi, E; Goletti, D; D' Amelio, R; Nisini, R

    2010-08-01

    Effective control of tuberculosis (TB) includes discrimination of subjects with active TB from individuals with latent TB infection (LTBI). As distinct interferon (IFN)-gamma and interleukin (IL)-2 profiles of antigen-specific T-cells have been associated with different clinical stages and antigen loads in several viral and bacterial diseases, we analysed these cytokines in TB using a modified QuantiFERON-TB Gold In Tube test. Detection of IL-2 in addition to IFN-gamma distinguishes not only Mycobacterium tuberculosis-infected subjects from healthy controls, but also individuals with LTBI from active TB patients. This may help to improve diagnostic tests for TB.

  14. Chronic stress, leukocyte subpopulations, and humoral response to latent viruses

    SciTech Connect

    McKinnon, W.; Weisse, C.S.; Reynolds, C.P.; Bowles, C.A.; Baum, A. )

    1989-01-01

    Psychological stress has been shown to affect immune system status and function, but most studies of this relationship have focused on acute stress and/or laboratory situations. The present study compared total numbers of leukocytes and lymphocyte subpopulations (determined by flow cytometry) and antibody titers to latent and nonlatent viruses among a group of chronically stressed individuals living near the damaged Three Mile Island (TMI) nuclear power plant with those of a demographically comparable control group. Urinary catecholamine and cortisol levels were also examined. Residents of the TMI area exhibited greater numbers of neutrophils, which were positively correlated with epinephrine levels. The TMI group also exhibited fewer B lymphocytes, T-suppressor/cytotoxic lymphocytes, and natural killer cells. Antibody titers to herpes simplex were significantly different across groups as well, whereas titers to nonlatent rubella virus as well as IgG and IgM levels were comparable.

  15. Human Cytomegalovirus Manipulation of Latently Infected Cells

    PubMed Central

    Sinclair, John H.; Reeves, Matthew B.

    2013-01-01

    Primary infection with human cytomegalovirus (HCMV) results in the establishment of a lifelong infection of the host which is aided by the ability of HCMV to undergo a latent infection. One site of HCMV latency in vivo is in haematopoietic progenitor cells, resident in the bone marrow, with genome carriage and reactivation being restricted to the cells of the myeloid lineage. Until recently, HCMV latency has been considered to be relatively quiescent with the virus being maintained essentially as a “silent partner” until conditions are met that trigger reactivation. However, advances in techniques to study global changes in gene expression have begun to show that HCMV latency is a highly active process which involves expression of specific latency-associated viral gene products which orchestrate major changes in the latently infected cell. These changes are argued to help maintain latent infection and to modulate the cellular environment to the benefit of latent virus. In this review, we will discuss these new findings and how they impact not only on our understanding of the biology of HCMV latency but also how they could provide tantalising glimpses into mechanisms that could become targets for the clearance of latent HCMV. PMID:24284875

  16. Cellular Localization of Latent Murine Cytomegalovirus

    PubMed Central

    Koffron, Alan J.; Hummel, Mary; Patterson, Bruce K.; Yan, Shixian; Kaufman, Dixon B.; Fryer, Jonathan P.; Stuart, Frank P.; Abecassis, Michael I.

    1998-01-01

    Herpesviruses typically establish latent infection in their hosts. The cell(s) responsible for harboring latent virus, in most cases, is not known. Using immunofluorescence and PCR-in situ hybridization (PISH), a technique which combines the sensitivity of PCR with the localization and specificity of in situ hybridization, we provide the first direct evidence that endothelial cells are a major site of murine cytomegalovirus (MCMV) DNA in latently infected animals. These findings are consistent with existing knowledge of the biological behavior of CMV, in particular the transmission of latent CMV by solid organ and bone marrow transplantation, in both human and animal models. In addition, we have localized MCMV DNA in the lung alveolar macrophage and in bone marrow cells. Our findings confirm that bone marrow-derived hematopoietic cells are a site of CMV latency and further suggest that bone marrow may be a reservoir of infected progeny capable of migrating into the circulation and establishing latency in various tissues. These findings provide clearly needed insight into the site of latent infection which is central to an understanding of the mechanisms of reactivation. PMID:9420204

  17. Robust Latent Subspace Learning for Image Classification.

    PubMed

    Fang, Xiaozhao; Teng, Shaohua; Lai, Zhihui; He, Zhaoshui; Xie, Shengli; Wong, Wai Keung

    2017-05-10

    This paper proposes a novel method, called robust latent subspace learning (RLSL), for image classification. We formulate an RLSL problem as a joint optimization problem over both the latent SL and classification model parameter predication, which simultaneously minimizes: 1) the regression loss between the learned data representation and objective outputs and 2) the reconstruction error between the learned data representation and original inputs. The latent subspace can be used as a bridge that is expected to seamlessly connect the origin visual features and their class labels and hence improve the overall prediction performance. RLSL combines feature learning with classification so that the learned data representation in the latent subspace is more discriminative for classification. To learn a robust latent subspace, we use a sparse item to compensate error, which helps suppress the interference of noise via weakening its response during regression. An efficient optimization algorithm is designed to solve the proposed optimization problem. To validate the effectiveness of the proposed RLSL method, we conduct experiments on diverse databases and encouraging recognition results are achieved compared with many state-of-the-arts methods.

  18. Neural antigen-specific autoimmune disorders.

    PubMed

    Iorio, Raffaele; Lennon, Vanda A

    2012-07-01

    Neural-specific autoantibodies have been documented and their diagnostic utility validated in diseases affecting the neuraxis from cerebral cortex to the somatic, autonomic, and enteric nervous system and skeletal muscle. These neurological disorders occur both idiopathically and in a paraneoplastic context. Molecular identification of the antigens has expedited development of confirmatory and high-throughput tests for serum and cerebrospinal fluid, which permit early diagnosis and reveal the underlying molecular pathogenic mechanisms. The autoantibodies are classifiable on the basis of antigen location: intracellular (nuclear or cytoplasmic) or plasma membrane. Immunohistopathological studies of patients' biopsied and autopsied tissues suggest that effector T cells mediate the autoimmune neurological disorders for which defining autoantibodies recognize intracellular antigens. Antigens within intact cells are inaccessible to circulating antibody, and the associated neurological deficits rarely improve with antibody-depleting therapies. Tumoricidal therapies may arrest neurological progression, but symptom reversal is rare. In contrast, autoantibodies specific for plasma membrane antigens have pathogenic potential, and the associated neurological deficits are often amenable to antibody-depleting immunotherapy, such as plasma exchange and anti-B-cell monoclonal antibody therapy. These reversible neurological disorders are frequently misdiagnosed as neurodegenerative. The focus of this review is the immunobiology, pathophysiology, and clinical spectrum of autoimmune neurological disorders accompanied by neural-specific IgGs.

  19. Microarray analysis in the HSV-1 latently infected mouse trigeminal ganglion.

    PubMed

    Higaki, Shiro; Deai, Tatsunori; Fukuda, Masahiko; Shimomura, Yoshikazu

    2004-11-01

    To review our previous studies regarding alterations in gene expression in HSV-1 latently infected mouse trigeminal ganglia (TGs) following treatment with immunosuppressants and hyperthermia. Uninfected and HSV-1 latently infected mice were treated with immunosuppressants or heat stressed (43 degrees C for 10 minutes). In the immunosuppressant study, 4 groups of animals were examined: (1) uninfected, not treated; (2) uninfected, drug-treated; (3) latently infected, not treated; and (4) latently infected, drug-treated. In the hyperthermia study, TG from 6 groups of mice were studied: (1) uninfected, not stressed; (2) uninfected, heat-stressed; killed at 6 hours after hyperthermia; (3) uninfected, heat-stressed, killed at 24 hours after hyperthermia; (4) latently infected, not stressed; (5) latently infected, heat-stressed, killed at 6 hours after hyperthermia; and (6) latently infected, heat-stressed, killed at 24 hours after hyperthermia. PolyA mRNA from the TGs of each group was reverse-transcribed, labeled with P, incubated on a gene array membrane, and analyzed by phosphorimaging. As a comparison and to confirm microarray results, semiquantitative RT-PCR for selected genes was also performed. The immunosuppressive drugs significantly increased expression of two genes--calpactin 1 light chain and guanine nucleotide-binding protein alpha stimulating activity polypeptide (GNAS)--in the ganglia of uninfected mice compared with untreated, uninfected mice. Ten genes were shown to be significantly increased in the latent TGs from mice treated with the immunosuppressants compared with latently infected untreated mice. These genes were prostaglandin E2 receptor EP4 subtype (PTGER4), insulin promoter factor 1 (IPF1), glutathione S-transferase mu2, cyclin D2, peripherin, plasma glutathione peroxidase, methyl CpG-binding protein 2, retinal S-antigen, ErbB2 protooncogene, and GNAS. Eight genes were shown to be significantly decreased in the HSV-1 latent TGs treated with the

  20. Causal Inference in Latent Class Analysis

    PubMed Central

    Lanza, Stephanie T.; Coffman, Donna L.; Xu, Shu

    2014-01-01

    The integration of modern methods for causal inference with latent class analysis (LCA) allows social, behavioral, and health researchers to address important questions about the determinants of latent class membership. In the present article, two propensity score techniques, matching and inverse propensity weighting, are demonstrated for conducting causal inference in LCA. The different causal questions that can be addressed with these techniques are carefully delineated. An empirical analysis based on data from the National Longitudinal Survey of Youth 1979 is presented, where college enrollment is examined as the exposure (i.e., treatment) variable and its causal effect on adult substance use latent class membership is estimated. A step-by-step procedure for conducting causal inference in LCA, including multiple imputation of missing data on the confounders, exposure variable, and multivariate outcome, is included. Sample syntax for carrying out the analysis using SAS and R is given in an appendix. PMID:25419097

  1. CD4 and CD8 T cell responses to tumour-associated Epstein-Barr virus antigens in nasopharyngeal carcinoma patients.

    PubMed

    Lin, Xiaorong; Gudgeon, Nancy H; Hui, Edwin P; Jia, Hui; Qun, Xue; Taylor, Graham S; Barnardo, Martin C N M; Lin, C Kit; Rickinson, Alan B; Chan, Anthony T C

    2008-07-01

    Nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-associated tumour common in Southern Chinese populations, is a potentially important target for T cell-based immunotherapy. The tumour cells are HLA class I- and II-positive and express a limited subset of EBV latent proteins, namely the nuclear antigen EBNA1 and the latent membrane proteins LMP2 and (in some cases) LMP1. To ask whether the tumour develops in the presence of a potentially protective host response or in its absence, we set out to determine the prevailing levels of CD4+ and CD8+ T cell memory to these proteins in NPC patients at tumour diagnosis. We first screened healthy Chinese donors against Chinese strain EBNA1, LMP1 and LMP2 sequences in Elispot assays of interferon-gamma release and identified the immunodominant CD4+ and CD8+ epitope peptides presented by common Chinese HLA alleles. Then, comparing 60 patients with >70 healthy controls on peptide epitope mini-panels, we found that T cell memory to CD4 epitopes in all three proteins was unimpaired in the blood of patients at diagnosis. In most cases NPC patients also showed detectable responses to CD8 epitopes relevant to their HLA type, the one consistent exception being the absence in patients of a B*4001-restricted response to LMP2. We infer that NPC arises in patients whose prevailing levels of T cell memory to tumour-associated EBV proteins is largely intact; the therapeutic goal must therefore be to re-direct the existing memory repertoire more effectively against antigen-expressing tumour cells.

  2. Role for a region of helically unstable DNA within the Epstein-Barr virus latent cycle origin of DNA replication oriP in origin function

    SciTech Connect

    Polonskaya, Zhanna; Benham, Craig J.; Hearing, Janet . E-mail: jhearing@ms.cc.sunysb.edu

    2004-10-25

    The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component of oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication.

  3. Antigen injection (image)

    MedlinePlus

    Leprosy is caused by the organism Mycobacterium leprae . The leprosy test involves injection of an antigen just under ... if your body has a current or recent leprosy infection. The injection site is labeled and examined ...

  4. Tensor Decompositions for Learning Latent Variable Models

    DTIC Science & Technology

    2012-12-08

    of a tensor, 2011. arXiv:1004.4953. [CSC+12] S. B. Cohen, K. Stratos, M. Collins, D. P. Foster, and L. Ungar . Spectral learning of latent-variable...12] P. S. Dhillon, J. Rodu, M. Collins, D. P. Foster, and L. H. Ungar . Spectral dependency parsing with latent variables. In EMNLP-CoNLL, 2012. [DS07...Foster, J. Rodu, and L. H. Ungar . Spectral dimensionality reduction for HMMs, 2012. arXiv:1203.6130. [GvL96] G. H. Golub and C. F. van Loan. Matrix

  5. Intractable diarrhoea of infancy and latent otomastoiditis.

    PubMed Central

    Salazar de Sousa, J; da Silva, A; da Costa Ribeiro, V

    1980-01-01

    In 16 infants with intractable diarrhoea, latent otomastoiditis was found in 9 (3 at necropsy and 6 at myringotomy-antrotomy). In 5 of the 6 operated group, surgery was followed by a striking cessation of the diarrhoea and with weight gain. It is concluded that (1) latent otomastoiditis may be a perpetuating factor in intractable diarrhoea; (2) myringotomy-antrotomy should be considered if other forms of treatment have failed, and especially if there is leucocytosis; (3) mastoiditis with diffuse osteitis seems to be associated with a poor prognosis. PMID:7458392

  6. Disulfiram reactivates latent HIV-1 expression through depletion of the phosphatase and tensin homolog.

    PubMed

    Doyon, Geneviève; Zerbato, Jennifer; Mellors, John W; Sluis-Cremer, Nicolas

    2013-01-14

    Disulfiram (DSF), an inhibitor of acetaldehyde dehydrogenase that is used for the treatment of alcoholism, was shown to reactivate latent HIV-1 expression in a primary cell model of virus latency and is currently being assessed in a clinical trial for its potential to deplete the latent HIV-1 reservoir in patients on combination antiretroviral therapy. The mechanism by which DSF reactivates latent HIV-1 expression, however, is not known and was the focus of this study. The impact of DSF treatment on HIV-1 latency was assessed in the ACH2, J89GFP and U1 cell line models of HIV-1 latency and in resting CD4 T cells isolated from HIV-negative donors. DSF reactivated latent HIV-1 expression in the U1 cell line, but not in the J89GFP or ACH2 cell lines. Interestingly, we found that DSF significantly reduced phosphatase and tensin homolog (PTEN) protein levels in U1 cells and in resting CD4 T cells from HIV-negative donors. Decreased PTEN resulted in increased phosphorylation of protein kinase B (Akt) and activation of the Akt signaling pathway. Consistent with these finding, pharmacological inhibitors of Akt and nuclear factor-kappaB (NF-κB) block the latent HIV-1-reactivating activity of DSF. Furthermore, we show that HIV-1 expression in the U1 cell line could be activated by a small molecule inhibitor of PTEN or by siRNA knockdown of PTEN expression. Neither the J89GFP nor ACH2 cells express PTEN, explaining the lack of DSF effect on HIV-1 expression in both these cell lines. DSF reactivates latent HIV-1 expression via the Akt signaling pathway through depletion of PTEN.

  7. Association of autophagy-related IRGM polymorphisms with latent versus active tuberculosis infection in a Chinese population.

    PubMed

    Lu, Yanjun; Li, Qian; Peng, Jing; Zhu, Yaowu; Wang, Feng; Wang, Chunyu; Wang, Xiong

    2016-03-01

    The autophagy-related immunity-related GTPase family M protein, IRGM, plays an important role in the defense against tuberculosis (TB) infection. IRGM polymorphisms are associated with TB infection susceptibility, and recent studies demonstrate host genetic differences between active and latent TB. Here, we investigated the association between IRGM polymorphisms and TB infection type in a Chinese population. We recruited 268 and 321 patients with confirmed or latent TB, respectively, and 475 TB-free healthy controls. Three single nucleotide polymorphisms, rs10065172, rs10051924, and rs13361189 within IRGM were genotyped using TaqMan-based assays. Interferon-gamma release levels were tested by T-SPOT. rs10065172 (P = 0.024, OR 0.67 (95% CI 0.48-0.95)), rs10051924 (P = 0.01, OR 0.64 (95% CI 0.46-0.90)), and rs13361189 (P = 0.055, OR 0.72 (95% CI 0.51-1.01)) were associated with a protective role against latent TB progression. Haplotype analysis showed that TCC was protective for latent TB (P = 0.022, OR 0.74 (95% CI 0.57-0.96)) whereas TTC conferred a higher risk of active TB. Additionally, patients with the rs10065172 TT genotype had a higher response to TB specific antigens. Thus, IRGM polymorphism differences between latent and active TB suggests that genetic differences in autophagy might partly affect host TB infection status.

  8. Comparative Study of Kaposi's Sarcoma-Associated Herpesvirus Serological Assays Using Clinically and Serologically Defined Reference Standards and Latent Class Analysis▿

    PubMed Central

    Nascimento, Maria Claudia; de Souza, Vanda Akico; Sumita, Laura Masami; Freire, Wilton; Munoz, Fernando; Kim, Joseph; Pannuti, Claudio S.; Mayaud, Philippe

    2007-01-01

    Accurate determination of infection with Kaposi's sarcoma-associated herpesvirus (KSHV) has been hindered by the lack of a “gold standard” for comparison of serological assays used to estimate KSHV prevalence in serosurveys conducted in different settings. We have evaluated the performance of five in-house (developed at University College London [UCL], United Kingdom, and at the virology laboratory of the Instituto de Medicine Tropical [IMT] in Sao Paulo, Brazil) and two commercial (ABI and DIAVIR) serological assays to detect antibodies to latency-associated nuclear antigen (LANA) and to lytic KSHV antigens. We used a variety of serum samples assembled to represent populations likely to be at high, intermediate, and low risk of KSHV infection in Brazil. Composite reference standard panels were prepared based on clinical and serological parameters, against which assay performances were assessed using conventional Bayesian statistics and latent class analysis (LCA). Against the clinical reference standard, in-house immunofluorescence assays to detect anti-LANA antibodies (IFA-LANA) produced at UCL and IMT had similar performances, with sensitivities of 61% (95% confidence interval [CI], 48% to 74%) and 72% (95% CI, 58% to 83%) and specificities of 99% (95% CI, 94% to 100%) and 100% (95% CI, 96% to 100%), respectively, and only the IMT IFA-LANA was included in LCA, together with the IMT IFA-lytic and four enzyme-linked immunosorbent assays (ELISAs). The LCA indicated that the IMT whole-virus ELISA performed best (sensitivity, 87% [95% CI, 81% to 91%]; and specificity, 100% [95% CI, 98% to 100%]), confirming the results obtained with the conventional statistical approach. Commercially available ELISA-based tests yielded the lowest specificities using a spectrum of serum samples. The evaluation of KSHV serological assays is warranted before planning serosurveys in various settings. PMID:17182752

  9. Comparative study of Kaposi's sarcoma-associated herpesvirus serological assays using clinically and serologically defined reference standards and latent class analysis.

    PubMed

    Nascimento, Maria Claudia; de Souza, Vanda Akico; Sumita, Laura Masami; Freire, Wilton; Munoz, Fernando; Kim, Joseph; Pannuti, Claudio S; Mayaud, Philippe

    2007-03-01

    Accurate determination of infection with Kaposi's sarcoma-associated herpesvirus (KSHV) has been hindered by the lack of a "gold standard" for comparison of serological assays used to estimate KSHV prevalence in serosurveys conducted in different settings. We have evaluated the performance of five in-house (developed at University College London [UCL], United Kingdom, and at the virology laboratory of the Instituto de Medicine Tropical [IMT] in Sao Paulo, Brazil) and two commercial (ABI and DIAVIR) serological assays to detect antibodies to latency-associated nuclear antigen (LANA) and to lytic KSHV antigens. We used a variety of serum samples assembled to represent populations likely to be at high, intermediate, and low risk of KSHV infection in Brazil. Composite reference standard panels were prepared based on clinical and serological parameters, against which assay performances were assessed using conventional Bayesian statistics and latent class analysis (LCA). Against the clinical reference standard, in-house immunofluorescence assays to detect anti-LANA antibodies (IFA-LANA) produced at UCL and IMT had similar performances, with sensitivities of 61% (95% confidence interval [CI], 48% to 74%) and 72% (95% CI, 58% to 83%) and specificities of 99% (95% CI, 94% to 100%) and 100% (95% CI, 96% to 100%), respectively, and only the IMT IFA-LANA was included in LCA, together with the IMT IFA-lytic and four enzyme-linked immunosorbent assays (ELISAs). The LCA indicated that the IMT whole-virus ELISA performed best (sensitivity, 87% [95% CI, 81% to 91%]; and specificity, 100% [95% CI, 98% to 100%]), confirming the results obtained with the conventional statistical approach. Commercially available ELISA-based tests yielded the lowest specificities using a spectrum of serum samples. The evaluation of KSHV serological assays is warranted before planning serosurveys in various settings.

  10. Distinct Effector Memory CD4+ T Cell Signatures in Latent Mycobacterium tuberculosis Infection, BCG Vaccination and Clinically Resolved Tuberculosis

    PubMed Central

    Adekambi, Toidi; Ibegbu, Chris C.; Kalokhe, Ameeta S.; Yu, Tianwei; Ray, Susan M.; Rengarajan, Jyothi

    2012-01-01

    Two billion people worldwide are estimated to be latently infected with Mycobacterium tuberculosis (Mtb) and are at risk for developing active tuberculosis since Mtb can reactivate to cause TB disease in immune-compromised hosts. Individuals with latent Mtb infection (LTBI) and BCG-vaccinated individuals who are uninfected with Mtb, harbor antigen-specific memory CD4+ T cells. However, the differences between long-lived memory CD4+ T cells induced by latent Mtb infection (LTBI) versus BCG vaccination are unclear. In this study, we characterized the immune phenotype and functionality of antigen-specific memory CD4+ T cells in healthy BCG-vaccinated individuals who were either infected (LTBI) or uninfected (BCG) with Mtb. Individuals were classified into LTBI and BCG groups based on IFN-γ ELISPOT using cell wall antigens and ESAT-6/CFP-10 peptides. We show that LTBI individuals harbored high frequencies of late-stage differentiated (CD45RA−CD27−) antigen-specific effector memory CD4+ T cells that expressed PD-1. In contrast, BCG individuals had primarily early-stage (CD45RA−CD27+) cells with low PD-1 expression. CD27+ and CD27− as well as PD-1+ and PD-1− antigen-specific subsets were polyfunctional, suggesting that loss of CD27 expression and up-regulation of PD-1 did not compromise their capacity to produce IFN-γ, TNF-α and IL-2. PD-1 was preferentially expressed on CD27− antigen-specific CD4+ T cells, indicating that PD-1 is associated with the stage of differentiation. Using statistical models, we determined that CD27 and PD-1 predicted LTBI versus BCG status in healthy individuals and distinguished LTBI individuals from those who had clinically resolved Mtb infection after anti-tuberculosis treatment. This study shows that CD4+ memory responses induced by latent Mtb infection, BCG vaccination and clinically resolved Mtb infection are immunologically distinct. Our data suggest that differentiation into CD27−PD-1+ subsets in LTBI is driven by Mtb

  11. Binding of antibodies to the extractable nuclear antigens SS-A/Ro and SS-B/La is induced on the surface of human keratinocytes by ultraviolet light (UVL): Implications for the pathogenesis of photosensitive cutaneous lupus

    SciTech Connect

    Furukawa, F.; Kashihara-Sawami, M.; Lyons, M.B.; Norris, D.A. )

    1990-01-01

    Autoantibodies to the non-histone nucleoprotein antigens SS-A/Ro, SS-B/La, and RNP are highly associated with photosensitive cutaneous lupus erythematosus (LE). In order to better understand the potential mechanisms of ultraviolet (UV) light on photosensitivity in patients with cutaneous LE, we designed immunopathologic in vitro and in vivo experiments to evaluate the effects of UV on the binding of such autoantibodies to the surface of human keratinocytes, one major target of immunologic damage in photosensitive LE. Short-term 2% paraformaldehyde fixation of suspensions of cultured human keratinocytes previously incubated with monospecific antiserum probes enabled the detection of ENA expression on the cell surface by flow-cytometry analysis. UVB light (280-320 nm) induced the binding of monospecific antibody probes for SS-A/Ro and SS-B/La on keratinocytes in a dose-dependent pattern with maximal induction observed at the dose of 200 mJ/cm2 UVB. Binding of SS-A/Ro, SS-B/La, and RNP antibody was augmented strongly, but binding of anti-Sm was very weak. In contrast, UVA (320-400 nm) light had no effect on the induction of binding of these antibody probes. Identical results were seen by standard immunofluorescence techniques. Hydroxyurea-treated keratinocytes showed similar induction of those antigens by UVB irradiation, which suggested that ENA expression on cultured keratinocytes by UVB were cell-cycle independent. Tunicamycin, an inhibitor of glycosylation of proteins, reduced UVB light effect on the SS-A/Ro and SS-B/La antigen's expression. These in vitro FACS analyses revealed that ENA augmentation on the keratinocyte cell surface was dose dependent, UVB dependent, glycosylation dependent, and cell-cycle independent. In vivo ENA augmentation on the keratinocyte surface was examined in suction blister epidermal roofs.

  12. Generalized Structured Component Analysis with Latent Interactions

    ERIC Educational Resources Information Center

    Hwang, Heungsun; Ho, Moon-Ho Ringo; Lee, Jonathan

    2010-01-01

    Generalized structured component analysis (GSCA) is a component-based approach to structural equation modeling. In practice, researchers may often be interested in examining the interaction effects of latent variables. However, GSCA has been geared only for the specification and testing of the main effects of variables. Thus, an extension of GSCA…

  13. Thermally Stable, Latent Olefin Metathesis Catalysts

    PubMed Central

    Thomas, Renee M.; Fedorov, Alexey; Keitz, Benjamin K.

    2011-01-01

    Highly thermally stable N-aryl,N-alkyl N-heterocyclic carbene (NHC) ruthenium catalysts were designed and synthesized for latent olefin metathesis. These catalysts showed excellent latent behavior toward metathesis reactions, whereby the complexes were inactive at ambient temperature and initiated at elevated temperatures, a challenging property to achieve with second generation catalysts. A sterically hindered N-tert-butyl substituent on the NHC ligand of the ruthenium complex was found to induce latent behavior toward cross-metathesis reactions, and exchange of the chloride ligands for iodide ligands was necessary to attain latent behavior during ring-opening metathesis polymerization (ROMP). Iodide-based catalysts showed no reactivity toward ROMP of norbornene-derived monomers at 25 °C, and upon heating to 85 °C gave complete conversion of monomer to polymer in less than 2 hours. All of the complexes were very stable to air, moisture, and elevated temperatures up to at least 90 °C, and exhibited a long catalyst lifetime in solution at elevated temperatures. PMID:22282652

  14. Dish-mounted latent heat buffer storage

    NASA Technical Reports Server (NTRS)

    Manvi, R.

    1981-01-01

    Dish-mounted latent heat storage subsystems for Rankine, Brayton, and Stirling engines operating at 427 C, 816 C, and 816 C respectively are discussed. Storage requirements definition, conceptual design, media stability and compatibility tests, and thermal performance analyses are considered.

  15. Immune Function and Reactivation of Latent Viruses

    NASA Technical Reports Server (NTRS)

    Butel, Janet S.

    1999-01-01

    A major concern associated with long-duration space flight is the possibility of infectious diseases posing an unacceptable medical risk to crew members. One major hypothesis addressed in this project is that space flight will cause alterations in the immune system that will allow latent viruses that are endogenous in the human population to reactivate and shed to higher levels than normal, which may affect the health of crew members. The second major hypothesis being examined is that the effects of space flight will alter the mucosal immune system, the first line of defense against many microbial infections, including herpesviruses, polyomaviruses, and gastroenteritis viruses, rendering crew members more susceptible to virus infections across the mucosa. We are focusing the virus studies on the human herpesviruses and polyomaviruses, important pathogens known to establish latent infections in most of the human population. Both primary infection and reactivation from latent infection with these groups of viruses (especially certain herpesviruses) can cause a variety of illnesses that result in morbidity and, occasionally, mortality. Both herpesviruses and polyomaviruses have been associated with human cancer, as well. Effective vaccines exist for only one of the eight known human herpesviruses and available antivirals are of limited use. Whereas normal individuals display minimal consequences from latent viral infections, events which alter immune function (such as immunosuppressive therapy following solid organ transplantation) are known to increase the risk of complications as a result of viral reactivations.

  16. Forensic Chemistry: The Revelation of Latent Fingerprints

    ERIC Educational Resources Information Center

    Friesen, J. Brent

    2015-01-01

    The visualization of latent fingerprints often involves the use of a chemical substance that creates a contrast between the fingerprint residues and the surface on which the print was deposited. The chemical-aided visualization techniques can be divided into two main categories: those that chemically react with the fingerprint residue and those…

  17. Immune Function and Reactivation of Latent Viruses

    NASA Technical Reports Server (NTRS)

    Butel, Janet S.

    1999-01-01

    A major concern associated with long-duration space flight is the possibility of infectious diseases posing an unacceptable medical risk to crew members. One major hypothesis addressed in this project is that space flight will cause alterations in the immune system that will allow latent viruses that are endogenous in the human population to reactivate and shed to higher levels than normal, which may affect the health of crew members. The second major hypothesis being examined is that the effects of space flight will alter the mucosal immune system, the first line of defense against many microbial infections, including herpesviruses, polyomaviruses, and gastroenteritis viruses, rendering crew members more susceptible to virus infections across the mucosa. We are focusing the virus studies on the human herpesviruses and polyomaviruses, important pathogens known to establish latent infections in most of the human population. Both primary infection and reactivation from latent infection with these groups of viruses (especially certain herpesviruses) can cause a variety of illnesses that result in morbidity and, occasionally, mortality. Both herpesviruses and polyomaviruses have been associated with human cancer, as well. Effective vaccines exist for only one of the eight known human herpesviruses and available antivirals are of limited use. Whereas normal individuals display minimal consequences from latent viral infections, events which alter immune function (such as immunosuppressive therapy following solid organ transplantation) are known to increase the risk of complications as a result of viral reactivations.

  18. Extended Generalized Linear Latent and Mixed Model

    ERIC Educational Resources Information Center

    Segawa, Eisuke; Emery, Sherry; Curry, Susan J.

    2008-01-01

    The generalized linear latent and mixed modeling (GLLAMM framework) includes many models such as hierarchical and structural equation models. However, GLLAMM cannot currently accommodate some models because it does not allow some parameters to be random. GLLAMM is extended to overcome the limitation by adding a submodel that specifies a…

  19. An Introduction to Latent Semantic Analysis.

    ERIC Educational Resources Information Center

    Landauer, Thomas K; Foltz, Peter W.; Laham, Darrell

    1998-01-01

    Offers an introduction to the theory and implementation of Latent Semantic Analysis (LSA), a theory and method for extracting and representing the contextual-usage meaning of words by statistical computations applied to a large corpus of text. Gives an overview of applications and modeling of human knowledge to which LSA has been applied. (SR)

  20. Essay Assessment with Latent Semantic Analysis

    ERIC Educational Resources Information Center

    Miller, Tristan

    2003-01-01

    Latent semantic analysis (LSA) is an automated, statistical technique for comparing the semantic similarity of words or documents. In this article, I examine the application of LSA to automated essay scoring. I compare LSA methods to earlier statistical methods for assessing essay quality, and critically review contemporary essay-scoring systems…

  1. Latent Functions of Enfranchising the Disenfranchised Griever.

    ERIC Educational Resources Information Center

    Kamerman, Jack

    1993-01-01

    Notes increasing prominence of grief felt by people with no socially legitimate right to grieve which stems from extrafamilial relationships that constitute one's personal life. Explores latent functions of enfranchising the disenfranchised griever as specific case of more general problem: allocation of sympathy and support in any person's dying.…

  2. CICATRIZATION OF WOUNDS : XI. LATENT PERIOD.

    PubMed

    Carrel, A; du Noüy, P L

    1921-09-30

    1. The latent period of cicatrization varies generally from 5 to 7 days. 2. It stops abruptly and contraction starts with its maximum velocity. 3. The formula of du Noüy applies to the beginning of the contraction period as well as to the subsequent periods.

  3. Detection of latent prints by Raman imaging

    DOEpatents

    Lewis, Linda Anne [Andersonville, TN; Connatser, Raynella Magdalene [Knoxville, TN; Lewis, Sr., Samuel Arthur

    2011-01-11

    The present invention relates to a method for detecting a print on a surface, the method comprising: (a) contacting the print with a Raman surface-enhancing agent to produce a Raman-enhanced print; and (b) detecting the Raman-enhanced print using a Raman spectroscopic method. The invention is particularly directed to the imaging of latent fingerprints.

  4. Forensic Chemistry: The Revelation of Latent Fingerprints

    ERIC Educational Resources Information Center

    Friesen, J. Brent

    2015-01-01

    The visualization of latent fingerprints often involves the use of a chemical substance that creates a contrast between the fingerprint residues and the surface on which the print was deposited. The chemical-aided visualization techniques can be divided into two main categories: those that chemically react with the fingerprint residue and those…

  5. Dish-mounted latent heat buffer storage

    NASA Technical Reports Server (NTRS)

    Manvi, R.

    1981-01-01

    Dish-mounted latent heat storage subsystems for Rankine, Brayton, and Stirling engines operating at 427 C, 816 C, and 816 C respectively are discussed. Storage requirements definition, conceptual design, media stability and compatibility tests, and thermal performance analyses are considered.

  6. Correcting for Nonresponse in Latent Class Analysis.

    ERIC Educational Resources Information Center

    Kolb, Rita R.; Dayton, C. Mitchell

    1996-01-01

    Monte Carlo methods were used to evaluate an EM algorithm used for the correction of missing data in latent class analysis. Findings regarding bias in parameter estimates suggest practical limits for the utility of the EM algorithm in terms of sample size and nonresponse rate. (SLD)

  7. Latent TGF-β-binding proteins

    PubMed Central

    Robertson, Ian B.; Horiguchi, Masahito; Zilberberg, Lior; Dabovic, Branka; Hadjiolova, Krassimira; Rifkin, Daniel B.

    2016-01-01

    The LTBPs (or latent transforming growth factor β binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, -2, -3, and -4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here. The LTBPs were first identified as forming latent complexes with TGFβ by covalently binding the TGFβ propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFβ precursor in the trans golgi network but LAP and TGFβ remain strongly bound through non-covalent interactions. LAP, TGFβ, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFβ latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFβ activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFβ regulation, TGFβ-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly. PMID:25960419

  8. Generalized Structured Component Analysis with Latent Interactions

    ERIC Educational Resources Information Center

    Hwang, Heungsun; Ho, Moon-Ho Ringo; Lee, Jonathan

    2010-01-01

    Generalized structured component analysis (GSCA) is a component-based approach to structural equation modeling. In practice, researchers may often be interested in examining the interaction effects of latent variables. However, GSCA has been geared only for the specification and testing of the main effects of variables. Thus, an extension of GSCA…

  9. Residual Structures in Latent Growth Curve Modeling

    ERIC Educational Resources Information Center

    Grimm, Kevin J.; Widaman, Keith F.

    2010-01-01

    Several alternatives are available for specifying the residual structure in latent growth curve modeling. Two specifications involve uncorrelated residuals and represent the most commonly used residual structures. The first, building on repeated measures analysis of variance and common specifications in multilevel models, forces residual variances…

  10. Generation of specific cytotoxic T cells with a fragment of the Epstein-Barr virus-encoded p63/latent membrane protein.

    PubMed Central

    Thorley-Lawson, D A; Israelsohn, E S

    1987-01-01

    Human B lymphocytes, transformed by the herpesvirus Epstein-Barr virus, are known to express a characteristic antigen(s) recognized by the cellular immune response. This structure has been termed lymphocyte-determined membrane antigen. Because of the significance of this structure in controlling Epstein-Barr virus infection in vivo, the molecular nature of lymphocyte-determined membrane antigen has been long sought. In this paper, we show that a sequence of 10 amino acids (residues 43-53) from the Epstein-Barr virus-encoded membrane protein p63/latent membrane protein can induce Epstein-Barr virus-specific cytotoxic T cells and, therefore, bears at least one of the lymphocyte-determined membrane antigenic determinants. PMID:3037547

  11. Latent mnemonic strengths are latent: a comment on Mickes, Wixted, and Wais (2007).

    PubMed

    Rouder, Jeffrey N; Pratte, Michael S; Morey, Richard D

    2010-06-01

    Mickes, Wixted, and Wais (2007) proposed a simple test of latent strength variability in recognition memory. They asked participants to rate their confidence using either a 20-point or a 99-point strength scale and plotted distributions of the resulting ratings. They found 25% more variability in ratings for studied than for new items, which they interpreted as providing evidence that latent mnemonic strength distributions are 25% more variable for studied than for new items. We show here that this conclusion is critically dependent on assumptions--so much so that these assumptions determine the conclusions. In fact, opposite conclusions, such that study does not affect the variability of latent strength, may be reached by making different but equally plausible assumptions. Because all measurements of mnemonic strength variability are critically dependent on untestable assumptions, all are arbitrary. Hence, there is no principled method for assessing the relative variability of latent mnemonic strength distributions.

  12. Reactions of latent prints exposed to blood.

    PubMed

    Praska, Nicole; Langenburg, Glenn

    2013-01-10

    We explored whether an undeveloped latent print (fingermark) exposed to blood and later developed by enhancement with blood reagents such as amido black (AB) or leucocrystal violet (LCV) could appear as a genuine blood mark. We examined three different experimental conditions. In Experiment I, fingermark residue only was tested, as a control to confirm that fingermark residue alone does not react with the blood reagents AB and LCV. Experiment II investigated whether latent fingermarks exposed to blood dilutions could be treated with AB or LCV and subsequently appear as a genuine blood mark enhanced with AB or LCV. Experiment III tested whether latent fingermarks exposed to whole blood could be processed with AB or LCV and subsequently appear as a genuine blood mark enhanced with AB or LCV. The present study found that indeed, fingermark residue alone does not react with the blood reagents AB and LCV. In Experiment II, an interaction occurred between the fingermark residue and the diluted blood that caused the ridges to appear a red color. In the present study, this interaction is called a faux blood mark. While the faux blood mark phenomenon occurred most often following exposure to diluted blood, it did not occur consistently, and a predictable pattern could not be established. However, the reaction occurred more frequently following extended fingermark residue drying times. Faux blood marks are distinguishable from genuine blood marks prior to enhancement with blood reagents. Following treatment with blood reagents, it became increasingly difficult to determine whether the enhanced mark was a genuine blood print or a latent fingermark exposed to diluted blood. Latent fingermarks exposed to whole blood often resulted in a void prior to enhancement, but following treatment with blood reagents, were difficult to distinguish from a genuine blood mark enhanced with blood reagents.

  13. AntigenMap 3D: an online antigenic cartography resource

    PubMed Central

    Barnett, J. Lamar; Yang, Jialiang; Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2012-01-01

    Summary: Antigenic cartography is a useful technique to visualize and minimize errors in immunological data by projecting antigens to 2D or 3D cartography. However, a 2D cartography may not be sufficient to capture the antigenic relationship from high-dimensional immunological data. AntigenMap 3D presents an online, interactive, and robust 3D antigenic cartography construction and visualization resource. AntigenMap 3D can be applied to identify antigenic variants and vaccine strain candidates for pathogens with rapid antigenic variations, such as influenza A virus. Availability and implementation: http://sysbio.cvm.msstate.edu/AntigenMap3D Contact: wan@cvm.msstate.edu; wanhenry@yahoo.com PMID:22399675

  14. The latent cytomegalovirus decreases telomere length by microcompetition

    PubMed Central

    Javaherian, Adrian

    2015-01-01

    Reduced telomere length has been associated with aging and age-related diseases. Latent infection with the Cytomegalovirus (CMV) induces telomere shortening in the infected cells. Latent CMV infection may cause reduced telomere length via GABP transcription factor deficiency, according to the Microcompetition Theory. Microcompetition and viral-induced transcription factor deficiency is important since most people harbor a latent viral infection.

  15. Stochastic Approximation Methods for Latent Regression Item Response Models

    ERIC Educational Resources Information Center

    von Davier, Matthias; Sinharay, Sandip

    2010-01-01

    This article presents an application of a stochastic approximation expectation maximization (EM) algorithm using a Metropolis-Hastings (MH) sampler to estimate the parameters of an item response latent regression model. Latent regression item response models are extensions of item response theory (IRT) to a latent variable model with covariates…

  16. Using Latent Class Analysis To Set Academic Performance Standards.

    ERIC Educational Resources Information Center

    Brown, Richard S.

    The use of latent class analysis for establishing student performance standards was studied. Latent class analysis (LCA) is an established procedure for investigating the latent structure of a set of data. LCA presumes that groups, classes, or respondents differ qualitatively from one another, and that these differences account for all of the…

  17. Modeling Interaction Effects in Latent Growth Curve Models.

    ERIC Educational Resources Information Center

    Li, Fuzhong; Duncan, Terry E.; Acock, Alan

    2000-01-01

    Presents an extension of the method of estimating interaction effects among latent variables to latent growth curve models developed by K. Joreskog and F. Yang (1996). Illustrates the procedure and discusses results in terms of practical and statistical problems associated with interaction analyses in latent curve models and structural equation…

  18. Skills Diagnosis Using IRT-Based Continuous Latent Trait Models

    ERIC Educational Resources Information Center

    Stout, William

    2007-01-01

    This article summarizes the continuous latent trait IRT approach to skills diagnosis as particularized by a representative variety of continuous latent trait models using item response functions (IRFs). First, several basic IRT-based continuous latent trait approaches are presented in some detail. Then a brief summary of estimation, model…

  19. A Latent Variable Approach to the Simple View of Reading

    ERIC Educational Resources Information Center

    Kershaw, Sarah; Schatschneider, Chris

    2012-01-01

    The present study utilized a latent variable modeling approach to examine the Simple View of Reading in a sample of students from 3rd, 7th, and 10th grades (N = 215, 188, and 180, respectively). Latent interaction modeling and other latent variable models were employed to investigate (a) the functional form of the relationship between decoding and…

  20. Bayesian Semiparametric Structural Equation Models with Latent Variables

    ERIC Educational Resources Information Center

    Yang, Mingan; Dunson, David B.

    2010-01-01

    Structural equation models (SEMs) with latent variables are widely useful for sparse covariance structure modeling and for inferring relationships among latent variables. Bayesian SEMs are appealing in allowing for the incorporation of prior information and in providing exact posterior distributions of unknowns, including the latent variables. In…

  1. Fingerprint Minutiae from Latent and Matching Tenprint Images

    National Institute of Standards and Technology Data Gateway

    NIST Fingerprint Minutiae from Latent and Matching Tenprint Images (PC database for purchase)   NIST Special Database 27 contains latent fingerprints from crime scenes and their matching rolled fingerprint mates. This database can be used to develop and test new fingerprint algorithms, test commercial and research AFIS systems, train latent examiners, and promote the ANSI/NIST file format standard.

  2. Stochastic Approximation Methods for Latent Regression Item Response Models

    ERIC Educational Resources Information Center

    von Davier, Matthias; Sinharay, Sandip

    2010-01-01

    This article presents an application of a stochastic approximation expectation maximization (EM) algorithm using a Metropolis-Hastings (MH) sampler to estimate the parameters of an item response latent regression model. Latent regression item response models are extensions of item response theory (IRT) to a latent variable model with covariates…

  3. A Note on Cluster Effects in Latent Class Analysis

    ERIC Educational Resources Information Center

    Kaplan, David; Keller, Bryan

    2011-01-01

    This article examines the effects of clustering in latent class analysis. A comprehensive simulation study is conducted, which begins by specifying a true multilevel latent class model with varying within- and between-cluster sample sizes, varying latent class proportions, and varying intraclass correlations. These models are then estimated under…

  4. Bayesian Semiparametric Structural Equation Models with Latent Variables

    ERIC Educational Resources Information Center

    Yang, Mingan; Dunson, David B.

    2010-01-01

    Structural equation models (SEMs) with latent variables are widely useful for sparse covariance structure modeling and for inferring relationships among latent variables. Bayesian SEMs are appealing in allowing for the incorporation of prior information and in providing exact posterior distributions of unknowns, including the latent variables. In…

  5. A General Approach to Defining Latent Growth Components

    ERIC Educational Resources Information Center

    Mayer, Axel; Steyer, Rolf; Mueller, Horst

    2012-01-01

    We present a 3-step approach to defining latent growth components. In the first step, a measurement model with at least 2 indicators for each time point is formulated to identify measurement error variances and obtain latent variables that are purged from measurement error. In the second step, we use contrast matrices to define the latent growth…

  6. Modeling Interaction Effects in Latent Growth Curve Models.

    ERIC Educational Resources Information Center

    Li, Fuzhong; Duncan, Terry E.; Acock, Alan

    2000-01-01

    Presents an extension of the method of estimating interaction effects among latent variables to latent growth curve models developed by K. Joreskog and F. Yang (1996). Illustrates the procedure and discusses results in terms of practical and statistical problems associated with interaction analyses in latent curve models and structural equation…

  7. A General Approach to Defining Latent Growth Components

    ERIC Educational Resources Information Center

    Mayer, Axel; Steyer, Rolf; Mueller, Horst

    2012-01-01

    We present a 3-step approach to defining latent growth components. In the first step, a measurement model with at least 2 indicators for each time point is formulated to identify measurement error variances and obtain latent variables that are purged from measurement error. In the second step, we use contrast matrices to define the latent growth…

  8. Latent Mycobacterium tuberculosis Infection and Interferon-Gamma Release Assays.

    PubMed

    Pai, Madhukar; Behr, Marcel

    2016-10-01

    The identification of individuals with latent tuberculosis infection (LTBI) is useful for both fundamental understanding of the pathogenesis of disease and for clinical and public health interventions (i.e., to prevent progression to disease). Basic research suggests there is a pathogenetic continuum from exposure to infection to disease, and individuals may advance or reverse positions within the spectrum, depending on changes in the host immunity. Unfortunately, there is no diagnostic test that resolves the various stages within the spectrum of Mycobacterium tuberculosis infection. Two main immune-based approaches are currently used for identification of LTBI: the tuberculin skin test (TST) and the interferon-gamma release assay (IGRA). TST can use either the conventional purified protein derivative or more specific antigens. Extensive research suggests that both TST and IGRA represent indirect markers of M. tuberculosis exposure and indicates a cellular immune response to M. tuberculosis. The imperfect concordance between these two tests suggests that neither test is perfect, presumably due to both technical and biological reasons. Neither test can accurately differentiate between LTBI and active TB. Both IGRA and TST have low sensitivity in a variety of immunocompromised populations. Cohort studies have shown that both TST and IGRA have low predictive value for progression from infection to active TB. For fundamental applications, basic research is necessary to identify those at highest risk of disease with a positive TST and/or IGRA. For clinical applications, the identification of such biomarkers can help prioritize efforts to interrupt progression to disease through preventive therapy.

  9. Fine structure of A and M antigens from Brucella biovars.

    PubMed

    Meikle, P J; Perry, M B; Cherwonogrodzky, J W; Bundle, D R

    1989-09-01

    Brucella A and M epitopes were found on single O-polysaccharide chains of all biotype strains of this species. Lipopolysaccharides from the type and reference strains of five of the six Brucella species, B. abortus, B. melitensis, B. suis, B. canis, and B. neotomae, were extracted and purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in conjunction with silver staining and immunoblotting developed by monoclonal antibodies, showed bands characteristic of A, M, or mixed A and M antigens. The A antigen previously described as an exclusively alpha 1,2-linked homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranose was shown by 1H and 13C nuclear magnetic resonance spectroscopy to possess a fine structure consistent with the low-frequency occurrence of alpha 1, 3-linked 4,6-dideoxy-4-formamido-D-mannopyranose residues. This feature was previously attributed only to the M antigen, which is also a homopolymer of the same sugar. B. melitensis biotype 3 and B. suis biotype 4 lipopolysaccharides showed characteristics of mixed A and M antigens. Immunoabsorption of these O polysaccharides on a column of immobilized A-antigen-specific monoclonal antibody enriched polymer chains with A-antigen characteristics but did not eliminate M epitopes. Composite A- and M-antigen characteristics resulted from O polysaccharides in which the frequency of alpha 1,3 linkages, and hence, M-antigen characteristics, varied. All biotypes assigned as A+ M- expressed one or two alpha 1,3-linked residues per polysaccharide O chain. M antigens (M+ A-) also possessed a unique M epitope as well as a tetrasaccharide determinant common to A-antigen structures. B. canis and B. abortus 45/20, both rough strains, expressed low-molecular-weight A antigen.

  10. Antineutrophil antibodies associated with ulcerative colitis interact with the antigen(s) during the process of apoptosis

    PubMed Central

    Mallolas, J; Esteve, M; Rius, E; Cabre, E; Gassull, M

    2000-01-01

    BACKGROUND—Cell death by apoptosis seems to be an important mechanism for translocation to the cell surface of a variety of intracellular components capable of inducing autoantibody production.
AIMS—To identify the cellular location of antigen (Ag)-antineutrophil cytoplasmic antibodies (ANCA) in non-apoptotic human neutrophils, and to assess if ANCA associated with ulcerative colitis reacts with neutrophil antigen(s) during neutrophil apoptosis. The cellular distribution of Ag-ANCA in apoptotic neutrophils was also investigated.
METHODS—Sera from 18 ulcerative colitis patients known to be positive for perinuclear IgG-ANCA (titre ⩾1/320), as assessed by indirect immunofluorescence (IIF), were analysed by immunofluorescent confocal laser scanning microscopy. ANCA were identified with fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) in non-apoptotic and apoptotic neutrophils, respectively. Apoptotic and non-apoptotic DNA was labelled with FITC and propidium iodide, respectively. Cycloheximide was added to polymorphonuclear leucocyte culture to induce apoptosis.
RESULTS—Three patterns of scanning laser immunofluorescence microscopy in non-apoptotic neutrophils were observed with respect to cellular ulcerative colitis associated ANCA distribution: (1) diffuse nuclear localisation (16.7%); (2) nuclear localisation in the nuclear periphery (50%); and (3) mixed nuclear and cytoplasmic localisation (33.4%). In all sera ANCA fluorescence colocalised almost completely with apoptotic DNA, with persistence of a diffuse and intense fluorescence. No significant changes in ANCA titres were found in non-apoptotic neutrophils.
CONCLUSIONS—The antigen(s) of ANCA associated with ulcerative colitis seems to be localised in most cases in the neutrophil nucleus. The almost identical colocalisation of ANCA and apoptotic cleaved DNA suggests that intracellular DNA redistribution during neutrophil apoptosis may play a role in antigen

  11. Modeling Nonlinear Change via Latent Change and Latent Acceleration Frameworks: Examining Velocity and Acceleration of Growth Trajectories

    ERIC Educational Resources Information Center

    Grimm, Kevin; Zhang, Zhiyong; Hamagami, Fumiaki; Mazzocco, Michele

    2013-01-01

    We propose the use of the latent change and latent acceleration frameworks for modeling nonlinear growth in structural equation models. Moving to these frameworks allows for the direct identification of "rates of change" and "acceleration" in latent growth curves--information available indirectly through traditional growth…

  12. Modeling Nonlinear Change via Latent Change and Latent Acceleration Frameworks: Examining Velocity and Acceleration of Growth Trajectories

    ERIC Educational Resources Information Center

    Grimm, Kevin; Zhang, Zhiyong; Hamagami, Fumiaki; Mazzocco, Michele

    2013-01-01

    We propose the use of the latent change and latent acceleration frameworks for modeling nonlinear growth in structural equation models. Moving to these frameworks allows for the direct identification of "rates of change" and "acceleration" in latent growth curves--information available indirectly through traditional growth…

  13. Epstein–Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-κB pathway

    PubMed Central

    Gatto, Graziana; Rossi, Annalisa; Rossi, Daniela; Kroening, Sven; Bonatti, Stefano; Mallardo, Massimo

    2008-01-01

    The Epstein–Barr virus (EBV)-encoded latent membrane protein-1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, substantially contributes to EBV's oncogenic potential by activating nuclear factor-κB (NF-κB). miR-155 is an oncogenic miRNA critical for B-cell maturation and immunoglobulin production in response to antigen. We report that miR-155 expression is much higher in EBV-immortalized B cells than in EBV-negative B cells. LMP1, but not LMP2, up-regulated the expression of miR-155, when transfected in EBV-negative B cells. We analyzed two putative NF-κB binding sites in the miR-155 promoter; both sites recruited NF-κB complex, in nuclear extract from EBV-immortalized cells. The exogenous expression of LMP1, in EBV-negative background, is temporally correlated to induction of p65 with binding on both NF-κB sites and with miR-155 overexpression. The induction of p65 binding together with increased RNA polymerase II binding, confirms that LMP1-mediated activation of miR-155 occurs transcriptionally. In reporter assays, miR-155 promoter lacking NF-κB binding sites was no longer activated by LMP1 expression and an intact AP1 site is needed to attain maximum activation. Finally, we demonstrate that LMP1-mediated activation of miR-155 in an EBV-negative background correlates with reduction of protein PU.1, which is a possible miR target. PMID:18940871

  14. Engineering Chimeric Antigen Receptors

    PubMed Central

    Kulemzin, S. V.; Kuznetsova, V. V.; Mamonkin, M.; Taranin, A. V.; Gorchakov, A. A.

    2017-01-01

    Chimeric antigen receptors (CARs) are recombinant protein molecules that redirect cytotoxic lymphocytes toward malignant and other target cells. The high feasibility of manufacturing CAR-modified lymphocytes for the therapy of cancer has spurred the development and optimization of new CAR T cells directed against a broad range of target antigens. In this review, we describe the main structural and functional elements constituting a CAR, discuss the roles of these elements in modulating the anti-tumor activity of CAR T cells, and highlight alternative approaches to CAR engineering. PMID:28461969

  15. Two Studies of Specification Error in Models for Categorical Latent Variables

    ERIC Educational Resources Information Center

    Kaplan, David; Depaoli, Sarah

    2011-01-01

    This article examines the problem of specification error in 2 models for categorical latent variables; the latent class model and the latent Markov model. Specification error in the latent class model focuses on the impact of incorrectly specifying the number of latent classes of the categorical latent variable on measures of model adequacy as…

  16. Two Studies of Specification Error in Models for Categorical Latent Variables

    ERIC Educational Resources Information Center

    Kaplan, David; Depaoli, Sarah

    2011-01-01

    This article examines the problem of specification error in 2 models for categorical latent variables; the latent class model and the latent Markov model. Specification error in the latent class model focuses on the impact of incorrectly specifying the number of latent classes of the categorical latent variable on measures of model adequacy as…

  17. The algebraic theory of latent projectors in lambda matrices

    NASA Technical Reports Server (NTRS)

    Denman, E. D.; Leyva-Ramos, J.; Jeon, G. J.

    1981-01-01

    Multivariable systems such as a finite-element model of vibrating structures, control systems, and large-scale systems are often formulated in terms of differential equations which give rise to lambda matrices. The present investigation is concerned with the formulation of the algebraic theory of lambda matrices and the relationship of latent roots, latent vectors, and latent projectors to the eigenvalues, eigenvectors, and eigenprojectors of the companion form. The chain rule for latent projectors and eigenprojectors for the repeated latent root or eigenvalues is given.

  18. The impact of HLA class I and EBV latency-II antigen-specific CD8(+) T cells on the pathogenesis of EBV(+) Hodgkin lymphoma.

    PubMed

    Jones, K; Wockner, L; Brennan, R M; Keane, C; Chattopadhyay, P K; Roederer, M; Price, D A; Cole, D K; Hassan, B; Beck, K; Gottlieb, D; Ritchie, D S; Seymour, J F; Vari, F; Crooks, P; Burrows, S R; Gandhi, M K

    2016-02-01

    In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein-Barr virus (EBV) latency-II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV(+) cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA-A*02 is protective in EBV(+) cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA-A*02(-) versus HLA-A*02(+) EBV(+) cHL patients, suggesting that LMP2A-specific CD8(+) T cell anti-tumoral immunity may be relatively ineffective in HLA-A*02(-) EBV(+) cHL. To ascertain the impact of HLA class I on EBV latency antigen-specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV(+) cHL, the magnitude of ex-vivo LMP1/2A-specific CD8(+) T cell responses was elevated in HLA-A*02(+) patients. Furthermore, in a controlled in-vitro assay, LMP2A-specific CD8(+) T cells from healthy HLA-A*02 heterozygotes expanded to a greater extent with HLA-A*02-restricted compared to non-HLA-A*02-restricted cell lines. In an extensive analysis of HLA class I-restricted immunity, immunodominant EBNA3A/3B/3C-specific CD8(+) T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A-specific responses were confined largely to HLA-A*02. Our results demonstrate that HLA-A*02 mediates a modest, but none the less stronger, EBV-specific CD8(+) T cell response than non-HLA-A*02 alleles, an effect confined to EBV latency-II antigens. Thus, the protective effect of HLA-A*02 against EBV(+) cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency-II antigen-specific CD8(+) T cell hierarchies. © 2015 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on

  19. Hetero-organic thymus antigens.

    PubMed

    Beletskaya, L V; Gnezditskaya, E V

    1985-01-01

    The use of sera containing antibodies to tissue-specific antigens of highly specialized organs (skeletal muscles, heart, skin, excretory glands) enabled us to detect, by immunofluorescence, cells capable of synthesizing analogous antigens (i.e. hetero-organic thymus antigens) in human and animal thymus. Detection of hetero-organic antigens in the thymus is the basis for the hypothesis that natural immunological tolerance to tissue self antigens is formed within the thymus in the course of T-lymphocyte maturation, with thymus antigens taking part in the process.

  20. Blocking of potentiation of latent inhibition.

    PubMed

    Hall, Geoffrey; Rodriguez, Gabriel

    2011-01-01

    We present a theory of latent inhibition based on the Pearce-Hall (Pearce & Hall, 1980) model for classical conditioning. Its central features are (1) that the associability of a stimulus declines as it comes to predict its consequences and (2) that nonreinforced exposure to a stimulus engages an associative learning process that makes the stimulus an accurate predictor of its consequences (in this case, the occurrence of no event). A formalization of this theory is shown to accommodate the finding that preexposure in compound with another cue can potentiate latent inhibition to the target cue. It further predicts that preexposure to the added cue will eliminate the potentiation effect. An experiment using rats and the flavor-aversion procedure confirmed this prediction.

  1. Prospects for treatment of latent HIV.

    PubMed

    Barton, K M; Burch, B D; Soriano-Sarabia, N; Margolis, D M

    2013-01-01

    Recent advances in antiretroviral therapy (ART) have drastically improved the quality of life for people with HIV infection. However, owing to the persistence of latent HIV in the presence of therapy, patients must remain on therapy indefinitely. Currently, the solution to the HIV pandemic rests on the prevention of new infections and many decades of ART for the steadily expanding number of people infected worldwide. ART is costly, requires ongoing medical care, and can have side effects, thereby preventing its universal availability. Therefore, to escape the ironic burdens of therapy, efforts have begun to develop treatments for latent HIV infection. Current approaches propose either complete eradication of infection or induction of a state of stringent control over viral replication without ART. This review will discuss these strategies in detail and their potential for clinical development.

  2. Antigen smuggling in tuberculosis.

    PubMed

    Hudrisier, Denis; Neyrolles, Olivier

    2014-06-11

    The importance of CD4 T lymphocytes in immunity to M. tuberculosis is well established; however, how dendritic cells activate T cells in vivo remains obscure. In this issue of Cell Host & Microbe, Srivastava and Ernst (2014) report a mechanism of antigen transfer for efficient activation of antimycobacterial T cells.

  3. Antigen detection systems

    USDA-ARS?s Scientific Manuscript database

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

  4. Antigen detection systems

    USDA-ARS?s Scientific Manuscript database

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissue using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular methodology is chosen ...

  5. Probabilistic Latent Variable Models as Nonnegative Factorizations

    PubMed Central

    Shashanka, Madhusudana; Raj, Bhiksha; Smaragdis, Paris

    2008-01-01

    This paper presents a family of probabilistic latent variable models that can be used for analysis of nonnegative data. We show that there are strong ties between nonnegative matrix factorization and this family, and provide some straightforward extensions which can help in dealing with shift invariances, higher-order decompositions and sparsity constraints. We argue through these extensions that the use of this approach allows for rapid development of complex statistical models for analyzing nonnegative data. PMID:18509481

  6. Latent common genetic components of obesity traits

    PubMed Central

    Harders, R; Luke, A; Zhu, X; Cooper, RS

    2008-01-01

    Background Obesity is rapidly becoming a global epidemic. Unlike many complex human diseases, obesity is defined not just by a single trait or phenotype, but jointly by measures of anthropometry and metabolic status. Methods We applied maximum likelihood factor analysis to identify common latent factors underlying observed covariance in multiple obesity-related measures. Both the genetic components and the mode of inheritance of the common factors were evaluated. A total of 1775 participants from 590 families for whom measures on obesity-related traits were available were included in this study. Results The average age of participants was 37 years, 39% of the participants were obese (body mass index ≥ 30.0 kg/m2) and 26% were overweight (body mass index 25.0 - 29.9 kg/m2). Two latent common factors jointly accounting for over 99% of the correlations among obesity-related traits were identified. Complex segregation analysis of the age and sex-adjusted latent factors provide evidence for a Mendelian mode of inheritance of major genetic effect with heritability estimates of 40.4% and 47.5% for the first and second factors, respectively. Conclusions These findings provide a support for multivariate-based approach for investigating pleiotropic effects on obesity-related traits which can be applied in both genetic linkage and association mapping. PMID:18936762

  7. The latent class multitrait-multimethod model.

    PubMed

    Oberski, Daniel L; Hagenaars, Jacques A P; Saris, Willem E

    2015-12-01

    A latent class multitrait-multimethod (MTMM) model is proposed to estimate random and systematic measurement error in categorical survey questions while making fewer assumptions than have been made so far in such evaluations, allowing for possible extreme response behavior and other nonmonotone effects. The method is a combination of the MTMM research design of Campbell and Fiske (1959), the basic response model for survey questions of Saris and Andrews (19