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Sample records for 14-3-3 protein epsilon

  1. Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis

    SciTech Connect

    Han, Dingding; Ye, Guangming; Liu, Tingting; Chen, Cong; Yang, Xianmei; Wan, Bo; Pan, Yuanwang; Yu, Long

    2010-05-28

    14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1' insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal {alpha}-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3 epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.

  2. Molecular evolution of the 14-3-3 protein family.

    PubMed

    Wang, W; Shakes, D C

    1996-10-01

    Members of the highly conserved and ubiquitous 14-3-3 protein family modulate a wide variety of cellular processes. To determine the evolutionary relationships among specific 14-3-3 proteins in different plant, animal, and fungal species and to initiate a predictive analysis of isoform-specific differences in light of the latest functional and structural studies of 14-3-3, multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases and a newly identified second 14-3-3 gene from Caenorhabditis elegans. The alignment revealed five highly conserved sequence blocks. Blocks 2-5 correlate well with the alpha helices 3, 5, 7, and 9 which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer. Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified which may account for functional diversity amongst isoforms. Protein phylogenic trees were constructed using both the maximum parsimony and neighbor joining methods of the PHYLIP(3.5c) package; 14-3-3 proteins from Entamoeba histolytica, an amitochondrial protozoa, were employed as an outgroup in our analysis. Epsilon isoforms from the animal lineage form a distinct grouping in both trees, which suggests an early divergence from the other animal isoforms. Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein. The known invertebrate proteins group with the nonepsilon mammalian isoforms. Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms. Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma (stratifin/HME1) occurred before the divergence of mammalian and perhaps

  3. Neuroprotective Function of 14-3-3 Proteins in Neurodegeneration

    PubMed Central

    Shimada, Tadayuki; Fournier, Alyson E.; Yamagata, Kanato

    2013-01-01

    14-3-3 proteins are abundantly expressed adaptor proteins that interact with a vast number of binding partners to regulate their cellular localization and function. They regulate substrate function in a number of ways including protection from dephosphorylation, regulation of enzyme activity, formation of ternary complexes and sequestration. The diversity of 14-3-3 interacting partners thus enables 14-3-3 proteins to impact a wide variety of cellular and physiological processes. 14-3-3 proteins are broadly expressed in the brain, and clinical and experimental studies have implicated 14-3-3 proteins in neurodegenerative disease. A recurring theme is that 14-3-3 proteins play important roles in pathogenesis through regulating the subcellular localization of target proteins. Here, we review the evidence that 14-3-3 proteins regulate aspects of neurodegenerative disease with a focus on their protective roles against neurodegeneration. PMID:24364034

  4. Transcriptional increase and misexpression of 14-3-3 epsilon in sea urchin embryos exposed to UV-B

    PubMed Central

    Russo, Roberta; Zito, Francesca; Costa, Caterina; Bonaventura, Rosa

    2010-01-01

    Members of the 14-3-3 protein family are involved in many important cellular events, including stress response, survival and apoptosis. Genes of the 14-3-3 family are conserved from plants to humans, and some members are responsive to UV radiation. Here, we report the isolation of the complete cDNA encoding the 14-3-3 epsilon isoform from Paracentrotus lividus sea urchin embryos, referred to as Pl14-3-3ε, and the phylogenetic relationship with other homologues described in different phyla. Pl14-3-3ε mRNA levels were measured by QPCR during development and found to increase from the mesenchyme blastula to the prism stage. In response to UV-B (312 nm) exposure, early stage embryos collected 2 h later showed a 2.3-fold (at 400 J/m2) and a 2.7-fold (at 800 J/m2) increase in Pl14-3-3ε transcript levels compared with controls. The spatial expression of Pl14-3-3ε mRNA, detected by whole mount in situ hybridization in both control and UV-B exposed embryos, harvested at late developmental stages, showed transcripts to be located in the archenteron of gastrula stage and widely distributed in all germ layers, respectively. The Pl14-3-3ε mRNA delocalization parallels the failure in archenteron elongation observed morphologically, as well as the lack of specific endoderm markers, investigated by indirect immuno-fluorescence on whole mount embryos. Results confirm the involvement of 14-3-3ε in the stress response elicited by UV-B and demonstrate, for the first time, its contribution at the transcriptional level in the sea urchin embryo. PMID:20607471

  5. Clinical implication of 14-3-3 epsilon expression in gastric cancer

    PubMed Central

    Leal, Mariana Ferreira; Calcagno, Danielle Queiroz; Demachki, Sâmia; Assumpção, Paulo Pimentel; Chammas, Roger; Burbano, Rommel Rodríguez; Smith, Marília de Arruda Cardoso

    2012-01-01

    AIM: To evaluate for the first time the protein and mRNA expression of 14-3-3ε in gastric carcinogenesis. METHODS: 14-3-3ε protein expression was determined by western blotting, and mRNA expression was examined by real-time quantitative RT-PCR in gastric tumors and their matched non-neoplastic gastric tissue samples. RESULTS: Authors observed a significant reduction of 14-3-3ε protein expression in gastric cancer (GC) samples compared to their matched non-neoplastic tissue. Reduced levels of 14-3-3ε were also associated with diffuse-type GC and early-onset of this pathology. Our data suggest that reduced 14-3-3ε may have a role in gastric carcinogenesis process. CONCLUSION: Our results reveal that the reduced 14-3-3ε expression in GC and investigation of 14-3-3ε interaction partners may help to elucidate the carcinogenesis process. PMID:22509086

  6. Regulation of starch accumulation by granule-associated plant 14-3-3 proteins.

    PubMed

    Sehnke, P C; Chung, H J; Wu, K; Ferl, R J

    2001-01-16

    In higher plants the production of starch is orchestrated by chloroplast-localized biosynthetic enzymes, namely starch synthases, ADP-glucose pyrophosphorylase, and starch branching and debranching enzymes. Diurnal regulation of these enzymes, as well as starch-degrading enzymes, influences both the levels and composition of starch, and is dependent in some instances upon phosphorylation-linked regulation. The phosphoserine/threonine-binding 14-3-3 proteins participate in environmentally responsive phosphorylation-related regulatory functions in plants, and as such are potentially involved in starch regulation. We report here that reduction of the epsilon subgroup of Arabidopsis 14-3-3 proteins by antisense technology resulted in a 2- to 4-fold increase in leaf starch accumulation. Dark-governed starch breakdown was unaffected in these "antisense plants," indicating an unaltered starch-degradation pathway and suggesting a role for 14-3-3 proteins in regulation of starch synthesis. Absorption spectra and gelatinization properties indicate that the starch from the antisense plants has an altered branched glucan composition. Biochemical characterization of protease-treated starch granules from both Arabidopsis leaves and maize endosperm showed that 14-3-3 proteins are internal intrinsic granule proteins. These data suggest a direct role for 14-3-3 proteins in starch accumulation. The starch synthase III family is a possible target for 14-3-3 protein regulation because, uniquely among plastid-localized starch metabolic enzymes, all members of the family contain the conserved 14-3-3 protein phosphoserine/threonine-binding consensus motif. This possibility is strengthened by immunocapture using antibodies to DU1, a maize starch synthase III family member, and direct interaction with biotinylated 14-3-3 protein, both of which demonstrated an association between 14-3-3 proteins and DU1 or DU1-like proteins. PMID:11149942

  7. 14-3-3 Proteins in Guard Cell Signaling

    PubMed Central

    Cotelle, Valérie; Leonhardt, Nathalie

    2016-01-01

    Guard cells are specialized cells located at the leaf surface delimiting pores which control gas exchanges between the plant and the atmosphere. To optimize the CO2 uptake necessary for photosynthesis while minimizing water loss, guard cells integrate environmental signals to adjust stomatal aperture. The size of the stomatal pore is regulated by movements of the guard cells driven by variations in their volume and turgor. As guard cells perceive and transduce a wide array of environmental cues, they provide an ideal system to elucidate early events of plant signaling. Reversible protein phosphorylation events are known to play a crucial role in the regulation of stomatal movements. However, in some cases, phosphorylation alone is not sufficient to achieve complete protein regulation, but is necessary to mediate the binding of interactors that modulate protein function. Among the phosphopeptide-binding proteins, the 14-3-3 proteins are the best characterized in plants. The 14-3-3s are found as multiple isoforms in eukaryotes and have been shown to be involved in the regulation of stomatal movements. In this review, we describe the current knowledge about 14-3-3 roles in the regulation of their binding partners in guard cells: receptors, ion pumps, channels, protein kinases, and some of their substrates. Regulation of these targets by 14-3-3 proteins is discussed and related to their function in guard cells during stomatal movements in response to abiotic or biotic stresses. PMID:26858725

  8. Isoform-specific cleavage of 14-3-3 proteins in apoptotic JURL-MK1 cells.

    PubMed

    Kuzelová, Katerina; Grebenová, Dana; Pluskalová, Michaela; Kavan, Daniel; Halada, Petr; Hrkal, Zbynek

    2009-03-01

    The proteins of 14-3-3 family are substantially involved in the regulation of many biological processes including the apoptosis. We studied the changes in the expression of five 14-3-3 isoforms (beta, gamma, epsilon, tau, and zeta) during the apoptosis of JURL-MK1 and K562 cells. The expression level of all these proteins markedly decreased in relation with the apoptosis progression and all isoforms underwent truncation, which probably corresponds to the removal of several C-terminal amino acids. The observed 14-3-3 modifications were partially blocked by caspase-3 inhibition. In addition to caspases, a non-caspase protease is likely to contribute to 14-3-3's cleavage in an isoform-specific manner. While 14-3-3 gamma seems to be cleaved mainly by caspase-3, the alternative mechanism is essentially involved in the case of 14-3-3 tau, and a combined effect was observed for the isoforms epsilon, beta, and zeta. We suggest that the processing of 14-3-3 proteins could form an integral part of the programmed cell death or at least of some apoptotic pathways. PMID:19173300

  9. Up-regulated 14-3-3β and 14-3-3ζ proteins in prefrontal cortex of subjects with schizophrenia: effect of psychotropic treatment.

    PubMed

    Rivero, Guadalupe; Gabilondo, Ane M; García-Sevilla, Jesús A; La Harpe, Romano; Morentín, Benito; Meana, J Javier

    2015-02-01

    14-3-3 is a family of conserved regulatory proteins that bind to a multitude of functionally diverse signalling proteins. Various genetic studies and gene expression and proteomic analyses have involved 14-3-3 proteins in schizophrenia (SZ). On the other hand, studies about the status of these proteins in major depressive disorder (MD) are still missing. Immunoreactivity values of cytosolic 14-3-3β and 14-3-3ζ proteins were evaluated by Western blot in prefrontal cortex (PFC) of subjects with schizophrenia (SZ; n=22), subjects with major depressive disorder (MD; n=21) and age-, gender- and postmortem delay-matched control subjects (n=52). The modulation of 14-3-3β and 14-3-3ζ proteins by psychotropic medication was also assessed. The analysis of both proteins in SZ subjects with respect to matched control subjects showed increased 14-3-3β (Δ=33±10%, p<0.05) and 14-3-3ζ (Δ=29±6%, p<0.05) immunoreactivity in antipsychotic-free but not in antipsychotic-treated SZ subjects. Immunoreactivity values of 14-3-3β and 14-3-3ζ were not altered in MD subjects. These results show the specific up-regulation of 14-3-3β and 14-3-3ζ proteins in PFC of SZ subjects and suggest a possible down-regulation of both proteins by antipsychotic treatment. PMID:25549848

  10. Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein, its heterodimerization profile with endogenous 14-3-3 isoforms, and effect on mammalian DNA replication in vitro.

    PubMed

    Alvarez, David; Callejo, Mario; Shoucri, Rami; Boyer, Lee; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2003-06-17

    The human cruciform binding protein (CBP), a member of the 14-3-3 protein family, has been recently identified as an origin of DNA replication binding protein and involved in DNA replication. Here, pure recombinant 14-3-3zeta tagged with maltose binding protein (r14-3-3zeta-MBP) at its N-terminus was tested for binding to cruciform DNA either in the absence or presence of F(TH), a CBP-enriched fraction, by electromobility shift assay (EMSA), followed by Western blot analysis of the electroeluted CBP-cruciform DNA complex. The r14-3-3zeta-MBP was found to have cruciform binding activity only after preincubation with F(TH). Anti-MBP antibody immunoprecipitation of F(TH) preincubated with r14-3-3zeta-MBP, followed by Western blot analysis with antibodies specific to the beta, gamma, epsilon, zeta, and sigma 14-3-3 isoforms showed that r14-3-3zeta-MBP heterodimerized with the endogenous beta, epsilon, and zeta isoforms present in the F(TH) but not with the gamma or sigma isoforms. Immunoprecipitation of endogenous 14-3-3zeta from nuclear extracts (NE) of HeLa cells that were either serum-starved (s-s) or blocked at the G(1)/S or G(2)/M phases of the cell cycle revealed that at G(1)/S and G(2)/M, the zeta isoform heterodimerized only with the beta and epsilon isoforms, while in s-s extracts, the 14-3-3zeta/epsilon heterodimer was never detected, and the 14-3-3zeta/beta heterodimer was seldom detected. Furthermore, addition of r14-3-3zeta-MBP to HeLa cell extracts used in a mammalian in vitro replication system increased the replication level of p186, a plasmid bearing the minimal 186-bp origin of the monkey origin of DNA replication ors8, by approximately 3.5-fold. The data suggest that specific dimeric combinations of the 14-3-3 isoforms have CBP activity and that upregulation of this activity leads to an increase in DNA replication. PMID:12795617

  11. 14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication.

    PubMed

    Alvarez, David; Novac, Olivia; Callejo, Mario; Ruiz, Marcia T; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2002-01-01

    A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding. PMID:12244572

  12. Involvement of 14-3-3 Proteins in Regulating Tumor Progression of Hepatocellular Carcinoma.

    PubMed

    Wu, Yi-Ju; Jan, Yee-Jee; Ko, Bor-Sheng; Liang, Shu-Man; Liou, Jun-Yang

    2015-01-01

    There are seven mammalian isoforms of the 14-3-3 protein, which regulate multiple cellular functions via interactions with phosphorylated partners. Increased expression of 14-3-3 proteins contributes to tumor progression of various malignancies. Several isoforms of 14-3-3 are overexpressed and associate with higher metastatic risks and poorer survival rates of hepatocellular carcinoma (HCC). 14-3-3β and 14-3-3ζ regulate HCC cell proliferation, tumor growth and chemosensitivity via modulating mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and p38 signal pathways. Moreover, 14-3-3ε suppresses E-cadherin and induces focal adhesion kinase (FAK) expression, thereby enhancing epithelial-mesenchymal transition (EMT) and HCC cell migration. 14-3-3ζ forms complexes with αB-crystallin, which induces EMT and is the cause of sorafenib resistance in HCC. Finally, a recent study has indicated that 14-3-3σ induces heat shock protein 70 (HSP70) expression, which increases HCC cell migration. These results suggest that selective 14-3-3 isoforms contribute to cell proliferation, EMT and cell migration of HCC by regulating distinct targets and signal pathways. Targeting 14-3-3 proteins together with specific downstream effectors therefore has potential to be therapeutic and prognostic factors of HCC. In this article, we will overview 14-3-3's regulation of its downstream factors and contributions to HCC EMT, cell migration and proliferation. PMID:26083935

  13. Determining novel functions of Arabidopsis 14-3-3 proteins in central metabolic processes

    PubMed Central

    2011-01-01

    Background 14-3-3 proteins are considered master regulators of many signal transduction cascades in eukaryotes. In plants, 14-3-3 proteins have major roles as regulators of nitrogen and carbon metabolism, conclusions based on the studies of a few specific 14-3-3 targets. Results In this study, extensive novel roles of 14-3-3 proteins in plant metabolism were determined through combining the parallel analyses of metabolites and enzyme activities in 14-3-3 overexpression and knockout plants with studies of protein-protein interactions. Decreases in the levels of sugars and nitrogen-containing-compounds and in the activities of known 14-3-3-interacting-enzymes were observed in 14-3-3 overexpression plants. Plants overexpressing 14-3-3 proteins also contained decreased levels of malate and citrate, which are intermediate compounds of the tricarboxylic acid (TCA) cycle. These modifications were related to the reduced activities of isocitrate dehydrogenase and malate dehydrogenase, which are key enzymes of TCA cycle. In addition, we demonstrated that 14-3-3 proteins interacted with one isocitrate dehydrogenase and two malate dehydrogenases. There were also changes in the levels of aromatic compounds and the activities of shikimate dehydrogenase, which participates in the biosynthesis of aromatic compounds. Conclusion Taken together, our findings indicate that 14-3-3 proteins play roles as crucial tuners of multiple primary metabolic processes including TCA cycle and the shikimate pathway. PMID:22104211

  14. The role of the 14-3-3 protein family in health, disease, and drug development.

    PubMed

    Aghazadeh, Yasaman; Papadopoulos, Vassilios

    2016-02-01

    14-3-3 proteins regulate intracellular signaling pathways, such as signal transduction, protein trafficking, cell cycle, and apoptosis. In addition to the ubiquitous roles of 14-3-3 isoforms, unique tissue-specific functions are also described for each isoform. Owing to their role in regulating cell cycle, protein trafficking, and steroidogenesis, 14-3-3 proteins are prevalent in human diseases, such as cancer, neurodegeneration, and reproductive disorders, and, therefore, serve as valuable drug targets. In this review, we summarize the role of 14-3-3 proteins in normal and disease states, with a focus on 14-3-3γ and ɛ. We also discuss drug compounds targeting 14-3-3 proteins and their potential therapeutic uses. PMID:26456530

  15. 14-3-3 proteins regulate Tctp-Rheb interaction for organ growth in Drosophila.

    PubMed

    Le, Thao Phuong; Vuong, Linh Thuong; Kim, Ah-Ram; Hsu, Ya-Chieh; Choi, Kwang-Wook

    2016-01-01

    14-3-3 family proteins regulate multiple signalling pathways. Understanding biological functions of 14-3-3 proteins has been limited by the functional redundancy of conserved isotypes. Here we provide evidence that 14-3-3 proteins regulate two interacting components of Tor signalling in Drosophila, translationally controlled tumour protein (Tctp) and Rheb GTPase. Single knockdown of 14-3-3ɛ or 14-3-3ζ isoform does not show obvious defects in organ development but causes synergistic genetic interaction with Tctp and Rheb to impair tissue growth. 14-3-3 proteins physically interact with Tctp and Rheb. Knockdown of both 14-3-3 isoforms abolishes the binding between Tctp and Rheb, disrupting organ development. Depletion of 14-3-3s also reduces the level of phosphorylated S6 kinase, phosphorylated Thor/4E-BP and cyclin E (CycE). Growth defects from knockdown of 14-3-3 and Tctp are suppressed by CycE overexpression. This study suggests a novel mechanism of Tor regulation mediated by 14-3-3 interaction with Tctp and Rheb. PMID:27151460

  16. 14-3-3 proteins regulate Tctp–Rheb interaction for organ growth in Drosophila

    PubMed Central

    Le, Thao Phuong; Vuong, Linh Thuong; Kim, Ah-Ram; Hsu, Ya-Chieh; Choi, Kwang-Wook

    2016-01-01

    14-3-3 family proteins regulate multiple signalling pathways. Understanding biological functions of 14-3-3 proteins has been limited by the functional redundancy of conserved isotypes. Here we provide evidence that 14-3-3 proteins regulate two interacting components of Tor signalling in Drosophila, translationally controlled tumour protein (Tctp) and Rheb GTPase. Single knockdown of 14-3-3ɛ or 14-3-3ζ isoform does not show obvious defects in organ development but causes synergistic genetic interaction with Tctp and Rheb to impair tissue growth. 14-3-3 proteins physically interact with Tctp and Rheb. Knockdown of both 14-3-3 isoforms abolishes the binding between Tctp and Rheb, disrupting organ development. Depletion of 14-3-3s also reduces the level of phosphorylated S6 kinase, phosphorylated Thor/4E-BP and cyclin E (CycE). Growth defects from knockdown of 14-3-3 and Tctp are suppressed by CycE overexpression. This study suggests a novel mechanism of Tor regulation mediated by 14-3-3 interaction with Tctp and Rheb. PMID:27151460

  17. A fusicoccin binding protein belongs to the family of 14-3-3 brain protein homologs.

    PubMed Central

    Korthout, H A; de Boer, A H

    1994-01-01

    The fusicoccin binding protein (FCBP) is a highly conserved plasma membrane protein present in all higher plants tested thus far. It exhibits high- and low-affinity binding for the fungal toxin fusicoccin (FC). We purified the active FCBP from a fraction highly enriched in plasma membrane by selective precipitation and anion exchange chromatography. After SDS-PAGE, the two FCBP subunits of 30 and 31 kD were detected as major bands. Amino acid sequence analysis of the 31-kD polypeptide displayed a high degree of identity with so-called 14-3-3 proteins, a class of mammalian brain proteins initially described as regulators of neurotransmitter synthesis and protein kinase C inhibitors. Thereafter, we affinity purified the 30- and 31-kD FCBP subunits, using biotinylated FC in combination with a monomeric avidin column. Immunodecoration of these 30- and 31-kD FCBP subunits with polyclonal antibodies raised against a 14-3-3 homolog from yeast confirmed the identity of the FCBP as a 14-3-3 homolog. Similar to all 14-3-3 protein homologs, the FCBP seems to exist as a dimer in native form. Thus far, the FCBP is the only 14-3-3 homolog with a receptor-like function. The conserved structure of the 14-3-3 protein family is a further indication that the FCBP plays an important role in the physiology of higher plants. PMID:7827499

  18. Differential neuroprotective effects of 14-3-3 proteins in models of Parkinson's disease.

    PubMed

    Yacoubian, T A; Slone, S R; Harrington, A J; Hamamichi, S; Schieltz, J M; Caldwell, K A; Caldwell, G A; Standaert, D G

    2010-01-01

    14-3-3 proteins are important negative regulators of cell death pathways. Recent studies have revealed alterations in 14-3-3s in Parkinson's disease (PD) and the ability of 14-3-3s to interact with alpha-synuclein (α-syn), a protein central to PD pathophysiology. In a transgenic α-syn mouse model, we found reduced expression of 14-3-3θ, ε, and γ. These same isoforms prevent α-syn inclusion formation in an H4 neuroglioma cell model. Using dopaminergic cell lines stably overexpressing each 14-3-3 isoform, we found that overexpression of 14-3-3θ, ε, or γ led to resistance to both rotenone and 1-methyl-4-phenylpyridinium (MPP(+)), while other isoforms were not protective against both toxins. Inhibition of a single protective isoform, 14-3-3θ, by shRNA did not increase vulnerability to neurotoxic injury, but toxicity was enhanced by broad-based inhibition of 14-3-3 action with the peptide inhibitor difopein. Using a transgenic C. elegans model of PD, we confirmed the ability of both human 14-3-3θ and a C. elegans 14-3-3 homolog (ftt-2) to protect dopaminergic neurons from α-syn toxicity. Collectively, these data show a strong neuroprotective effect of enhanced 14-3-3 expression - particularly of the 14-3-3θ, ε, and γ isoforms - in multiple cellular and animal models of PD, and point to the potential value of these proteins in the development of neuroprotective therapies for human PD. PMID:21152247

  19. Hyperglycemia decreases expression of 14-3-3 proteins in an animal model of stroke.

    PubMed

    Jeon, Seong-Jun; Sung, Jin-Hee; Koh, Phil-Ok

    2016-07-28

    Diabetes is a severe metabolic disorder and a major risk factor for stroke. Stroke severity is worse in patients with diabetes compared to the non-diabetic population. The 14-3-3 proteins are a family of conserved acidic proteins that are ubiquitously expressed in cells and tissues. These proteins are involved in many cellular processes including metabolic pathways, signal transduction, protein trafficking, protein synthesis, and cell cycle control. This study investigated 14-3-3 proteins expression in the cerebral cortex of animals with diabetes, cerebral ischemic injury and a combination of both diabetes and cerebral ischemic injury. Diabetes was induced by intraperitoneal injection of streptozotocin (40mg/kg) in adult male rats. After 4 weeks of treatment, middle cerebral artery occlusion (MCAO) was performed for the induction of focal cerebral ischemia and cerebral cortex tissue was collected 24h after MCAO. We confirmed that diabetes increases infarct volume following MCAO compared to non-diabetic animals. In diabetic animals with MCAO injury, reduction of 14-3-3 β/α, 14-3-3 ζ/δ, 14-3-3 γ, and 14-3-3 ε isoforms was detected. The expression of these proteins was significantly decreased in diabetic animals with MCAO injury compared to diabetic-only and MCAO-only animals. Moreover, Western blot analysis ascertained the decreased expression of 14-3-3 family proteins in diabetic animals with MCAO injury, including β/α, ζ/δ, γ, ε, τ, and η isoforms. These results show the changes of 14-3-3 proteins expression in streptozotocin-induced diabetic animals with MCAO injury. Thus, these findings suggest that decreases in 14-3-3 proteins might be involved in the regulation of 14-3-3 proteins under the presence of diabetes following MCAO. PMID:27177727

  20. Identification of a redox-modulatory interaction between selenoprotein W and 14-3-3 protein.

    PubMed

    Jeon, Yeong Ha; Ko, Kwan Young; Lee, Jea Hwang; Park, Ki Jun; Jang, Jun Ki; Kim, Ick Young

    2016-01-01

    Selenoprotein W (SelW) contains a selenocysteine (Sec, U) in a conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin, suggesting a putative redox function of SelW. We have previously reported that the binding of 14-3-3 protein to its target proteins, including CDC25B, Rictor and TAZ, is inhibited by the interaction of 14-3-3 protein with SelW. However, the binding mechanism is unclear. In this study, we sought to determine the binding site of SelW to understand the regulatory mechanism of the interaction between SelW and 14-3-3 and its biological effects. Phosphorylated Ser(pS) or Thr(pT) residues in RSXpSXP or RXXXp(S/T)XP motifs are well-known common 14-3-3-binding sites, but Thr41, Ser59, and T69 of SelW, which are computationally predicted to serve are phosphorylation sites, were neither phosphorylation sites nor sites involved in the interaction. A mutant SelW in which Sec13 is changed to Ser (U13S) was unable to interact with 14-3-3 protein and thus did not inhibit the interaction of 14-3-3 to other target proteins. However, other Cys mutants of SelW(C10S, C33S and C37S) normally interacted with 14-3-3 protein. The interaction of SelW to 14-3-3 protein was enhanced by diamide or H2O2 and decreased by dithiothreitol (DTT). Taken together, these findings demonstrate that the Sec of SelW is involved in its interaction with 14-3-3 protein and that this interaction is increased under oxidative stress conditions. Thus, SelW may have a regulatory function in redox cell signaling by interacting with 14-3-3 protein. PMID:26474786

  1. Identification of 14-3-3zeta associated protein networks in oral cancer.

    PubMed

    Matta, Ajay; Masui, Olena; Siu, K W Michael; Ralhan, Ranju

    2016-04-01

    Advancements in genomics, proteomics, and bioinformatics have improved our understanding of gene/protein networks involved in intra- and intercellular communication and tumor-host interactions. Using proteomics integrated with bioinformatics, previously we reported overexpression of 14-3-3ζ in premalignant oral lesions and oral squamous cell carcinoma tissues in comparison with normal oral epithelium. 14-3-3ζ emerged as a novel molecular target for therapeutics and a potential prognostic marker in oral squamous cell carcinoma patients. However, the role of 14-3-3ζ in development and progression of oral cancer is not known yet. This study aimed to identify the 14-3-3ζ associated protein networks in oral cancer cell lines using IP-MS/MS and bioinformatics. A total of 287 binding partners of 14-3-3ζ were identified in metastatic (MDA1986) and nonmetastatic (SCC4) oral cancer cell lines including other 14-3-3 isoforms (2%), proteins involved in apoptosis (2%), cytoskeleton (9%), metabolism (16%), and maintenance of redox potential (2%). Our bioinformatics analysis revealed involvement of 14-3-3ζ in protein networks regulating cell cycle, proliferation, apoptosis, cellular trafficking, and endocytosis in oral cancer. In conclusion, our data revealed several novel protein interaction networks involving 14-3-3ζ in oral cancer progression and metastasis. PMID:26857332

  2. Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity.

    PubMed

    Chi, Jen-Chih; Roeper, Juliane; Schwarz, Guenter; Fischer-Schrader, Katrin

    2015-03-01

    14-3-3 proteins represent a family of ubiquitous eukaryotic proteins involved in numerous signal transduction processes and metabolic pathways. One important 14-3-3 target in higher plants is nitrate reductase (NR), whose activity is regulated by different physiological conditions. Intra-molecular electron transfer in NR is inhibited following 14-3-3 binding to a conserved phospho-serine motif located in hinge 1, a surface exposed loop between the catalytic molybdenum and central heme domain. Here we describe a novel 14-3-3 binding site within the NR N-terminus, an acidic motif conserved in NRs of higher plants, which significantly contributes to 14-3-3-mediated inhibition of NR. Deletion or mutation of the N-terminal acidic motif resulted in a significant loss of 14-3-3 mediated inhibition of Ser534 phosphorylated NR-Mo-heme (residues 1-625), a previously established model of NR regulation. Co-sedimentation and crosslinking studies with NR peptides comprising each of the two binding motifs demonstrated direct binding of either peptide to 14-3-3. Surface plasmon resonance spectroscopy disclosed high-affinity binding of 14-3-3ω to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3ω variant retained interaction to the acidic motif, but lost binding to the phospho-hinge motif. To our knowledge, NR is the first enzyme that harbors two independent 14-3-3 binding sites with different affinities, which both need to be occupied by 14-3-3ω to confer full inhibition of NR activity under physiological conditions. PMID:25578809

  3. 14-3-3β protein expression in eosinophilic meningitis caused by Angiostrongylus cantonensis infection

    PubMed Central

    2014-01-01

    Background Angiostrongylus cantonensis is a parasite endemic in the Southeast Asian and Pacific regions. Humans are incidentally infected either by eating uncooked intermediate hosts or by consuming vegetables containing the living third-stage larvae. The 14-3-3β protein is a cerebrospinal fluid (CSF) marker of neuronal damage during the development of Creutzfeldt-Jakob disease. In addition, increased 14-3-3β protein is also found in CSF from patients with a variety of neurological disorders. The goal of this study is to determine the roles of serum/CSF14-3-3β protein in patients with eosinophilic meningitis. Methods In a cohort study among nine Thai laborers with eosinophilic meningitis due to eating raw snails (Pomacea canaliculata), we examined the CSF weekly while patients were still hospitalized and followed up the serum for 6 months. The levels of 14-3-3β protein in CSF were analyzed by western blot and an in-house 14-3-3β enzyme-linked immunosorbent assay (ELISA) measurement was established and tested in an animal model of eosinophilic meningitis. Results The elevated 14-3-3β level was detected in the CSF from eight out of nine (81%) patients After 2 weeks of treatment, all patients showed a declined level or cleared of 14-3-3β protein in the CSF. By developing an in-house ELISA for measurement of 14-3-3β protein, it was found that the serum 14-3-3β level was significantly increased in patients during initial visit. . This finding was consistent to the animal experiment result in which there was severe blood brain barrier damage three weeks after infection and increased 14-3-3β protein expression in the CSF and serum by western blot and in house ELISA. After treatment, the serum 14-3-3β level in meningitis patients was rapidly returned to normal threshold. There was a correlation between initial CSF 14-3-3β level with severity of headache (r = 0.692, p = 0.039), CSF pleocytosis (r = 0.807, p = 0.009) and eosinophilia (r = 0

  4. 14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes

    PubMed Central

    Xu, Zhe; Graham, Kourtney; Foote, Molly; Liang, Fengshan; Rizkallah, Raed; Hurt, Myra; Wang, Yanchang; Wu, Yuying; Zhou, Yi

    2013-01-01

    Summary The aggresome is a key cytoplasmic organelle for sequestration and clearance of toxic protein aggregates. Although loading misfolded proteins cargos to dynein motors has been recognized as an important step in the aggresome formation process, the molecular machinery that mediates the association of cargos with the dynein motor is poorly understood. Here, we report a new aggresome-targeting pathway that involves isoforms of 14-3-3, a family of conserved regulatory proteins. 14-3-3 interacts with both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3), thereby recruiting chaperone-associated protein cargos to dynein motors for their transport to aggresomes. This molecular cascade entails functional dimerization of 14-3-3, which we show to be crucial for the formation of aggresomes in both yeast and mammalian cells. These results suggest that 14-3-3 functions as a molecular adaptor to promote aggresomal targeting of misfolded protein aggregates and may link such complexes to inclusion bodies observed in various neurodegenerative diseases. PMID:23843611

  5. Molecular tweezers modulate 14-3-3 protein-protein interactions.

    PubMed

    Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

    2013-03-01

    Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins--a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)--in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions. PMID:23422566

  6. Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana

    PubMed Central

    Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John; Mittler, Ron; Harmon, Alice; Harper, Jeffrey

    2014-01-01

    In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, a 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a “tandem affinity purification” (TAP) tag and expressed in transgenic plants. Purified complexes were analyzed by tandem mass spectrometry. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (1) Ion transport, such as a K+ channel (GORK), a Cl− channel (CLCg), Ca2+ channels belonging to the glutamate receptor family (GLRs 1.2, 2.1, 2.9, 3.4, 3.7); (2) hormone signaling, such as ACC synthase (isoforms ACS-6, 7 and 8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (3) transcription, such as 7 WRKY family transcription factors; (4) metabolism, such as phosphoenol pyruvate (PEP) carboxylase; and (5) lipid signaling, such as phospholipase D (β, and γ). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300. PMID:19452453

  7. Molecular tweezers modulate 14-3-3 protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

    2013-03-01

    Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins—a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)—in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

  8. 14-3-3zeta is indispensable for aggregate formation of polyglutamine-expanded huntingtin protein.

    PubMed

    Omi, Kazuya; Hachiya, Naomi S; Tanaka, Mayumi; Tokunaga, Katsushi; Kaneko, Kiyotoshi

    2008-01-24

    Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Htt) protein. A hallmark of HD is the presence of aggregates-predominantly composed of NH(2)-terminal fragments of polyQ-expanded Htt-in the nucleus and cytoplasm of affected neurons. We previously proposed that 14-3-3zeta might act as a sweeper of misfolded proteins by facilitating the formation of aggregates possibly for neuroprotection; these aggregates are referred to as inclusion bodies. However, evidence available in this regard is indirect and circumstantial. In this study, analysis of the aggregation-prone protein Htt encoded by HD gene exon 1 containing polyglutamine expansions (Htt86Q) revealed that 17 residues in the NH(2)-terminal of this protein are indispensable for its aggregate formation. Immunoprecipitation assays revealed that 14-3-3beta, gamma, eta, and zeta interact with Htt86Q transfected in N2a cells. Interestingly, the small interfering ribonucleic acid (siRNA) suppression of 14-3-3zeta exclusively abolished Htt86Q aggregate formation, whereas 14-3-3beta or eta siRNA suppression did not. This indicates that 14-3-3zeta participates in aggregate formation under nonnative conditions. Our data support a novel role for 14-3-3zeta in the aggregate formation of nonnative, aggregation-prone proteins. PMID:18078716

  9. Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum

    PubMed Central

    Bajaj Pahuja, Kanika; Wang, Jinzhi; Blagoveshchenskaya, Anastasia; Lim, Lillian; Madhusudhan, M. S.; Mayinger, Peter; Schekman, Randy

    2015-01-01

    Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII. PMID:26056309

  10. 14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells.

    PubMed

    Gómez-Suárez, M; Gutiérrez-Martínez, I Z; Hernández-Trejo, J A; Hernández-Ruiz, M; Suárez-Pérez, D; Candelario, A; Kamekura, R; Medina-Contreras, O; Schnoor, M; Ortiz-Navarrete, V; Villegas-Sepúlveda, N; Parkos, C; Nusrat, A; Nava, P

    2016-06-01

    Akt activation has been associated with proliferation, differentiation, survival and death of epithelial cells. Phosphorylation of Thr308 of Akt by phosphoinositide-dependent kinase 1 (PDK1) is critical for optimal stimulation of its kinase activity. However, the mechanism(s) regulating this process remain elusive. Here, we report that 14-3-3 proteins control Akt Thr308 phosphorylation during intestinal inflammation. Mechanistically, we found that IFNγ and TNFα treatment induce degradation of the PDK1 inhibitor, 14-3-3η, in intestinal epithelial cells. This mechanism requires association of 14-3-3ζ with raptor in a process that triggers autophagy and leads to 14-3-3η degradation. Notably, inhibition of 14-3-3 function by the chemical inhibitor BV02 induces uncontrolled Akt activation, nuclear Akt accumulation and ultimately intestinal epithelial cell death. Our results suggest that 14-3-3 proteins control Akt activation and regulate its biological functions, thereby providing a new mechanistic link between cell survival and apoptosis of intestinal epithelial cells during inflammation. PMID:26846144

  11. Ablation of the 14-3-3gamma Protein Results in Neuronal Migration Delay and Morphological Defects in the Developing Cerebral Cortex.

    PubMed

    Wachi, Tomoka; Cornell, Brett; Marshall, Courtney; Zhukarev, Vladimir; Baas, Peter W; Toyo-Oka, Kazuhito

    2016-06-01

    14-3-3 proteins are ubiquitously-expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14-3-3epsilon and 14-3-3zeta, are important for brain development, in particular, radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14-3-3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14-3-3gamma-deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14-3-3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time-lapse live imaging of brain slices revealed that the ablation of the 14-3-3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14-3-3gamma deficient neurons showed morphological abnormalities, including increased multipolar neurons with a thicker leading processes stem during migration. These results indicate that the 14-3-3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14-3-3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 600-614, 2016. PMID:26297819

  12. Diagnosing Sporadic Creutzfeldt-Jakob Disease: Accuracy of CSF 14-3-3 Protein Test of the Spinal Fluid

    MedlinePlus

    ... JAKOB DISEASE: ACCURACY OF THE 14-3-3 PROTEIN TEST OF THE SPINAL FLUID This information sheet ... help you understand how the 14-3-3 protein test helps in diagnosing sporadic Creutzfeldt-Jakob disease ( ...

  13. Alternations of 14-3-3 θ and β protein levels in brain during experimental sepsis.

    PubMed

    Memos, Nikolaos; Kataki, Agapi; Chatziganni, Emmy; Nikolopoulou, Marilena; Skoulakis, Euthimios; Consoulas, Christos; Zografos, George; Konstadoulakis, Manousos

    2011-09-01

    The 14-3-3 family members play a crucial role in the determination of cell fate, exerting their antiapoptotic activity through directly interfering with the critical function of the mitochondrial core proapoptotic machinery. Dimerization of 14-3-3 is vital for the interaction with many of its client proteins and is regulated by phosphorylation. In a previous study, we observed time-dependent neuronal apoptosis during sepsis. Therefore, in the present study, we sought to evaluate the expression of 14-3-3 θ and β isoforms in septic brain and their association with apoptosis. Sepsis was induced by a CLP model in Wistar rats that were sacrificed at predefined time points. Flow cytometric analysis showed a sepsis-induced, time-dependent alteration of 14-3-3 θ and β isoforms in both Neun(+) and GFAP(+) cells. 14-3-3 θ was linearly correlated with apoptosis, and stratified analysis for alive and apoptotic neuronal cells demonstrated a gradual down-regulation of θ isoform in alive neurons and astrocytes. The phospho-P38 (pP38) MAP kinase levels were altered in a time-dependent manner during sepsis, presenting a peak at 6 hr post-CLP. A significant correlation between the two isoforms of 14-3-3 was observed in septic rats, with the θ isoform predominant at all time points. The hippocampus, Purkinje cells, and glia-like cells showed intense immunohistochemical reactivity for 14-3-3 θ isoform, whereas the choroid plexus showed constantly increased β isoform expression. Our results showed that sepsis alters the expression of both 14-3-3 θ and β isoforms in a time-, cell-, and topography-dependent manner. PMID:21618583

  14. 14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling

    PubMed Central

    Grant, Michael P.; Cavanaugh, Alice; Breitwieser, Gerda E.

    2015-01-01

    Calcium sensing receptors (CaSR) interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium. PMID:26317416

  15. Regional rescue of spinocerebellar ataxia type 1 phenotypes by 14-3-3epsilon haploinsufficiency in mice underscores complex pathogenicity in neurodegeneration.

    PubMed

    Jafar-Nejad, Paymaan; Ward, Christopher S; Richman, Ronald; Orr, Harry T; Zoghbi, Huda Y

    2011-02-01

    Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by the expansion of a CAG repeat encoding a polyglutamine tract in Ataxin-1 (ATXN1). Both WT and mutant ATXN1 interact with 14-3-3 proteins, and 14-3-3 overexpression stabilizes ATXN1 levels in cells and increases ATXN1 toxicity in flies. To determine whether reducing 14-3-3 levels might mitigate SCA1 pathogenesis, we bred Sca1(154Q/+) mice to mice lacking one allele of 14-3-3ε. 14-3-3ε haploinsufficiency rescued cerebellar pathology and motor phenotypes but, surprisingly, not weight loss, respiratory dysfunction, or premature lethality. Biochemical studies revealed that reducing 14-3-3ε levels exerted different effects in two brain regions especially vulnerable in SCA1: Although diminishing levels of both WT and mutant ATXN1 in the cerebellum, 14-3-3ε haploinsufficiency did not alter ATXN1 levels in the brainstem. Furthermore, 14-3-3ε haploinsufficiency decreased the incorporation of expanded ATXN1 into its large toxic complexes in the cerebellum but not in the brainstem, and the distribution of ATXN1's small and large native complexes differed significantly between the two regions. These data suggest that distinct pathogenic mechanisms operate in different vulnerable brain regions, adding another level of complexity to SCA1 pathogenesis. PMID:21245341

  16. Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins

    PubMed Central

    Wilson, Rashaun S.; Swatek, Kirby N.; Thelen, Jay J.

    2016-01-01

    14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants. PMID:27242818

  17. Polycations Globally Enhance Binding of 14-3-3 omega to Target Proteins in Spinach Leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The binding of 14-3-3' to phosphorylated NR (pNR) is stimulated by cations such as Mg2+ or spermine, and decreased by 5'-AMP. In order to determine whether binding to other cellular proteins is affected similarly, Far-Western overlays of extracts prepared from light- or dark-treated spinach (Spinac...

  18. 14-3-3 proteins restrain the Exo1 nuclease to prevent overresection.

    PubMed

    Chen, Xiaoqing; Kim, In-Kwon; Honaker, Yuchi; Paudyal, Sharad C; Koh, Won Kyun; Sparks, Melanie; Li, Shan; Piwnica-Worms, Helen; Ellenberger, Tom; You, Zhongsheng

    2015-05-01

    The DNA end resection process dictates the cellular response to DNA double strand break damage and is essential for genome maintenance. Although insufficient DNA resection hinders homology-directed repair and ATR (ataxia telangiectasia and Rad3 related)-dependent checkpoint activation, overresection produces excessive single-stranded DNA that could lead to genomic instability. However, the mechanisms controlling DNA end resection are poorly understood. Here we show that the major resection nuclease Exo1 is regulated both positively and negatively by protein-protein interactions to ensure a proper level of DNA resection. We have shown previously that the sliding DNA clamp proliferating cell nuclear antigen (PCNA) associates with the C-terminal domain of Exo1 and promotes Exo1 damage association and DNA resection. In this report, we show that 14-3-3 proteins interact with a central region of Exo1 and negatively regulate Exo1 damage recruitment and subsequent resection. 14-3-3s limit Exo1 damage association, at least in part, by suppressing its association with PCNA. Disruption of the Exo1 interaction with 14-3-3 proteins results in elevated sensitivity of cells to DNA damage. Unlike Exo1, the Dna2 resection pathway is apparently not regulated by PCNA and 14-3-3s. Our results provide critical insights into the mechanism and regulation of the DNA end resection process and may have implications for cancer treatment. PMID:25833945

  19. Molecular Dynamics Simulations and Structural Analysis of Giardia duodenalis 14-3-3 Protein-Protein Interactions.

    PubMed

    Cau, Ylenia; Fiorillo, Annarita; Mori, Mattia; Ilari, Andrea; Botta, Maurizo; Lalle, Marco

    2015-12-28

    Giardiasis is a gastrointestinal diarrheal illness caused by the protozoan parasite Giardia duodenalis, which affects annually over 200 million people worldwide. The limited antigiardial drug arsenal and the emergence of clinical cases refractory to standard treatments dictate the need for new chemotherapeutics. The 14-3-3 family of regulatory proteins, extensively involved in protein-protein interactions (PPIs) with pSer/pThr clients, represents a highly promising target. Despite homology with human counterparts, the single 14-3-3 of G. duodenalis (g14-3-3) is characterized by a constitutive phosphorylation in a region critical for target binding, thus affecting the function and the conformation of g14-3-3/clients interaction. However, to approach the design of specific small molecule modulators of g14-3-3 PPIs, structural elucidations are required. Here, we present a detailed computational and crystallographic study exploring the implications of g14-3-3 phosphorylation on protein structure and target binding. Self-Guided Langevin Dynamics and classical molecular dynamics simulations show that phosphorylation affects locally and globally g14-3-3 conformation, inducing a structural rearrangement more suitable for target binding. Profitable features for g14-3-3/clients interaction were highlighted using a hydrophobicity-based descriptor to characterize g14-3-3 client peptides. Finally, the X-ray structure of g14-3-3 in complex with a mode-1 prototype phosphopeptide was solved and combined with structure-based simulations to identify molecular features relevant for clients binding to g14-3-3. The data presented herein provide a further and structural understanding of g14-3-3 features and set the basis for drug design studies. PMID:26551337

  20. Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor

    SciTech Connect

    Zhao, Jing; Du, Yuhong; Horton, John R.; Upadhyay, Anup K.; Lou, Bin; Bai, Yan; Zhang, Xing; Du, Lupei; Li, Minyong; Wang, Binghe; Zhang, Lixin; Barbieri, Joseph T.; Khuri, Fadlo R.; Cheng, Xiaodong; Fu, Haian

    2013-02-14

    The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kD{sub a} and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3{zeta} in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer.

  1. Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor.

    PubMed

    Zhao, Jing; Du, Yuhong; Horton, John R; Upadhyay, Anup K; Lou, Bin; Bai, Yan; Zhang, Xing; Du, Lupei; Li, Minyong; Wang, Binghe; Zhang, Lixin; Barbieri, Joseph T; Khuri, Fadlo R; Cheng, Xiaodong; Fu, Haian

    2011-09-27

    The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kD(a) and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3ζ in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer. PMID:21908710

  2. A novel sphingosine-dependent protein kinase (SDK1) specifically phosphorylates certain isoforms of 14-3-3 protein.

    PubMed

    Megidish, T; Cooper, J; Zhang, L; Fu, H; Hakomori, S

    1998-08-21

    Protein kinases activated by sphingosine or N,N'-dimethylsphingosine, but not by other lipids, have been detected and are termed sphingosine-dependent protein kinases (SDKs). These SDKs were previously shown to phosphorylate endogenous 14-3-3 proteins (Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1995) Biochem. Biophys. Res. Commun. 216, 739-747). We have now partially purified one SDK, termed SDK1, from cytosol of mouse Balb/c 3T3(A31) fibroblasts. SDK1 is a serine kinase with molecular mass 50-60 kDa that is strongly activated by N, N'-dimethylsphingosine and sphingosine, but not by ceramide, sphingosine 1-phosphate, or other sphingo-, phospho-, or glycerolipids tested. Its activity is inhibited by the protein kinase C activator phosphatidylserine. Activity of SDK1 is clearly distinct from other types of serine kinases tested, including casein kinase II, the alpha and zeta isoforms of protein kinase C, extracellular signal-regulated mitogene-activated protein kinase 1 (Erk-1), Erk-2, and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (eta, beta, zeta) but not others (sigma, tau). The phosphorylation site was identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3 beta. The sigma and tau isoforms of 14-3-3 lack serine at this position, potentially explaining their lack of phosphorylation by SDK1. Interestingly, the phosphorylation site is located on the dimer interface of 14-3-3. Phosphorylation of this site by SDK1 was studied in 14-3-3 mutants. Mutation of a lysine residue, located 9 amino acids N-terminal to the phosphorylation site, abolished 14-3-3 phosphorylation. Furthermore, co-immunoprecipitation experiments demonstrate an association between an SDK and 14-3-3 in situ. Exogenous N, N'-dimethylsphingosine stimulates 14-3-3 phosphorylation in Balb/c 3T3 fibroblasts, suggesting that SDK1 may phosphorylate 14-3-3 in situ. These data support a biological role of SDK1 activation and consequent

  3. 14-3-3 proteins: Macro-regulators with great potential for improving abiotic stress tolerance in plants.

    PubMed

    Liu, Qing; Zhang, Shaohong; Liu, Bin

    2016-08-12

    14-3-3 proteins (14-3-3s) are highly conserved regulatory proteins that are uniquely eukaryotic, and deeply involved in protein-protein interactions that mediate diverse signaling pathways. In plants, 14-3-3s have been validated to regulate many biological processes, such as metabolism, light and hormone signaling, cell-cycle control and protein trafficking. Recent years we have also witnessed an increasing number of reports describing the functions of 14-3-3s in plant stress responses through interactions with key proteins in both biotic and abiotic stresses. In this review, we highlight the advances that have been made in investigating the roles of 14-3-3s in plant abiotic stress tolerance. These advances provide a framework for our understanding of how signals are integrated to perceive and respond to the abiotic stresses in plants. PMID:27233603

  4. 14-3-3 Proteins Bind to the Brassinosteroid Receptor Kinase, BRI1 and are Positive Regulators of Brassinosteroid Signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiple members of the 14-3-3 protein family have been found in all eukaryotes, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client. Thus, 14-3-3s are involved in many processes. The plant brassinosteroid (BR) recepto...

  5. Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3{zeta} protein

    SciTech Connect

    Sadik, Golam; Tanaka, Toshihisa; Kato, Kiyoko; Yanagi, Kentaro; Kudo, Takashi; Takeda, Masatoshi

    2009-05-22

    Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3{zeta}. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3{zeta} is {approx}3-folds higher than that between unphosphorylated 4R-tau and 14-3-3{zeta}. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3{zeta} to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3{zeta}. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3{zeta} exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3{zeta} suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.

  6. Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics.

    PubMed

    Woodcock, Joanna M; Coolen, Carl; Goodwin, Katy L; Baek, Dong Jae; Bittman, Robert; Samuel, Michael S; Pitson, Stuart M; Lopez, Angel F

    2015-06-10

    14-3-3 proteins play a pivotal role in controlling cell proliferation and survival, two commonly dysregulated hallmarks of cancers. 14-3-3 protein expression is enhanced in many human cancers and correlates with more aggressive tumors and poor prognosis, suggesting a role for 14-3-3 proteins in tumorigenesis and/or progression. We showed previously that the dimeric state of 14-3-3 proteins is regulated by the lipid sphingosine, a physiological inducer of apoptosis. As the functions of 14-3-3 proteins are dependent on their dimeric state, this sphingosine-mediated 14-3-3 regulation provides a possible means to target dimeric 14-3-3 for therapeutic effect. However, sphingosine mimics are needed that are not susceptible to sphingolipid metabolism. We show here the identification and optimization of sphingosine mimetics that render dimeric 14-3-3 susceptible to phosphorylation at a site buried in the dimer interface and induce mitochondrial-mediated apoptosis. Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells. Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice. Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery. PMID:25971334

  7. Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic.

    PubMed

    Schmitz, Matthias; Ebert, Elisabeth; Stoeck, Katharina; Karch, André; Collins, Steven; Calero, Miguel; Sklaviadis, Theodor; Laplanche, Jean-Louis; Golanska, Ewa; Baldeiras, Ines; Satoh, Katsuya; Sanchez-Valle, Raquel; Ladogana, Anna; Skinningsrud, Anders; Hammarin, Anna-Lena; Mitrova, Eva; Llorens, Franc; Kim, Yong Sun; Green, Alison; Zerr, Inga

    2016-05-01

    At present, the testing of 14-3-3 protein in cerebrospinal fluid (CSF) is a standard biomarker test in suspected sporadic Creutzfeldt-Jakob disease (sCJD) diagnosis. Increasing 14-3-3 test referrals in CJD reference laboratories in the last years have led to an urgent need to improve established 14-3-3 test methods. The main result of our study was the validation of a commercially available 14-3-3 ELISA next to the commonly used Western blot method as a high-throughput screening test. Hereby, 14-3-3 protein expression was quantitatively analyzed in CSF of 231 sCJD and 2035 control patients. We obtained excellent sensitivity/specificity values of 88 and 96% that are comparable to the established Western blot method. Since standard protocols and preanalytical sample handling have become more important in routine diagnostic, we investigated in a further step the reproducibility and stability of 14-3-3 as a biomarker for human prion diseases. Ring trial data from 2009 to 2013 revealed an increase of Fleiss' kappa from 0.51 to 0.68 indicating an improving reliability of 14-3-3 protein detection. The stability of 14-3-3 protein under short-term and long-term storage conditions at various temperatures and after repeated freezing/thawing cycles was confirmed. Contamination of CSF samples with blood appears likely to be an important factor at a concentration of more than 2500 erythrocytes/μL. Hemolysis of erythrocytes with significant release of 14-3-3 protein started after 2 days at room temperature. We first define clear standards for the sample handling, short- and long-term storage of CSF samples as well as the handling of blood- contaminated samples which may result in artificially elevated CSF levels of 14-3-3. PMID:25947081

  8. Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins

    NASA Technical Reports Server (NTRS)

    Toroser, D.; Athwal, G. S.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    We report an Mg2+-dependent interaction between spinach leaf sucrose-phosphate synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.

  9. 14-3-3 proteins interact with the insulin-like growth factor receptor but not the insulin receptor.

    PubMed Central

    Furlanetto, R W; Dey, B R; Lopaczynski, W; Nissley, S P

    1997-01-01

    We have used a yeast two-hybrid system to identify proteins which bind to the cytosolic portion of the type 1 insulin-like growth factor (IGF) receptor (IGFIR) but not the insulin receptor (IR). This analysis identified 14-3-3beta and zeta proteins. 14-3-3beta also binds to the IGFIR but not the IR in vitro and 14-3-3-IGFIR complexes are present in insect cells overexpressing the IGFIR cytoplasmic domain. 14-3-3 proteins are substrates of the IGFIR in the yeast system and in vitro. The interaction of 14-3-3 with the IGFIR requires receptor-kinase activity and maps to the C-terminus of the receptor, but does not depend on tyrosine residues in this or the juxtamembrane regions. Instead, the binding maps to serine residue 1283 and requires phosphorylation of this residue. 14-3-3 proteins are phosphoserine-binding proteins which have been shown to interact directly with components of the mitogenic and apoptotic signalling pathways, suggesting that they participate in growth regulation. Our findings suggest that 14-3-3 proteins may play a role in IGFIR signal transduction and may contribute to the differences in IGF and IR signalling capabilities. PMID:9581554

  10. Small-Molecule Stabilization of the 14-3-3/Gab2 Protein-Protein Interaction (PPI) Interface.

    PubMed

    Bier, David; Bartel, Maria; Sies, Katharina; Halbach, Sebastian; Higuchi, Yusuke; Haranosono, Yu; Brummer, Tilman; Kato, Nobuo; Ottmann, Christian

    2016-04-19

    Small-molecule modulation of protein-protein interactions (PPIs) is one of the most promising new areas in drug discovery. In the vast majority of cases only inhibition or disruption of PPIs is realized, whereas the complementary strategy of targeted stabilization of PPIs is clearly under-represented. Here, we report the example of a semi-synthetic natural product derivative-ISIR-005-that stabilizes the cancer-relevant interaction of the adaptor protein 14-3-3 and Gab2. The crystal structure of ISIR-005 in complex with 14-3-3 and the binding motif of Gab2 comprising two phosphorylation sites (Gab2pS210pT391) showed how the stabilizing molecule binds to the rim-of-the-interface of the protein complex. Only in the direct vicinity of 14-3-3/Gab2pT391 site is a pre-formed pocket occupied by ISIR-005; binding of the Gab2pS210 motif to 14-3-3 does not create an interface pocket suitable for the molecule. Accordingly, ISIR-005 only stabilizes the binding of the Gab2pT391 but not the Gab2pS210 site. This study represents structural and biochemical proof of the druggability of the 14-3-3/Gab2 PPI interface with important implications for the development of PPI stabilizers. PMID:26644359

  11. Quantitative proteomic dissection of a native 14-3-3ε interacting protein complex associated with hepatocellular carcinoma.

    PubMed

    Bai, Chen; Tang, Siwei; Bai, Chen; Chen, Xian

    2014-04-01

    The 14-3-3 proteins regulate diverse biological processes that are implicated in cancer development, and seven 14-3-3 isoforms were identified with isoform-specific roles in different human tumors. In our previous work, we dissected the interactome of 14-3-3ε formed during the DNA damage response in a hepatocellular carcinoma (HCC) cell using an AACT/SILAC-based quantitative proteomic approach. In this study, we used a similar proteomic approach to profile/identify the 14-3-3ε interactome formed in native HCC cells. Functional categorization and data-dependent network analysis of the native HCC-specific 14-3-3ε interactome revealed that 14-3-3ε is involved in the regulation of multiple biological processes (BPs)/pathways, including cell cycle control, apoptosis, signal transduction, transport, cell adhesion, carbohydrate metabolism, and nucleic acid metabolism. Biological validation further supports that 14-3-3ε, via association with multiple BP/pathway-specific proteins, coordinates the regulation of proliferation, survival, and metastasis of HCC. The findings in this study, together with those of our previous study, provide an extensive profile of the 14-3-3ε interaction network in HCC cells, which should be valuable for understanding the pathology of HCC and HCC therapy. PMID:24363202

  12. Inhibition of the Arabidopsis Salt Overly Sensitive Pathway by 14-3-3 Proteins[C][W

    PubMed Central

    Zhou, Huapeng; Lin, Huixin; Chen, She; Becker, Katia; Yang, Yongqing; Zhao, Jinfeng; Kudla, Jörg; Schumaker, Karen S.; Guo, Yan

    2014-01-01

    The Salt Overly Sensitive (SOS) pathway regulates intracellular sodium ion (Na+) homeostasis and salt tolerance in plants. Until recently, little was known about the mechanisms that inhibit the SOS pathway when plants are grown in the absence of salt stress. In this study, we report that the Arabidopsis thaliana 14-3-3 proteins λ and κ interact with SOS2 and repress its kinase activity. Growth in the presence of salt decreases the interaction between SOS2 and the 14-3-3 proteins, leading to kinase activation in planta. 14-3-3 λ interacts with the SOS2 junction domain, which is important for its kinase activity. A phosphorylation site (Ser-294) is identified within this domain by mass spectrometry. Mutation of Ser-294 to Ala or Asp does not affect SOS2 kinase activity in the absence of the 14-3-3 proteins. However, in the presence of 14-3-3 proteins, the inhibition of SOS2 activity is decreased by the Ser-to-Ala mutation and enhanced by the Ser-to-Asp exchange. These results identify 14-3-3 λ and κ as important regulators of salt tolerance. The inhibition of SOS2 mediated by the binding of 14-3-3 proteins represents a novel mechanism that confers basal repression of the SOS pathway in the absence of salt stress. PMID:24659330

  13. The alternative role of 14-3-3 zeta as a sweeper of misfolded proteins in disease conditions.

    PubMed

    Kaneko, Kiyotoshi; Hachiya, Naomi S

    2006-01-01

    Here, we propose a novel hypothesis that 14-3-3 zeta might act as a sweeper of misfolded proteins by facilitating the formation of aggregates, which are referred to as inclusion bodies. Studies on the localization of the 14-3-3 proteins in different types of inclusion bodies in the brain including neurofibrillary tangle in Alzheimer's disease, pick bodies in Pick's disease, Lewy body-like hyaline inclusions in sporadic amyotrophic lateral sclerosis, prion/florid plaques in sporadic/variant Creutzfeldt-Jakob disease, nuclear inclusions in spinocerebellar ataxia-1, and possibly Lewy bodies in Parkinson's disease suggest a close association of these diseases with 14-3-3 zeta. The highly conserved hydrophobic surface of the amphipathic groove in 14-3-3 zeta represents a general mechanism with diverse cellular proteins, and it may also allow for the molecular recognition of misfolded proteins by hydrophobic interaction in disease conditions. When the abnormal processing of misfolded proteins overwhelms the quality control systems of the cell, it is likely that 14-3-3 zeta is recruited to form deposits of protein aggregates with nonnative, misfolded proteins in order to protect the cell against toxicity. Hence, 14-3-3 zeta may be considered as an auxiliary therapeutic tool in the protein aggregation disorders. PMID:16516399

  14. Proteomic analysis of media from lung cancer cells reveals role of 14-3-3 proteins in cachexia

    PubMed Central

    McLean, Julie B.; Moylan, Jennifer S.; Horrell, Erin M. W.; Andrade, Francisco H.

    2015-01-01

    Aims: At the time of diagnosis, 60% of lung cancer patients present with cachexia, a severe wasting syndrome that increases morbidity and mortality. Tumors secrete multiple factors that contribute to cachectic muscle wasting, and not all of these factors have been identified. We used Orbitrap electrospray ionization mass spectrometry to identify novel cachexia-inducing candidates in media conditioned with Lewis lung carcinoma cells (LCM). Results: One-hundred and 58 proteins were confirmed in three biological replicates. Thirty-three were identified as secreted proteins, including 14-3-3 proteins, which are highly conserved adaptor proteins known to have over 200 binding partners. We confirmed the presence of extracellular 14-3-3 proteins in LCM via western blot and discovered that LCM contained less 14-3-3 content than media conditioned with C2C12 myotubes. Using a neutralizing antibody, we depleted extracellular 14-3-3 proteins in myotube culture medium, which resulted in diminished myosin content. We identified the proposed receptor for 14-3-3 proteins, CD13, in differentiated C2C12 myotubes and found that inhibiting CD13 via Bestatin also resulted in diminished myosin content. Conclusions: Our novel findings show that extracellular 14-3-3 proteins may act as previously unidentified myokines and may signal via CD13 to help maintain muscle mass. PMID:25972815

  15. Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428

    PubMed Central

    Pozuelo Rubio, Mercedes; Campbell, David G.; Morrice, Nicholas A.; Mackintosh, Carol

    2005-01-01

    PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A–14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding. PMID:16153182

  16. Interaction of a 14-3-3 protein with the plant microtubule-associated protein EDE1

    PubMed Central

    Pignocchi, Cristina; Doonan, John H.

    2011-01-01

    Background and Aims The cell cycle-regulated protein ENDOSPERM DEFECTIVE 1 (EDE1) is a novel plant microtubule-associated protein essential for plant cell division and for microtubule organization in endosperm. EDE1 is only present on microtubules at mitosis and its expression is highly cell cycle regulated both at the protein and the transcript levels. Methods To search for EDE1-interacting proteins, a yeast two-hybrid screen was used in which EDE1 was fused with GAL4 DNA binding domain and expressed in a yeast strain that was then mated with a strain carrying a cDNA library fused to the GAL4 transactivation domain. Candidate interacting proteins were identified and confirmed in vitro. Key Results 14-3-3 upsilon was isolated several times from the library screen. In in vitro tests, it also interacted with EDE1: 14-3-3 upsilon most strongly associates with EDE1 in its free form, but also weakly when EDE1 is bound to microtubules. This study shows that EDE1 is a cyclin-dependent kinase substrate but that phosphorylation is not required for interaction with 14-3-3 upsilon. Conclusions The results suggest that 14-3-3 proteins may play a role in cytoskeletal organization of plant cells. The potential role of this interaction in the dynamics of EDE1 during the cell cycle is discussed. PMID:21558460

  17. Identification of 14-3-3 Proteins Phosphopeptide-Binding Specificity Using an Affinity-Based Computational Approach

    PubMed Central

    Li, Zhao; Tang, Jijun; Guo, Fei

    2016-01-01

    The 14-3-3 proteins are a highly conserved family of homodimeric and heterodimeric molecules, expressed in all eukaryotic cells. In human cells, this family consists of seven distinct but highly homologous 14-3-3 isoforms. 14-3-3σ is the only isoform directly linked to cancer in epithelial cells, which is regulated by major tumor suppressor genes. For each 14-3-3 isoform, we have 1,000 peptide motifs with experimental binding affinity values. In this paper, we present a novel method for identifying peptide motifs binding to 14-3-3σ isoform. First, we propose a sampling criteria to build a predictor for each new peptide sequence. Then, we select nine physicochemical properties of amino acids to describe each peptide motif. We also use auto-cross covariance to extract correlative properties of amino acids in any two positions. Finally, we consider elastic net to predict affinity values of peptide motifs, based on ridge regression and least absolute shrinkage and selection operator (LASSO). Our method tests on the 1,000 known peptide motifs binding to seven 14-3-3 isoforms. On the 14-3-3σ isoform, our method has overall pearson-product-moment correlation coefficient (PCC) and root mean squared error (RMSE) values of 0.84 and 252.31 for N–terminal sublibrary, and 0.77 and 269.13 for C–terminal sublibrary. We predict affinity values of 16,000 peptide sequences and relative binding ability across six permutated positions similar with experimental values. We identify phosphopeptides that preferentially bind to 14-3-3σ over other isoforms. Several positions on peptide motifs are in the same amino acid category with experimental substrate specificity of phosphopeptides binding to 14-3-3σ. Our method is fast and reliable and is a general computational method that can be used in peptide-protein binding identification in proteomics research. PMID:26828594

  18. Identification of 14-3-3 Proteins Phosphopeptide-Binding Specificity Using an Affinity-Based Computational Approach.

    PubMed

    Li, Zhao; Tang, Jijun; Guo, Fei

    2016-01-01

    The 14-3-3 proteins are a highly conserved family of homodimeric and heterodimeric molecules, expressed in all eukaryotic cells. In human cells, this family consists of seven distinct but highly homologous 14-3-3 isoforms. 14-3-3σ is the only isoform directly linked to cancer in epithelial cells, which is regulated by major tumor suppressor genes. For each 14-3-3 isoform, we have 1,000 peptide motifs with experimental binding affinity values. In this paper, we present a novel method for identifying peptide motifs binding to 14-3-3σ isoform. First, we propose a sampling criteria to build a predictor for each new peptide sequence. Then, we select nine physicochemical properties of amino acids to describe each peptide motif. We also use auto-cross covariance to extract correlative properties of amino acids in any two positions. Finally, we consider elastic net to predict affinity values of peptide motifs, based on ridge regression and least absolute shrinkage and selection operator (LASSO). Our method tests on the 1,000 known peptide motifs binding to seven 14-3-3 isoforms. On the 14-3-3σ isoform, our method has overall pearson-product-moment correlation coefficient (PCC) and root mean squared error (RMSE) values of 0.84 and 252.31 for N-terminal sublibrary, and 0.77 and 269.13 for C-terminal sublibrary. We predict affinity values of 16,000 peptide sequences and relative binding ability across six permutated positions similar with experimental values. We identify phosphopeptides that preferentially bind to 14-3-3σ over other isoforms. Several positions on peptide motifs are in the same amino acid category with experimental substrate specificity of phosphopeptides binding to 14-3-3σ. Our method is fast and reliable and is a general computational method that can be used in peptide-protein binding identification in proteomics research. PMID:26828594

  19. CSF Tau proteins reduce misdiagnosis of sporadic Creutzfeldt-Jakob disease suspected cases with inconclusive 14-3-3 result.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-09-01

    Cerebrospinal fluid (CSF) 14-3-3 protein supports sporadic Creutzfeldt-Jakob (sCJD) diagnosis, but often leads to weak-positive results and lacks standardization. In this study, we explored the added diagnostic value of Total Tau (t-Tau) and phosphorylated Tau (p-Tau) in sCJD diagnosis, particularly in the cases with inconclusive 14-3-3 result. 95 definite sCJD and 287 patients without prion disease (non-CJD) were included in this study. CSF samples were collected in routine clinical diagnosis and analysed for 14-3-3 detection by Western blot (WB). CSF t-Tau and p-Tau were quantified by commercial ELISA kits and PRNP and APOE genotyping assessed by PCR-RFLP. In a regression analysis of the whole cohort, 14-3-3 protein revealed an overall accuracy of 82 % (sensitivity = 96.7 %; specificity = 75.6 %) for sCJD. Regarding 14-3-3 clear positive results, we observed no added value either of t-Tau alone or p-Tau/t-Tau ratio in the model. On the other hand, considering 14-3-3 weak-positive cases, t-Tau protein increased the overall accuracy of 14-3-3 alone from 91 to 94 % and specificity from 74 to 93 % (p < 0.05), with no sensitivity improvement. However, inclusion of p-Tau/t-Tau ratio did not significantly improve the first model (p = 0.0595). Globally, t-Tau protein allowed a further discrimination of 65 % within 14-3-3 inconclusive results. Furthermore, PRNP MV genotype showed a trend to decrease 14-3-3 sensitivity (p = 0.051), but such effect was not seen on t-Tau protein. In light of these results, we suggest that t-Tau protein assay is of significant importance as a second marker in identifying 14-3-3 false-positive results among sCJD probable cases. PMID:27357003

  20. Modulation of GluK2a subunit-containing kainate receptors by 14-3-3 proteins.

    PubMed

    Sun, Changcheng; Qiao, Haifa; Zhou, Qin; Wang, Yan; Wu, Yuying; Zhou, Yi; Li, Yong

    2013-08-23

    Kainate receptors (KARs) are one of the ionotropic glutamate receptors that mediate excitatory postsynaptic currents (EPSCs) with characteristically slow kinetics. Although mechanisms for the slow kinetics of KAR-EPSCs are not totally understood, recent evidence has implicated a regulatory role of KAR-associated proteins. Here, we report that decay kinetics of GluK2a-containing receptors is modulated by closely associated 14-3-3 proteins. 14-3-3 binding requires PKC-dependent phosphorylation of serine residues localized in the carboxyl tail of the GluK2a subunit. In transfected cells, 14-3-3 binding to GluK2a slows desensitization kinetics of both homomeric GluK2a and heteromeric GluK2a/GluK5 receptors. Moreover, KAR-EPSCs at mossy fiber-CA3 synapses decay significantly faster in the 14-3-3 functional knock-out mice. Collectively, these results demonstrate that 14-3-3 proteins are an important regulator of GluK2a-containing KARs and may contribute to the slow decay kinetics of native KAR-EPSCs. PMID:23861400

  1. A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis

    PubMed Central

    Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Chen, Chao; Qin, Zhiwei; Yang, Kejun; Shen, Yang; Meiping, Zhang; Mingyang, Cong; Zhu, Yanming

    2015-01-01

    It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis. PMID:26717241

  2. 14-3-3 family members act coordinately to regulate mitotic progression.

    PubMed

    Dalal, Sorab N; Yaffe, Michael B; DeCaprio, James A

    2004-05-01

    The mitosis promoting phosphatase, cdc25C, is a target of both the DNA replication and DNA damage checkpoint pathways. These pathways regulate cdc25C function, in part, by promoting the association of cdc25C with 14-3-3 proteins, which results in the retention of cdc25C in the cytoplasm. To determine which 14-3-3 proteins were required to regulate cdc25C function, we tested the ability of various 14-3-3 family members to form a complex with and negatively regulate cdc25C in human cells. Two 14-3-3 family members, 14-3-3epsilon and 14-3-3gamma specifically formed a complex with cdc25C but not with the 14-3-3 binding defective cdc25C mutant, S216A. In addition, 14-3-3epsilon and 14-3-3gamma inhibited the ability of cdc25C, but not the S216A mutant, to induce premature chromatin condensation (PCC) in U-2OS cells. These results suggested that the reduction in PCC by 14-3-3epsilon and 14-3-3gamma was due to inhibition of cdc25C function. In contrast, 14-3-3sigma was unable to form a complex with cdc25C, but was able to inhibit the ability of both wild type cdc25C and S216A to induce PCC. This suggests that 14-3-3sigma regulates entry into mitosis independently of cdc25C and 14-3-3epsilon and 14-3-3gamma. Thus, specific members of the 14-3-3 family of proteins may act coordinately to maintain the DNA replication checkpoint by regulating the activity of different cell cycle proteins. PMID:15107609

  3. Interaction of 14-3-3 proteins with the Estrogen Receptor Alpha F domain provides a drug target interface

    PubMed Central

    De Vries-van Leeuwen, Ingrid J.; da Costa Pereira, Daniel; Flach, Koen D.; Piersma, Sander R.; Haase, Christian; Bier, David; Yalcin, Zeliha; Michalides, Rob; Feenstra, K. Anton; Jiménez, Connie R.; de Greef, Tom F. A.; Brunsveld, Luc; Ottmann, Christian; Zwart, Wilbert; de Boer, Albertus H.

    2013-01-01

    Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERα dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ERα dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ERα/14-3-3 interaction. Cocrystallization of the trimeric ERα/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERα-T594) for high-affinity interaction. We confirm that T594 is a distinct ERα phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T594–specific antibody and by mass spectrometry. In line with its ERα/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERα dimerization, inhibits ERα/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERα are provided, together with a small molecule that selectively targets this ERα/14-3-3 interface. PMID:23676274

  4. Revealing the binding modes and the unbinding of 14-3-3σ proteins and inhibitors by computational methods

    PubMed Central

    Hu, Guodong; Cao, Zanxia; Xu, Shicai; Wang, Wei; Wang, Jihua

    2015-01-01

    The 14-3-3σ proteins are a family of ubiquitous conserved eukaryotic regulatory molecules involved in the regulation of mitogenic signal transduction, apoptotic cell death, and cell cycle control. A lot of small-molecule inhibitors have been identified for 14-3-3 protein-protein interactions (PPIs). In this work, we carried out molecular dynamics (MD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) method to study the binding mechanism between a 14-3-3σ protein and its eight inhibitors. The ranking order of our calculated binding free energies is in agreement with the experimental results. We found that the binding free energies are mainly from interactions between the phosphate group of the inhibitors and the hydrophilic residues. To improve the binding free energy of Rx group, we designed the inhibitor R9 with group R9 = 4-hydroxypheny. However, we also found that the binding free energy of inhibitor R9 is smaller than that of inhibitor R1. By further using the steer molecular dynamics (SMD) simulations, we identified a new hydrogen bond between the inhibitor R8 and residue Arg64 in the pulling paths. The information obtained from this study may be valuable for future rational design of novel inhibitors, and provide better structural understanding of inhibitor binding to 14-3-3σ proteins. PMID:26568041

  5. Identification of a host 14-3-3 Protein that Interacts with Xanthomonas effector AvrRxv

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AvrRxv is a member of a family of pathogen effectors from both plant and mammalian pathogens. Using a yeast two hybrid screen, we identified a 14-3-3 protein from tomato that interacts with AvrRxv called AvrRxv Interactor 1 (ARI1). The interaction was confirmed in vitro with affinity chromatograph...

  6. Protein Modifications Regulate the Role of 14-3-3γ Adaptor Protein in cAMP-induced Steroidogenesis in MA-10 Leydig Cells*

    PubMed Central

    Aghazadeh, Yasaman; Ye, Xiaoying; Blonder, Josip; Papadopoulos, Vassilios

    2014-01-01

    The 14-3-3 protein family comprises adaptors and scaffolds that regulate intracellular signaling pathways. The 14-3-3γ isoform is a negative regulator of steroidogenesis that is hormonally induced and transiently functions at the initiation of steroidogenesis by delaying maximal steroidogenesis in MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with the cAMP analog 8-bromo-cAMP (8-Br-cAMP), which stimulates steroidogenesis, triggers the interaction of 14-3-3γ with the steroidogenic acute regulatory protein (STAR) in the cytosol, limiting STAR activity to basal levels. Over time, this interaction ceases, allowing for a 2-fold induction in STAR activity and maximal increase in the rate of steroid formation. The 14-3-3γ/STAR pattern of interaction was found to be opposite that of the 14-3-3γ homodimerization pattern. Phosphorylation and acetylation of 14-3-3γ showed similar patterns to homodimerization and STAR binding, respectively. 14-3-3γ Ser58 phosphorylation and 14-3-3γ Lys49 acetylation were blocked using trans-activator of HIV transcription factor 1 peptides coupled to 14-3-3γ sequences containing Ser58 or Lys49. Blocking either one of these modifications further induced 8-Br-cAMP-induced steroidogenesis while reducing lipid storage, suggesting that the stored cholesterol is used for steroid formation. Taken together, these results indicate that Ser58 phosphorylation and Lys49 acetylation of 14-3-3γ occur in a coordinated time-dependent manner to regulate 14-3-3γ homodimerization. 14-3-3γ Ser58 phosphorylation is required for STAR interactions under control conditions, and 14-3-3γ Lys49 acetylation is important for the cAMP-dependent induction of these interactions. PMID:25086053

  7. Binding and Transcriptional Regulation by 14-3-3 (Bmh) Proteins Requires Residues Outside of the Canonical Motif

    PubMed Central

    Parua, Pabitra K.

    2014-01-01

    Evolutionarily conserved 14-3-3 proteins have important functions as dimers in numerous cellular signaling processes, including regulation of transcription. Yeast 14-3-3 proteins, known as Bmh, inhibit a post-DNA binding step in transcription activation by Adr1, a glucose-regulated transcription factor, by binding to its regulatory domain, residues 226 to 240. The domain was originally defined by regulatory mutations, ADR1c alleles that alter activator-dependent gene expression. Here, we report that ADR1c alleles and other mutations in the regulatory domain impair Bmh binding and abolish Bmh-dependent regulation both directly and indirectly. The indirect effect is caused by mutations that inhibit phosphorylation of Ser230 and thus inhibit Bmh binding, which requires phosphorylated Ser230. However, several mutations inhibit Bmh binding without inhibiting phosphorylation and thus define residues that provide important interaction sites between Adr1 and Bmh. Our proposed model of the Adr1 regulatory domain bound to Bmh suggests that residues Ser238 and Tyr239 could provide cross-dimer contacts to stabilize the complex and that this might explain the failure of a dimerization-deficient Bmh mutant to bind Adr1 and to inhibit its activity. A bioinformatics analysis of Bmh-interacting proteins suggests that residues outside the canonical 14-3-3 motif might be a general property of Bmh target proteins and might help explain the ability of 14-3-3 to distinguish target and nontarget proteins. Bmh binding to the Adr1 regulatory domain, and its failure to bind when mutations are present, explains at a molecular level the transcriptional phenotype of ADR1c mutants. PMID:24142105

  8. Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7.

    PubMed

    Mohammad, Dara K; Nore, Beston F; Gustafsson, Manuela O; Mohamed, Abdalla J; Smith, C I Edvard

    2016-09-01

    The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK's interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK's binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK's interaction with Importin 7. PMID:27381982

  9. Proteomic screen in the simple metazoan Hydra identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Ca2+ signalling

    PubMed Central

    Pauly, Barbara; Lasi, Margherita; MacKintosh, Carol; Morrice, Nick; Imhof, Axel; Regula, Jörg; Rudd, Stephen; David, Charles N; Böttger, Angelika

    2007-01-01

    Background 14-3-3 proteins have been implicated in many signalling mechanisms due to their interaction with Ser/Thr phosphorylated target proteins. They are evolutionarily well conserved in eukaryotic organisms from single celled protozoans and unicellular algae to plants and humans. A diverse array of target proteins has been found in higher plants and in human cell lines including proteins involved in cellular metabolism, apoptosis, cytoskeletal organisation, secretion and Ca2+ signalling. Results We found that the simple metazoan Hydra has four 14-3-3 isoforms. In order to investigate whether the diversity of 14-3-3 target proteins is also conserved over the whole animal kingdom we isolated 14-3-3 binding proteins from Hydra vulgaris using a 14-3-3-affinity column. We identified 23 proteins that covered most of the above-mentioned groups. We also isolated several novel 14-3-3 binding proteins and the Hydra specific secreted fascin-domain-containing protein PPOD. In addition, we demonstrated that one of the 14-3-3 isoforms, 14-3-3 HyA, interacts with one Hydra-Bcl-2 like protein in vitro. Conclusion Our results indicate that 14-3-3 proteins have been ubiquitous signalling components since the start of metazoan evolution. We also discuss the possibility that they are involved in the regulation of cell numbers in response to food supply in Hydra. PMID:17651497

  10. ISOFORM-SPECIFIC BINDING OF 14-3-3 PROTEINS TO NITRATE REDUCTASE AND THE BRASSINOSTEROID INSENSITIVE 1 RECEPTOR KINASE SIGNALING COMPLEX

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 14-3-3 proteins are known to bind many different soluble protein clients, but less is known about binding to integral membrane proteins, and in both cases the issue of isoform specificity remains largely unexplored. Using an array of anti-14-3-3 antibodies and 2-dimensional electrophoresis (2-DE...

  11. Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins*

    PubMed Central

    Yasuda, Shigetaka; Sato, Takeo; Maekawa, Shugo; Aoyama, Shoki; Fukao, Yoichiro; Yamaguchi, Junji

    2014-01-01

    Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr209, Ser247, Ser270, and Ser303 as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr209 and Ser247 on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr209 peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response. PMID:24722992

  12. Functions of Saccharomyces cerevisiae 14-3-3 proteins in response to DNA damage and to DNA replication stress.

    PubMed Central

    Lottersberger, Francisca; Rubert, Fabio; Baldo, Veronica; Lucchini, Giovanna; Longhese, Maria Pia

    2003-01-01

    Two members of the 14-3-3 protein family, involved in key biological processes in different eukaryotes, are encoded by the functionally redundant Saccharomyces cerevisiae BMH1 and BMH2 genes. We produced and characterized 12 independent bmh1 mutant alleles, whose presence in the cell as the sole 14-3-3 source causes hypersensitivity to genotoxic agents, indicating that Bmh proteins are required for proper response to DNA damage. In particular, the bmh1-103 and bmh1-266 mutant alleles cause defects in G1/S and G2/M DNA damage checkpoints, whereas only the G2/M checkpoint is altered by the bmh1-169 and bmh1-221 alleles. Impaired checkpoint responses correlate with the inability to maintain phosphorylated forms of Rad53 and/or Chk1, suggesting that Bmh proteins might regulate phosphorylation/dephosphorylation of these checkpoint kinases. Moreover, several bmh1 bmh2Delta mutants are defective in resuming DNA replication after transient deoxynucleotide depletion, and all display synthetic effects when also carrying mutations affecting the polalpha-primase and RPA DNA replication complexes, suggesting a role for Bmh proteins in DNA replication stress response. Finally, the bmh1-169 bmh2Delta and bmh1-170 bmh2Delta mutants show increased rates of spontaneous gross chromosomal rearrangements, indicating that Bmh proteins are required to suppress genome instability. PMID:14704161

  13. The 14-3-3 protein PAR-5 regulates the asymmetric localization of the LET-99 spindle positioning protein.

    PubMed

    Wu, Jui-Ching; Espiritu, Eugenel B; Rose, Lesilee S

    2016-04-15

    PAR proteins play important roles in establishing cytoplasmic polarity as well as regulating spindle positioning during asymmetric division. However, the molecular mechanisms by which the PAR proteins generate asymmetry in different cell types are still being elucidated. Previous studies in Caenorhabditis elegans revealed that PAR-3 and PAR-1 regulate the asymmetric localization of LET-99, which in turn controls spindle positioning by affecting the distribution of the conserved force generating complex. In wild-type embryos, LET-99 is localized in a lateral cortical band pattern, via inhibition at the anterior by PAR-3 and at the posterior by PAR-1. In this report, we show that the 14-3-3 protein PAR-5 is also required for cortical LET-99 asymmetry. PAR-5 associated with LET-99 in pull-down assays, and two PAR-5 binding sites were identified in LET-99 using the yeast two-hybrid assay. Mutation of these sites abolished binding in yeast and altered LET-99 localization in vivo: LET-99 was present at the highest levels at the posterior pole of the embryo instead of a band in par-5 embryos. Together the results indicate that PAR-5 acts in a mechanism with PAR-1 to regulate LET-99 cortical localization. PMID:26921457

  14. Transcription variants of the prostate-specific PrLZ gene and their interaction with 14-3-3 proteins

    SciTech Connect

    Wang, Ruoxiang; He, Hui; Sun, Xiaojuan; Xu, Jianchun; Marshall, Fray F.; Zhau, Haiyen; Chung, Leland W.K.; Fu, Haian; He, Dalin

    2009-11-20

    We have reported isolation and characterization of the prostate-specific and androgen-regulated PrLZ gene abnormally expressed in prostate cancer. PrLZ is a potential biomarker for prostate cancer and a candidate oncogene promoting cell proliferation and survival in prostate cancer cells. A full delineation of the PrLZ gene and its gene products may provide clues to the mechanisms regulating its expression and function. In this report, we identified three additional exons in the PrLZ gene and recognized five transcript variants from alternative splicing that could be detected by RT-PCR and Western blotting. Structural comparison demonstrated that the PrLZ proteins are highly conserved among species. PrLZ contains multiple potential sites for interaction with other proteins. We used mammalian two-hybrid assays to demonstrate that PrLZ isoforms interact with 14-3-3 proteins, and multiple sites in the PrLZ may be involved in the interaction. Alternative splicing may contribute to abnormally enhanced PrLZ levels in prostate cancer, and interaction with 14-3-3 proteins may be a mechanism by which PrLZ promotes cell proliferation and survival during prostate cancer development and progression. This information is a valuable addition to the investigation of the oncogenic properties of the PrLZ gene.

  15. Spinach 14-3-3 protein interacts with the plasma membrane H(+)-ATPase and nitrate reductase in response to excess nitrate stress.

    PubMed

    Xu, Huini; Zhao, Xiuling; Guo, Chuanlong; Chen, Limei; Li, Kunzhi

    2016-09-01

    To investigate the function of 14-3-3 protein in response to excess nitrate stress, a 14-3-3 protein, designated as So14-3-3, was isolated from spinach. Phylogenetic analysis demonstrated that So14-3-3 belongs to non-ε group of 14-3-3 superfamily. Real time-quantitative RT-PCR and western blot analysis showed that So14-3-3 was induced by excess nitrate stress in spinach roots and leaves. After nitrate treatment, the phosphorylated H(+)-ATPase and nitrate reductase (NR) increased and decreased respectively. Co-Immunoprecipitation (Co-IP) suggested that the interaction of So14-3-3 with the phosphorylated H(+)-ATPase enhanced, but reduced with phosphorylated NR in spinach roots after nitrate treatment. Besides, 5 proteins interacted with So14-3-3 were found by Co-IP and LC-MS/MS analysis. So14-3-3 overexpressing transgenic tobacco plants showed enhanced tolerance to nitrate treatment at the germination and young seedlings stage. The transgenic plants showed longer root length, lower malondialdehyde (MDA), H2O2, protein carbonyl contents, relatively higher soluble sugar and protein contents, than the WT plants after nitrate treatment. The phosphorylation levels of H(+)-ATPase in transgenic plants were higher than the WT plants after nitrate treatment, whereas NR were lower. Additionally, in transgenic plants, the interaction of So14-3-3 with phosphorylated H(+)-ATPase and NR increased and decreased more than the WT plants under nitrate stress, leading to higher H(+)-ATPase and NR activities in transgenic plants. These data suggested that So14-3-3 might be involved in nitrate stress response by interacting with H(+)-ATPase and NR. PMID:27161584

  16. Overexpression of the 14-3-3gamma protein in embryonic mice results in neuronal migration delay in the developing cerebral cortex.

    PubMed

    Cornell, Brett; Wachi, Tomoka; Zhukarev, Vladimir; Toyo-Oka, Kazuhito

    2016-08-15

    The 14-3-3 protein family is a group of multifunctional proteins that are highly expressed in the brain; however, their functions in brain development are largely unknown. Williams Syndrome is a neurodevelopmental disorder caused by a deletion in the 7q11.23 chromosome locus, including the gene encoding 14-3-3gamma, resulting in developmental delay, intellectual disabilities and epilepsy. We have previously shown that knocking down the 14-3-3gamma protein in utero in mice results in delays in neuronal migration of pyramidal neurons in the cortex. Importantly, there is a reciprocal duplication syndrome to Williams Syndrome where the 7q11.23 locus is duplicated, resulting in epilepsy and intellectual disabilities. Thus, the deletion or the duplication of the 7q11.23 chromosome locus results in epilepsy. Taken together with the fact that defects in neuronal migration are one of main causes for epilepsy, we analyzed if the overexpression of 14-3-3gamma causes neuronal migration defects. In this work, we found that the overexpression of 14-3-3gamma in utero in the developing mouse cortex results in delays in pyramidal neuron migration, similar to what was previously observed when 14-3-3gamma was knocked down. These results, in conjunction with our previous research, indicate that a balance of 14-3-3gamma expression is required during cortical development to prevent delays in neuronal migration. This work provides clear evidence as to the involvement of 14-3-3gamma in neurodevelopmental disorders and how a disruption in 14-3-3gamma expression may contribute to the neurodevelopmental disorders that manifest when the 7q11.23 locus is altered. PMID:27288018

  17. 14-3-3 proteins sequester a pool of soluble TRIM32 ubiquitin ligase to repress autoubiquitylation and cytoplasmic body formation.

    PubMed

    Ichimura, Tohru; Taoka, Masato; Shoji, Ikuo; Kato, Hiroki; Sato, Tomonobu; Hatakeyama, Shigetsugu; Isobe, Toshiaki; Hachiya, Naomi

    2013-05-01

    Deregulated expression of tripartite motif-containing protein 32 (TRIM32, an E3 ubiquitin-protein ligase) contributes to various diseases. Here we report, using quantitative proteomics and biochemistry, that 14-3-3 proteins bind to phosphorylated TRIM32 and prevent TRIM32 autoubiquitylation and the formation of TRIM32-containing cytoplasmic bodies, which are potential autoregulatory mechanisms that can reduce the concentration of soluble free TRIM32. The 14-3-3-TRIM32 interaction is dependent on protein-kinase-A-catalyzed phosphorylation of TRIM32 at Ser651. We found that the inhibitory effect of 14-3-3 is, in part, a consequence of disrupting the propensity of TRIM32 to undergo higher-order self-association without affecting its dimerization. Consequently, dimerized TRIM32 bound to 14-3-3 was sequestered in a distinct cytoplasmic pool away from the microtubule network, whereas a TRIM32 mutant that cannot bind 14-3-3 underwent multimerization and was unavailable to facilitate cell growth. Our results reveal a novel connection between ubiquitylation and phosphorylation pathways, which could modulate a variety of cell events by stimulating the formation of the 14-3-3-TRIM32 signaling complex. PMID:23444366

  18. Association of 14-3-3 Proteins to β1-Adrenergic Receptors Modulates Kv11.1 K+ Channel Activity in Recombinant Systems

    PubMed Central

    Tutor, Antonio S.; Delpón, Eva; Caballero, Ricardo; Gómez, Ricardo; Núñez, Lucía; Vaquero, Miguel; Tamargo, Juan; Penela, Petronila

    2006-01-01

    We identify a new mechanism for the β1-adrenergic receptor (β1AR)-mediated regulation of human ether-a-go-go–related gene (HERG) potassium channel (Kv11.1). We find that the previously reported modulatory interaction between Kv11.1 channels and 14-3-3ε proteins is competed by wild type β1AR by means of a novel interaction between this receptor and 14-3-3ε. The association between β1AR and 14-3-3ε is increased by agonist stimulation in both transfected cells and heart tissue and requires cAMP-dependent protein kinase (PKA) activity. The β1AR/14-3-3ε association is direct, since it can be recapitulated using purified 14-3-3ε and β1AR fusion proteins and is abolished in cells expressing β1AR phosphorylation–deficient mutants. Biochemical and electrophysiological studies of the effects of isoproterenol on Kv11.1 currents recorded using the whole-cell patch clamp demonstrated that β1AR phosphorylation–deficient mutants do not recruit 14-3-3ε away from Kv11.1 and display a markedly altered agonist-mediated modulation of Kv11.1 currents compared with wild-type β1AR, increasing instead of inhibiting current amplitudes. Interestingly, such differential modulation is not observed in the presence of 14-3-3 inhibitors. Our results suggest that the dynamic association of 14-3-3 proteins to both β1AR and Kv11.1 channels is involved in the adrenergic modulation of this critical regulator of cardiac repolarization and refractoriness. PMID:16914520

  19. Identification of 14-3-3 proteins as a target of ATL31 ubiquitin ligase, a regulator of the C/N response in Arabidopsis.

    PubMed

    Sato, Takeo; Maekawa, Shugo; Yasuda, Shigetaka; Domeki, Yukie; Sueyoshi, Kuni; Fujiwara, Masayuki; Fukao, Yoichiro; Goto, Derek B; Yamaguchi, Junji

    2011-10-01

    The balance between carbon (C) and nitrogen (N) availability is an important determinant for various phases of plant growth; however, the detailed mechanisms regulating the C/N response are not well understood. We previously described two related ubiquitin ligases, ATL31 and ATL6, that function in the C/N response in Arabidopsis thaliana. Here, we used FLAG tag affinity purification and MS analysis to identify proteins targeted by ATL31, and thus likely to be involved in regulating the phase transition checkpoint based on C/N status. This analysis revealed that 14-3-3 proteins were associated with ATL31, and one of these, 14-3-3χ, was selected for detailed characterization. The interaction between ATL31 and 14-3-3χ was confirmed by yeast two-hybrid and co-immunoprecipitation analyses. In vitro assays showed that ubiquitination of 14-3-3χ is catalyzed by ATL31. Degradation of 14-3-3χin vivo was shown to be correlated with ATL31 activity, and to occur in a proteasome-dependent manner. Furthermore, 14-3-3 protein accumulation was induced by a shift to high-C/N stress conditions in Arabidopsis seedlings, and this regulated response required both ATL31 and ATL6. It was also shown that over-expression of 14-3-3χ leads to hypersensitivity of Arabidopsis seedlings to C/N stress conditions. These results indicate that ATL31 targets and ubiquitinates 14-3-3 proteins for degradation via the ubiquitin-proteasome system during the response to cellular C/N status. PMID:21668537

  20. Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein.

    PubMed Central

    Svennelid, F; Olsson, A; Piotrowski, M; Rosenquist, M; Ottman, C; Larsson, C; Oecking, C; Sommarin, M

    1999-01-01

    The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth. PMID:10590165

  1. Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family.

    PubMed

    Uhart, Marina; Flores, Gabriel; Bustos, Diego M

    2016-01-01

    Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

  2. Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family

    PubMed Central

    Uhart, Marina; Flores, Gabriel; Bustos, Diego M.

    2016-01-01

    Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

  3. 14-3-3 isoforms bind directly exon B of the 5′-UTR of human surfactant protein A2 mRNA

    PubMed Central

    Noutsios, Georgios T.; Ghattas, Paul; Bennett, Stephanie

    2015-01-01

    Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5′-untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722–L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5′-UTR mRNA. PMID:26001776

  4. Properties of the monomeric form of human 14-3-3ζ protein and its interaction with tau and HspB6.

    PubMed

    Sluchanko, Nikolai N; Sudnitsyna, Maria V; Seit-Nebi, Alim S; Antson, Alfred A; Gusev, Nikolai B

    2011-11-15

    Dimers formed by seven isoforms of the human 14-3-3 protein participate in multiple cellular processes. The dimeric form has been extensively characterized; however, little is known about the structure and properties of the monomeric form of 14-3-3. The monomeric form is involved in the assembly of homo- and heterodimers, which could partially dissociate back into monomers in response to phosphorylation at Ser58. To obtain monomeric forms of human 14-3-3ζ, we produced four protein constructs with different combinations of mutated (M) or wild-type (W) segments E(5), (12)LAE(14), and (82)YREKIE(87). Under a wide range of expression conditions in Escherichia coli, the MMM and WMM mutants were insoluble, whereas WMW and MMW mutants were soluble, highly expressed, and purified to homogeneity. WMW and MMW mutants remained monomeric over a wide range of concentrations while retaining the α-helical structure characteristic of wild-type 14-3-3. However, WMW and MMW mutants were highly susceptible to proteolysis and had much lower thermal stabilities than the wild-type protein. Using WMW and MMW mutants, we show that the monomeric form interacts with the tau protein and with the HspB6 protein, in both cases forming complexes with a 1:1 stoichiometry, in contrast to the 2:1 and/or 2:2 complexes formed by wild-type 14-3-3. Significantly, this interaction requires phosphorylation of tau protein and HspB6. Because of minimal changes in structure, MMW and especially WMW mutant proteins are promising candidates for analyzing the effect of monomerization on the physiologically important properties of 14-3-3ζ. PMID:21978388

  5. The Saccharomyces cerevisiae 14-3-3 proteins are required for the G1/S transition, actin cytoskeleton organization and cell wall integrity.

    PubMed

    Lottersberger, Francisca; Panza, Andrea; Lucchini, Giovanna; Piatti, Simonetta; Longhese, Maria Pia

    2006-06-01

    14-3-3 proteins are highly conserved polypeptides that participate in many biological processes by binding phosphorylated target proteins. The Saccharomyces cerevisiae BMH1 and BMH2 genes, whose concomitant deletion is lethal, encode two functionally redundant 14-3-3 isoforms. To gain insights into the essential function(s) shared by these proteins, we searched for high-dosage suppressors of the growth defects of temperature-sensitive bmh mutants. Both the protein kinase C1 (Pkc1) and its upstream regulators Wsc2 and Mid2 were found to act as high dosage suppressors of bmh mutants' temperature sensitivity, indicating a functional interaction between 14-3-3 and Pkc1. Consistent with a role of 14-3-3 proteins in Pkc1-dependent cellular processes, shift to the restrictive temperature of bmh mutants severely impaired initiation of DNA replication, polarization of the actin cytoskeleton, and budding, as well as cell wall integrity. Because Pkc1 acts in concert with the Swi4-Swi6 (SBF) transcriptional activator to control all these processes, the defective G(1)/S transition of bmh mutants might be linked to impaired SBF activity. Indeed, the levels of the G(1) cyclin CLN2 transcripts, which are positively regulated by SBF, were dramatically reduced in bmh mutants. Remarkably, budding and DNA replication defects of bmh mutants were suppressed by CLN2 expression from an SBF-independent promoter, suggesting that 14-3-3 proteins might contribute to regulating the late G(1) transcriptional program. PMID:16648583

  6. Observation of interaction between bid and 14-3-3 proteins by FRET in living cell during TNF-a-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Wang, Jinjun; Chen, Tongsheng; Xing, Da; Wang, Fang

    2005-01-01

    Caspase8 is activated and cleaves Bid into two fragments when cells are exposed to death-inducing molecules such as tumor necrosis factor-α (TNF-α). Then the C-terminal fragment relocates from cytosol to mitochondria and promotes the release of cytochrome c, in the final cellular apoptosis is induced. Despite recent progress in the study of Bid during apoptosis induction, it remains unclear how C-terminal fragment of Bid cleaved moves to mitochondria and then induces the release of cytochrome c and so on. The 14-3-3 proteins are known to sequester certain pro-apoptotic members of Bcl-2 family. In order to further study the biological action of Bid during apoptosis, especially under physiological condition of living cell, the plasmids pBid-CFP and pYFP-14-3-3 were constructed. By the transient transfection of pBid-CFP and pYFP-14-3-3, the dynamic process of interaction of Bid and 14-3-3 protein in individual living cell during the apoptosis was primarily investigated with FRET (fluorescent resonance energy transfer) technique by the use of fluorescence microscopy.

  7. Protein Kinase CK2 Interacts at the Neuromuscular Synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 Proteins and Phosphorylates the Latter Two*

    PubMed Central

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-01-01

    Previously, we demonstrated that the protein kinase CK2 associates with and phosphorylates the receptor tyrosine kinase MuSK (muscle specific receptor tyrosine kinase) at the neuromuscular junction (NMJ), thereby preventing fragmentation of the NMJs (Cheusova, T., Khan, M. A., Schubert, S. W., Gavin, A. C., Buchou, T., Jacob, G., Sticht, H., Allende, J., Boldyreff, B., Brenner, H. R., and Hashemolhosseini, S. (2006) Genes Dev. 20, 1800–1816). Here, we asked whether CK2 interacts with other proteins involved in processes at the NMJ, which would be consistent with the previous observation that CK2 appears enriched at the NMJ. We identified the following proteins to interact with protein kinase CK2: (a) the α and β subunits of the nicotinic acetylcholine receptors with weak interaction, (b) dishevelled (Dsh), and (c) another four proteins, Rapsyn, Rac1, 14-3-3γ, and Dok-7, with strong interaction. CK2 phosphorylated 14-3-3γ at serine residue 235 and Dok-7 at several serine residues but does not phosphorylate Rapsyn or Rac1. Furthermore, phosphomimetic Dok-7 mutants aggregated nicotinic acetylcholine receptors in C2C12 myotubes with significantly higher frequency than wild type Dok-7. Additionally, we mapped the interacting epitopes of all four binding partners to CK2 and thereby gained insights into the potential role of the CK2/Rapsyn interaction. PMID:26198629

  8. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    SciTech Connect

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.; E-mail: andy.blakely@vanderbilt.edu

    2005-08-05

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH{sub 2}-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.

  9. Rapid antidepressants stimulate the decoupling of GABAB receptors from GIRK/Kir3 channels through increased protein stability of 14-3-3η

    PubMed Central

    Workman, E R; Haddick, P C G; Bush, K; Dilly, G A; Niere, F; Zemelman, B V; Raab-Graham, K F

    2015-01-01

    A single injection of N-methyl-D-aspartate receptor (NMDAR) antagonists produces a rapid antidepressant response. Lasting changes in the synapse structure and composition underlie the effectiveness of these drugs. We recently discovered that rapid antidepressants cause a shift in the γ-aminobutyric acid receptor (GABABR) signaling pathway, such that GABABR activation shifts from opening inwardly rectifiying potassium channels (Kir/GIRK) to increasing resting dendritic calcium signal and mammalian Target of Rapamycin activity. However, little is known about the molecular and biochemical mechanisms that initiate this shift. Herein, we show that GABABR signaling to Kir3 (GIRK) channels decreases with NMDAR blockade. Blocking NMDAR signaling stabilizes the adaptor protein 14-3-3η, which decouples GABABR signaling from Kir3 and is required for the rapid antidepressant efficacy. Consistent with these results, we find that key proteins involved in GABABR signaling bidirectionally change in a depression model and with rapid antidepressants. In socially defeated rodents, a model for depression, GABABR and 14-3-3η levels decrease in the hippocampus. The NMDAR antagonists AP5 and Ro-25-6981, acting as rapid antidepressants, increase GABABR and 14-3-3η expression and decrease Kir3.2. Taken together, these data suggest that the shift in GABABR function requires a loss of GABABR-Kir3 channel activity mediated by 14-3-3η. Our findings support a central role for 14-3-3η in the efficacy of rapid antidepressants and define a critical molecular mechanism for activity-dependent alterations in GABABR signaling. PMID:25560757

  10. Anchoring of both PKA-RIIα and 14-3-3θ regulates retinoic acid induced 16 mediated phosphorylation of heat shock protein 70

    PubMed Central

    Tang, Hai-Lin; Zhu, Shi-Ying; Zhao, Lan-Juan; Ren, Hao; Zhao, Ping; Qi, Zhong-Tian; Wang, Wen

    2015-01-01

    Our previous study reported that retinoic acid induced 16 (RAI16) could enhance tumorigenesis in hepatocellular carcinoma (HCC). However, the cellular functions of RAI16 are still unclear. In this study, by immunoprecipitation and tandem (MS/MS) mass spectrometry analysis, we identified that RAI16 interacted with the type II regulatory subunit of PKA (PKA-RIIα), acting as a novel protein kinase A anchoring protein (AKAP). In addition, RAI16 also interacted with heat shock protein 70 (HSP70) and 14-3-3θ. Further studies indicated that RAI16 mediated PKA phosphorylation of HSP70 at serine 486, resulting in anti-apoptosis events. RAI16 was also phosphorylated by the anchored PKA at serine 325, which promoted the recruitment of 14-3-3θ, which, in turn, inhibited RAI16 mediated PKA phosphorylation of HSP70. These findings offer mechanism insight into RAI16 mediated anti-apoptosis signaling in HCC. PMID:25900241

  11. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization.

    PubMed

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-01

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement. PMID:26791570

  12. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization

    PubMed Central

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-01

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement. PMID:26791570

  13. 14-3-3 Proteins SGF14c and SGF14l Play Critical Roles during Soybean Nodulation1[W][OA

    PubMed Central

    Radwan, Osman; Wu, Xia; Govindarajulu, Manjula; Libault, Marc; Neece, David J.; Oh, Man-Ho; Berg, R. Howard; Stacey, Gary; Taylor, Christopher G.; Huber, Steven C.; Clough, Steven J.

    2012-01-01

    The soybean (Glycine max) genome contains 18 members of the 14-3-3 protein family, but little is known about their association with specific phenotypes. Here, we report that the Glyma0529080 Soybean G-box Factor 14-3-3c (SGF14c) and Glyma08g12220 (SGF14l) genes, encoding 14-3-3 proteins, appear to play essential roles in soybean nodulation. Quantitative reverse transcription-polymerase chain reaction and western-immunoblot analyses showed that SGF14c mRNA and protein levels were specifically increased in abundance in nodulated soybean roots 10, 12, 16, and 20 d after inoculation with Bradyrhizobium japonicum. To investigate the role of SGF14c during soybean nodulation, RNA interference was employed to silence SGF14c expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Due to the paleopolyploid nature of soybean, designing a specific RNA interference sequence that exclusively targeted SGF14c was not possible. Therefore, two highly similar paralogs (SGF14c and SGF14l) that have been shown to function as dimers were silenced. Transcriptomic and proteomic analyses showed that mRNA and protein levels were significantly reduced in the SGF14c/SGF14l-silenced roots, and these roots exhibited reduced numbers of mature nodules. In addition, SGF14c/SGF14l-silenced roots contained large numbers of arrested nodule primordia following B. japonicum inoculation. Transmission electron microscopy further revealed that the host cytoplasm and membranes, except the symbiosome membrane, were severely degraded in the failed nodules. Altogether, transcriptomic, proteomic, and cytological data suggest a critical role of one or both of these 14-3-3 proteins in early development stages of soybean nodules. PMID:23060368

  14. A rare case of rapidly progressive dementia with elevated RT-QuIC and negative 14-3-3 and tau proteins.

    PubMed

    Trikamji, Bhavesh; Hamlin, Clive; Baldwin, Kelly J

    2016-05-01

    Creutzfeldt-Jakob disease (CJD) is characterized by rapidly progressing dementia with death usually occurring within 6 months. There is no verified disease-specific pre-mortem diagnostic test besides brain biopsy. We describe a 66 y old previously high functioning male who presented with a 5 month history of rapidly progressive dementia. Neurological examination revealed a score of 19/30 on MOCA testing. An extensive workup into various causes of dementia including electroencephalography and imaging studies was unremarkable. The cerebrospinal fluid was sent to National Prion Disease Center and it revealed elevated RT-QuIC levels with negative 14-3-3 and T tau proteins. Based on literature review, our case is one of few living subjects with elevated RT-QuIC levels and negative 14-3-3 and tau proteins. PMID:27249661

  15. 14-3-3 and aggresome formation

    PubMed Central

    Jia, Baohui; Wu, Yuying; Zhou, Yi

    2014-01-01

    Protein misfolding and aggregation underlie the pathogenesis of many neurodegenerative diseases. In addition to chaperone-mediated refolding and proteasomal degradation, the aggresome-macroautophagy pathway has emerged as another defense mechanism for sequestration and clearance of toxic protein aggregates in cells. Previously, the 14-3-3 proteins were shown to be indispensable for the formation of aggresomes induced by mutant huntingtin proteins. In a recent study, we have determined that 14-3-3 functions as a molecular adaptor to recruit chaperone-associated misfolded proteins to dynein motors for transport to aggresomes. This molecular complex involves a dimeric binding of 14-3-3 to both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3). As 14-3-3 has been implicated in various neurodegenerative diseases, our findings may provide mechanistic insights into its role in managing misfolded protein stress during the process of neurodegeneration. PMID:24549097

  16. The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins

    PubMed Central

    Shinn-Thomas, Jessica H.; del Campo, Jacob J.; Wang, Jianjun; Mohler, William A.

    2016-01-01

    Background Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform’s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1. Methodology/Principal Findings Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis. Conclusions/Significance Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions in vivo. However

  17. A Glycine soja 14-3-3 protein GsGF14o participates in stomatal and root hair development and drought tolerance in Arabidopsis thaliana.

    PubMed

    Sun, Xiaoli; Luo, Xiao; Sun, Mingzhe; Chen, Chao; Ding, Xiaodong; Wang, Xuedong; Yang, Shanshan; Yu, Qingyue; Jia, Bowei; Ji, Wei; Cai, Hua; Zhu, Yanming

    2014-01-01

    It is well established that 14-3-3 proteins are key regulators of multiple stress signal transduction cascades. However, the biological functions of soybean 14-3-3 proteins, especially in plant drought response, are not yet known. In this study, we characterized a Glycine soja 14-3-3 gene, GsGF14o, which is involved in plant development and drought response. GsGF14o expression was greatly induced by drought stress, as evidenced by the quantitative real-time PCR and β-glucuronidase (GUS) activity analysis. GsGF14o overexpression in Arabidopsis thaliana resulted in decreased drought tolerance during seed germination and seedling growth. Furthermore, silencing of AtGF14µ, the most homologous 14-3-3 gene of GsGF14o, led to enhanced drought tolerance at both the seed germination and seedling stage. Unexpectedly, GsGF14o transgenic lines showed reduced water loss and transpiration rates compared with wild-type plants, which was demonstrated to be the consequence of the decreased stomatal size. At the same time, the smaller stomata due to GsGF14o overexpression led to a relatively slow net photosynthesis rate, which led to a growth penalty under drought stress. We further demonstrated that GsGF14o overexpression caused deficits in root hair formation and development, and thereby reduced the water intake capacity of the transgenic root system. In addition, GsGF14o overexpression down-regulated the transcript levels of drought-responsive marker genes. Finally, we also investigated the tissue-specific accumulation of GsGF14o by using a GUS activity assay. Collectively, the results presented here confirm that GsGF14o plays a dual role in drought stress responses through its involvement in the regulation of stomatal size and root hair development. PMID:24272249

  18. Specificity of ε and Non-ε Isoforms of Arabidopsis 14-3-3 Proteins Towards the H+-ATPase and Other Targets

    PubMed Central

    Pallucca, Roberta; Visconti, Sabina; Camoni, Lorenzo; Cesareni, Giovanni; Melino, Sonia; Panni, Simona; Torreri, Paola; Aducci, Patrizia

    2014-01-01

    14-3-3 proteins are a family of ubiquitous dimeric proteins that modulate many cellular functions in all eukaryotes by interacting with target proteins. 14-3-3s exist as a number of isoforms that in Arabidopsis identifies two major groups named ε and non-ε. Although isoform specificity has been demonstrated in many systems, the molecular basis for the selection of specific sequence contexts has not been fully clarified. In this study we have investigated isoform specificity by measuring the ability of different Arabidopsis 14-3-3 isoforms to activate the H+-ATPase. We observed that GF14 isoforms of the non-ε group were more effective than ε group isoforms in the interaction with the H+-ATPase and in the stimulation of its activity. Kinetic and thermodynamic parameters of the binding of GF14ε and GF14ω isoforms, representative of ε and non-ε groups respectively, with the H+-ATPase, have been determined by Surface Plasmon Resonance analysis demonstrating that the higher affinity of GF14ω is mainly due to slower dissociation. The role of the C-terminal region and of a Gly residue located in the loop 8 and conserved in all non-ε isoforms has also been studied by deletion and site-specific mutagenesis. The C-terminal domains, despite their high divergence, play an auto-inhibitory role in both isoforms and they, in addition to a specific residue located in the loop 8, contribute to isoform specificity. To investigate the generality of these findings, we have used the SPOT-synthesis technology to array a number of phosphopeptides matching known or predicted 14-3-3 binding sites present in a number of clients. The results of this approach confirmed isoform specificity in the recognition of several target peptides, suggesting that the isoform specificity may have an impact on the modulation of a variety of additional protein activities, as suggested by probing of a phosphopeptide array with members of the two 14-3-3 groups. PMID:24603559

  19. Involvement of 14-3-3 protein GRF9 in root growth and response under polyethylene glycol-induced water stress.

    PubMed

    He, Yuchi; Wu, Jingjing; Lv, Bing; Li, Jia; Gao, Zhiping; Xu, Weifeng; Baluška, František; Shi, Weiming; Shaw, Pang Chui; Zhang, Jianhua

    2015-04-01

    Plant 14-3-3 proteins are phosphoserine-binding proteins that regulate a wide array of targets via direct protein-protein interactions. In this study, the role of a 14-3-3 protein, GRF9, in plant response to water stress was investigated. Arabidopsis wild-type, GRF9-deficient mutant (grf9), and GRF9-overexpressing (OE) plants were treated with polyethylene glycol (PEG) to induce mild water stress. OE plant showed better whole-plant growth and root growth than the wild type under normal or water stress conditions while the grf9 mutant showed worse growth. In OE plants, GRF9 favours the allocation of shoot carbon to roots. In addition, GRF9 enhanced proton extrusion, mainly in the root elongation zone and root hair zone, and maintained root growth under mild water stress. Grafting among the wild type, OE, and grf9 plants showed that when OE plants were used as the scion and GRF9 was overexpressed in the shoot, it enhanced sucrose transport into the root, and when OE plants were used as rootstock and GRF9 was overexpressed in the root, it caused more release of protons into the root surface under water stress. Taken together, the results suggest that under PEG-induced water stress, GRF9 is involved in allocating more carbon from the shoot to the root and enhancing proton secretion in the root growing zone, and this process is important for root response to mild water stress. PMID:25873671

  20. Chloride intracellular channel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms.

    PubMed Central

    Suginta, W; Karoulias, N; Aitken, A; Ashley, R H

    2001-01-01

    Mammalian chloride intracellular channel (CLIC) (p64-related) proteins are widely expressed, with an unusual dual localization as both soluble and integral membrane proteins. The molecular basis for their cellular localization and ion channel activity remains unclear. To help in addressing these problems, we identified novel rat brain CLIC4 (p64H1) binding partners by affinity chromatography, mass spectrometric analysis and microsequencing. Brain CLIC4 binds dynamin I, alpha-tubulin, beta-actin, creatine kinase and two 14-3-3 isoforms; the interactions are confirmed in vivo by immunoprecipitation. Gel overlay and reverse pull-down assays indicate that the binding of CLIC4 to dynamin I and 14-3-3zeta is direct. In HEK-293 cells, biochemical and immunofluorescence analyses show partial co-localization of recombinant CLIC4 with caveolin and with functional caveolae, which is consistent with a dynamin-associated role for CLIC4 in caveolar endocytosis. We speculate that brain CLIC4 might be involved in the dynamics of neuronal plasma membrane microdomains (micropatches) containing caveolin-like proteins and might also have other cellular roles related to membrane trafficking. Our results provide the basis for new hypotheses concerning novel ways in which CLIC proteins might be associated with cell membrane remodelling, the control of cell shape, and anion channel activity. PMID:11563969

  1. Involvement of 14-3-3 protein GRF9 in root growth and response under polyethylene glycol-induced water stress

    PubMed Central

    He, Yuchi; Wu, Jingjing; Lv, Bing; Li, Jia; Gao, Zhiping; Xu, Weifeng; Baluška, František; Shi, Weiming; Shaw, Pang Chui; Zhang, Jianhua

    2015-01-01

    Plant 14-3-3 proteins are phosphoserine-binding proteins that regulate a wide array of targets via direct protein–protein interactions. In this study, the role of a 14-3-3 protein, GRF9, in plant response to water stress was investigated. Arabidopsis wild-type, GRF9-deficient mutant (grf9), and GRF9-overexpressing (OE) plants were treated with polyethylene glycol (PEG) to induce mild water stress. OE plant showed better whole-plant growth and root growth than the wild type under normal or water stress conditions while the grf9 mutant showed worse growth. In OE plants, GRF9 favours the allocation of shoot carbon to roots. In addition, GRF9 enhanced proton extrusion, mainly in the root elongation zone and root hair zone, and maintained root growth under mild water stress. Grafting among the wild type, OE, and grf9 plants showed that when OE plants were used as the scion and GRF9 was overexpressed in the shoot, it enhanced sucrose transport into the root, and when OE plants were used as rootstock and GRF9 was overexpressed in the root, it caused more release of protons into the root surface under water stress. Taken together, the results suggest that under PEG-induced water stress, GRF9 is involved in allocating more carbon from the shoot to the root and enhancing proton secretion in the root growing zone, and this process is important for root response to mild water stress. PMID:25873671

  2. Positive 14-3-3 and tau proteins in a sporadic Creutzfeldt-Jakob disease case and a brief perspective of prion diseases in Colombia.

    PubMed

    Escandón-Vargas, Kevin; Zorrilla-Vaca, Andrés; Corral-Prado, Raúl Heli

    2016-01-01

    Prion diseases are rare neurodegenerative disorders occurring worldwide and affecting both humans and animals. Herein, we present the case of a patient diagnosed with definite sporadic Creutzfeldt-Jakob disease in Cali, Colombia. Besides neurological examination, 14-3-3 and tau proteins were valuable tools supporting the diagnosis. We also present a brief perspective of the prion diseases reported in Colombia to date. Although the incidence of prion diseases is unknown in Colombia, our literature review revealed that one case of scrapie in 1981 and 29 human sporadic cases of Creutzfeldt-Jakob disease have been documented and published in our country. PMID:27622622

  3. Phospho-specific recognition by 14-3-3 proteins and antibodies monitored by a high throughput label-free optical biosensor.

    PubMed

    Wu, Meng; Coblitz, Brian; Shikano, Sojin; Long, Shunyou; Spieker, Matt; Frutos, Anthony G; Mukhopadhyay, Sunil; Li, Min

    2006-10-16

    Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs. Using a resonant waveguide grating biosensor, the Epic mark System, we have developed a generic assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with a solution-based fluorescence anisotropy assay, our results support that the high throughput resonant waveguide grating biosensor system has favorable technical profiles in detecting protein-protein interactions that recognize phosphorylated motifs. Hence it provides a new generic HTS platform for phospho-detection. PMID:17011553

  4. A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor.

    PubMed Central

    van der Hoeven, P C; Siderius, M; Korthout, H A; Drabkin, A V; de Boer, A H

    1996-01-01

    A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed. PMID:8754686

  5. 14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides

    PubMed Central

    Madeira, Fábio; Tinti, Michele; Murugesan, Gavuthami; Berrett, Emily; Stafford, Margaret; Toth, Rachel; Cole, Christian; MacKintosh, Carol; Barton, Geoffrey J.

    2015-01-01

    Motivation: The 14-3-3 family of phosphoprotein-binding proteins regulates many cellular processes by docking onto pairs of phosphorylated Ser and Thr residues in a constellation of intracellular targets. Therefore, there is a pressing need to develop new prediction methods that use an updated set of 14-3-3-binding motifs for the identification of new 14-3-3 targets and to prioritize the downstream analysis of >2000 potential interactors identified in high-throughput experiments. Results: Here, a comprehensive set of 14-3-3-binding targets from the literature was used to develop 14-3-3-binding phosphosite predictors. Position-specific scoring matrix, support vector machines (SVM) and artificial neural network (ANN) classification methods were trained to discriminate experimentally determined 14-3-3-binding motifs from non-binding phosphopeptides. ANN, position-specific scoring matrix and SVM methods showed best performance for a motif window spanning from −6 to +4 around the binding phosphosite, achieving Matthews correlation coefficient of up to 0.60. Blind prediction showed that all three methods outperform two popular 14-3-3-binding site predictors, Scansite and ELM. The new methods were used for prediction of 14-3-3-binding phosphosites in the human proteome. Experimental analysis of high-scoring predictions in the FAM122A and FAM122B proteins confirms the predictions and suggests the new 14-3-3-predictors will be generally useful. Availability and implementation: A standalone prediction web server is available at http://www.compbio.dundee.ac.uk/1433pred. Human candidate 14-3-3-binding phosphosites were integrated in ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome database. Contact: cmackintosh@dundee.ac.uk or gjbarton@dundee.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25735772

  6. 14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein.

    PubMed

    Raychaudhuri, Kumarkrishna; Chaudhary, Neelam; Gurjar, Mansa; D'Souza, Roseline; Limzerwala, Jazeel; Maddika, Subbareddy; Dalal, Sorab N

    2016-07-29

    Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ(-/-) cells. 14-3-3σ(-/-) cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene. PMID:27261462

  7. Identification of 14-3-3 Family in Common Bean and Their Response to Abiotic Stress

    PubMed Central

    Dhaubhadel, Sangeeta; Bian, Shaomin; Li, Xuyan

    2015-01-01

    14-3-3s are a class of conserved regulatory proteins ubiquitously found in eukaryotes, which play important roles in a variety of cellular processes including response to diverse stresses. Although much has been learned about 14-3-3s in several plant species, it remains unknown in common bean. In this study, 9 common bean 14-3-3s (PvGF14s) were identified by exhaustive data mining against the publicly available common bean genomic database. A phylogenetic analysis revealed that each predicted PvGF14 was clustered with two GmSGF14 paralogs from soybean. Both epsilon-like and non-epsilon classes of PvGF14s were found in common bean, and the PvGF14s belonging to each class exhibited similar gene structure. Among 9 PvGF14s, only 8 are transcribed in common bean. Expression patterns of PvGF14s varied depending on tissue type, developmental stage and exposure of plants to stress. A protein-protein interaction study revealed that PvGF14a forms dimer with itself and with other PvGF14 isoforms. This study provides a first comprehensive look at common bean 14-3-3 proteins, a family of proteins with diverse functions in many cellular processes, especially in response to stresses. PMID:26599110

  8. Development of a dot blot assay with antibodies to recombinant “core” 14-3-3 protein: Evaluation of its usefulness in diagnosis of Creutzfeldt–Jakob disease

    PubMed Central

    Subramanian, Sarada; Mahadevan, Anita; Satishchandra, Parthasarathy; Shankar, Susarla Krishna

    2016-01-01

    Background and Purpose: Definitive diagnosis of Creutzfeldt–Jakob disease (CJD) requires demonstration of infective prion protein (PrPSc) in brain tissues by immunohistochemistry or immunoblot, making antemortem diagnosis of CJD difficult. The World Health Organization (WHO) recommends detection of 14-3-3 protein in cerebrospinal fluid (CSF) in cases of dementia, with clinical correlation, as a useful diagnostic marker for CJD, obviating the need for brain biopsy. This facility is currently available in only a few specialized centers in the West and no commercial kit is available for clinical diagnostic use in India. Hence the objective of this study was to develop an in-house sensitive assay for quantitation of 14-3-3 protein and to evaluate its diagnostic potential to detect 14-3-3 proteins in CSF as a biomarker in suspected cases of CJD. Materials and Methods: A minigene expressing the “core” 14-3-3 protein was synthesized by overlapping polymerase chain reaction (PCR) and the recombinant protein was produced by employing a bacterial expression system. Polyclonal antibodies raised in rabbit against the purified recombinant protein were used for developing a dot blot assay with avidin-biotin technology for signal amplification and quantitation of 14-3-3 protein in CSF. Results: The results in the present study suggest the diagnostic potential of the dot blot method with about 10-fold difference (P< 0.001) in the CSF levels of 14-3-3 protein between the CJD cases (N= 50) and disease controls (N= 70). The receiver operating characteristic (ROC) analysis of the results suggested an optimal cutoff value of 2 ng/mL. Conclusions: We have developed an indigenous, economical, and sensitive dot blot method for the quantitation of 14-3-3 protein in CSF. PMID:27293331

  9. The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

    PubMed

    Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

    2016-07-01

    Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. PMID:27001512

  10. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

    PubMed Central

    da Silva, Julhiany de Fátima; Vicentim, Juliana; de Oliveira, Haroldo Cesar; Marcos, Caroline Maria; Assato, Patricia Akemi; Andreotti, Patrícia Ferrari; da Silva, Juliana Leal Monteiro; Soares, Christiane Pienna; Benard, Gil; Almeida, Ana Marisa Fusco; Mendes-Giannini, Maria José Soares

    2015-01-01

    The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis. PMID:26038961

  11. The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae

    PubMed Central

    Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

    2016-01-01

    Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. PMID:27001512

  12. The chaperone-like protein 14-3-3η interacts with human α-synuclein aggregation intermediates rerouting the amyloidogenic pathway and reducing α-synuclein cellular toxicity.

    PubMed

    Plotegher, Nicoletta; Kumar, Dhruv; Tessari, Isabella; Brucale, Marco; Munari, Francesca; Tosatto, Laura; Belluzzi, Elisa; Greggio, Elisa; Bisaglia, Marco; Capaldi, Stefano; Aioanei, Daniel; Mammi, Stefano; Monaco, Hugo L; Samo, Bruno; Bubacco, Luigi

    2014-11-01

    Familial and idiopathic Parkinson's disease (PD) is associated with the abnormal neuronal accumulation of α-synuclein (aS) leading to β-sheet-rich aggregates called Lewy Bodies (LBs). Moreover, single point mutation in aS gene and gene multiplication lead to autosomal dominant forms of PD. A connection between PD and the 14-3-3 chaperone-like proteins was recently proposed, based on the fact that some of the 14-3-3 isoforms can interact with genetic PD-associated proteins such as parkin, LRRK2 and aS and were found as components of LBs in human PD. In particular, a direct interaction between 14-3-3η and aS was reported when probed by co-immunoprecipitation from cell models, from parkinsonian brains and by surface plasmon resonance in vitro. However, the mechanisms through which 14-3-3η and aS interact in PD brains remain unclear. Herein, we show that while 14-3-3η is unable to bind monomeric aS, it interacts with aS oligomers which occur during the early stages of aS aggregation. This interaction diverts the aggregation process even when 14-3-3η is present in sub-stoichiometric amounts relative to aS. When aS level is overwhelmingly higher than that of 14-3-3η, the fibrillation process becomes a sequestration mechanism for 14-3-3η, undermining all processes governed by this protein. Using a panel of complementary techniques, we single out the stage of aggregation at which the aS/14-3-3η interaction occurs, characterize the products of the resulting processes, and show how the processes elucidated in vitro are relevant in cell models. Our findings constitute a first step in elucidating the molecular mechanism of aS/14-3-3η interaction and in understanding the critical aggregation step at which 14-3-3η has the potential to rescue aS-induced cellular toxicity. PMID:24895406

  13. 14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase

    SciTech Connect

    Mizuno, Emi; Kitamura, Naomi; Komada, Masayuki

    2007-10-01

    The deubiquitinating enzyme UBPY, also known as USP8, regulates cargo sorting and membrane traffic at early endosomes. Here we demonstrate the regulatory mechanism of the UBPY catalytic activity. We identified 14-3-3 {epsilon}, {gamma}, and {zeta} as UBPY-binding proteins using co-immunoprecipitation followed by mass spectrometric analysis. The 14-3-3 binding of UBPY was inhibited by mutating the consensus 14-3-3-binding motif RSYS{sup 680}SP, by phosphatase treatment, and by competition with the Ser{sup 680}-phosphorylated RSYS{sup 680}SP peptide. Metabolic labeling with [{sup 32}P]orthophosphate and immunoblotting using antibody against the phosphorylated 14-3-3-binding motif showed that Ser{sup 680} is a major phosphorylation site in UBPY. These results indicated that 14-3-3s bind to the region surrounding Ser{sup 680} in a phosphorylation-dependent manner. The mutation at Ser{sup 680} led to enhanced ubiquitin isopeptidase activity of UBPY toward poly-ubiquitin chains and a cellular substrate, epidermal growth factor receptor, in vitro and in vivo. Moreover, addition of 14-3-3{epsilon} inhibited the UBPY activity in vitro. Finally, UBPY was dephosphorylated at Ser{sup 680} and dissociated from 14-3-3s in the M phase, resulting in enhanced activity of UBPY during cell division. We conclude that UBPY is catalytically inhibited in a phosphorylation-dependent manner by 14-3-3s during the interphase, and this regulation is cancelled in the M phase.

  14. 14-3-3ζ coordinates adipogenesis of visceral fat

    PubMed Central

    Lim, Gareth E.; Albrecht, Tobias; Piske, Micah; Sarai, Karnjit; Lee, Jason T. C; Ramshaw, Hayley S.; Sinha, Sunita; Guthridge, Mark A.; Acker-Palmer, Amparo; Lopez, Angel F.; Clee, Susanne M.; Nislow, Corey; Johnson, James D.

    2015-01-01

    The proteins that coordinate complex adipogenic transcriptional networks are poorly understood. 14-3-3ζ is a molecular adaptor protein that regulates insulin signalling and transcription factor networks. Here we report that 14-3-3ζ-knockout mice are strikingly lean from birth with specific reductions in visceral fat depots. Conversely, transgenic 14-3-3ζ overexpression potentiates obesity, without exacerbating metabolic complications. Only the 14-3-3ζ isoform is essential for adipogenesis based on isoform-specific RNAi. Mechanistic studies show that 14-3-3ζ depletion promotes autophagy-dependent degradation of C/EBP-δ, preventing induction of the master adipogenic factors, Pparγ and C/EBP-α. Transcriptomic data indicate that 14-3-3ζ acts upstream of hedgehog signalling-dependent upregulation of Cdkn1b/p27Kip1. Indeed, concomitant knockdown of p27Kip1 or Gli3 rescues the early block in adipogenesis induced by 14-3-3ζ knockdown in vitro. Adipocyte precursors in 14-3-3ζKO embryos also appear to have greater Gli3 and p27Kip1 abundance. Together, our in vivo and in vitro findings demonstrate that 14-3-3ζ is a critical upstream driver of adipogenesis. PMID:26220403

  15. Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 α-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein.

    PubMed

    Llopis-Torregrosa, Vicent; Ferri-Blázquez, Alba; Adam-Artigues, Anna; Deffontaines, Emilie; van Heusden, G Paul H; Yenush, Lynne

    2016-07-15

    Cell viability requires adaptation to changing environmental conditions. Ubiquitin-mediated endocytosis plays a crucial role in this process, because it provides a mechanism to remove transport proteins from the membrane. Arrestin-related trafficking proteins are important regulators of the endocytic pathway in yeast, facilitating selective ubiquitylation of target proteins by the E3 ubiquitin ligase, Rsp5. Specifically, Rod1 (Art4) has been reported to regulate the endocytosis of both the Hxt1, Hxt3, and Hxt6 glucose transporters and the Jen1 lactate transporter. Also, the AMP kinase homologue, Snf1, and 14-3-3 proteins have been shown to regulate Jen1 via Rod1. Here, we further characterized the role of Rod1, Snf1, and 14-3-3 in the signal transduction route involved in the endocytic regulation of the Hxt6 high affinity glucose transporter by showing that Snf1 interacts specifically with Rod1 and Rog3 (Art7), that the interaction between the Bmh2 and several arrestin-related trafficking proteins may be modulated by carbon source, and that both the 14-3-3 protein Bmh2 and the Snf1 regulatory domain interact with the arrestin-like domain containing the N-terminal half of Rod1 (amino acids 1-395). Finally, using both co-immunoprecipitation and bimolecular fluorescence complementation, we demonstrated the interaction of Rod1 with Hxt6 and showed that the localization of the Rod1-Hxt6 complex at the plasma membrane is affected by carbon source and is reduced upon overexpression of SNF1 and BMH2. PMID:27261460

  16. Akirin interacts with Bap60 and 14-3-3 proteins to regulate the expression of antimicrobial peptides in the kuruma shrimp (Marsupenaeus japonicus).

    PubMed

    Liu, Ning; Wang, Xian-Wei; Sun, Jie-Jie; Wang, Lei; Zhang, Hong-Wei; Zhao, Xiao-Fan; Wang, Jin-Xing

    2016-02-01

    Akirin is a recently discovered nuclear factor that plays important roles in innate immune responses. Akirin is a positive regulator of the NF-κB factor of the Drosophila immune deficiency (IMD) pathway, which shares extensive similarities with the mammalian tumor necrosis factor receptor (TNFR) signaling pathway. However, some studies found that the NF-κB transcriptional targets were also strongly repressed in akirin2 knockout mice following TLR, IL-1β and TNFα treatment. Therefore, the function of Akirin in the immune response requires further clarification. In this study, an Akirin homolog in the kuruma shrimp (Marsupenaeus japonicus) was identified. It was mainly expressed in hemocytes, heart and intestines. The expression of Akirin was upregulated by challenge with the Gram-negative bacterium Vibrio anguillarum, but was not significantly influenced by challenge with the Gram-positive bacterium Staphylococcus aureus. Knockdown of Akirin suppressed the expression of several IMD-Relish target effectors (antimicrobial peptides, AMPs). The limited regulating spectrum of Akirin might be associated with Bap60, a component of the Brahma (SWI/SNF) ATP-dependent chromatin-remodeling complex. In addition, Akirin also interacts with 14-3-3, which inhibited the expression of Akirin-target AMPs. The results suggested that Akirin is involved in the IMD-Relish pathway by interacting with Relish. The interaction of Akirin with Bap60 positively regulated the Akirin-Relish function, and its interaction with 14-3-3 negatively regulated the Akirin-Relish function. PMID:26493016

  17. Characterization of the Interactome of the Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 2 Reveals the Hyper Variable Region as a Binding Platform for Association with 14-3-3 Proteins.

    PubMed

    Xiao, Yihong; Wu, Weining; Gao, Jiming; Smith, Nikki; Burkard, Christine; Xia, Dong; Zhang, Minxia; Wang, Chengbao; Archibald, Alan; Digard, Paul; Zhou, En-Min; Hiscox, Julian A

    2016-05-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry worldwide and hence global food security, exacerbated by a newly emerged highly pathogenic (HP-PRRSV) strain from China. PRRSV nonstructural protein 2 (nsp2) is a multifunctional polypeptide with strain-dependent influences on pathogenicity. A number of discrete functional regions have been identified on the protein. Quantitative label free proteomics was used to identify cellular binding partners of nsp2 expressed by HP-PRRSV. This allowed the identification of potential cellular interacting partners and the discrimination of nonspecific interactions. The interactome data were further investigated and validated using biological replicates and also compared with nsp2 from a low pathogenic (LP) strain of PRRSV. Validation included both forward and reverse pulldowns and confocal microscopy. The data indicated that nsp2 interacted with a number of cellular proteins including 14-3-3, CD2AP, and other components of cellular aggresomes. The hyper-variable region of nsp2 protein was identified as a binding platform for association with 14-3-3 proteins. PMID:26709850

  18. Sphingosine induces apoptosis in hippocampal neurons and astrocytes by activating caspase-3/-9 via a mitochondrial pathway linked to SDK/14-3-3 protein/Bax/cytochrome c.

    PubMed

    Kanno, Takeshi; Nishizaki, Tomoyuki

    2011-09-01

    The present study examined sphingosine-induced apoptosis in cultured rat hippocampal neurons and astrocytes. Sphingosine induced apoptosis in a concentration (1-100 µM)-dependent manner, that is inhibited by the PKC-δ inhibitor rottlerin, and a similar effect was obtained with the sphingosine kinase inhibitors, to raise intracellular sphingosine concentrations. Sphingosine increased presence of sphingosine-dependent protein kinase (SDK), and the effect was suppressed by rottlerin. Sphingosine increased phosphorylated 14-3-3 protein, thereby transforming the protein from a dimeric structure into a monomeric structure. Sphingosine accumulated Bax in the mitochondria and stimulated cytochrome c release into the cytosol, and those effects were inhibited by rottlerin. Sphingosine disrupted mitochondrial membrane potentials, that was abolished by silencing the PKC-δ-targeted gene. Moreover, sphingosine activated caspase-9 and the effector caspase-3 in a PKC-δ-dependent manner. Taken together, the results of the present study indicate that sphingosine activates SDK, produced through proteolytic processing of an active form of PKC-δ, to phosphorylate 14-3-3 protein and transform into a monomeric structure, causing Bax dissociation from 14-3-3 protein and accumulation in the mitochondria, which perturbs mitochondrial membrane potentials allowing cytochrome c release into the cytosol, to activate caspase-9 and the effector caspase-3, responsible for apoptosis in hippocampal neurons and astrocytes. PMID:21660956

  19. Characteristics of Korean patients with suspected Creutzfeldt-Jakob disease with 14-3-3 protein in cerebrospinal fluid: Preliminary study of the Korean Creutzfeldt-Jakob disease active surveillance program

    PubMed Central

    Lim, Jae-Sung; Kwon, Hyung-Min; Jang, Jae-Won; Ju, Young-Ran; Kim, SuYeon; Park, Young Ho; Park, So Young; Kim, SangYun

    2015-01-01

    Abstract Although Korea had a national surveillance system for Creutzfeldt-Jakob disease (CJD), it was mainly dependent on attending physician's reports. Thus, little prospective data about the epidemiology, characteristics, and final diagnoses of suspected patients were available. We have established a nationwide network for the active surveillance of patients with suspected CJD. When the requested cerebrospinal fluid (CSF) samples tested positive for 14-3-3 protein, we investigated the clinical characteristics of the corresponding patients and followed them until their final diagnoses were confirmed. A total of 218 samples were requested for CSF assays from May 2010 to August 2012, and 106 (48.6%) were positive for 14-3-3 protein. In 89 patients with complete clinical data, 38 (42.7%) were diagnosed with probable CJD and the estimated annual occurrence of CJD was 16.3 persons-per-year. The most common diagnoses of the remainder were central nervous system infection and any-cause encephalopathy. Non-CJD subjects showed worse initial consciousness levels than CJD patients. This preliminary study showed that the number of reported cases of CJD and the true positivity rates of CSF 14-3-3 protein assays were both low in Korea. An active surveillance system is urgently needed to provide the latest nationwide epidemiological data of CJD. PMID:25996401

  20. Exon B of human surfactant protein A2 mRNA, alone or within its surrounding sequences, interacts with 14-3-3; role of cis-elements and secondary structure

    PubMed Central

    Noutsios, Georgios T.; Silveyra, Patricia; Bhatti, Faizah

    2013-01-01

    Human surfactant protein A, an innate immunity molecule, is encoded by two genes: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5′ untranslated (5′UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis-regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441 proteins to wild-type eB, eB mutant, AD, and ABD 5′UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found that 1) proteins bind eB mRNA in a sequence-specific manner, with two cis-elements identified within eB to be important; 2) eB secondary structure is necessary for binding; 3) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; 4) other ribosomal and cytoskeletal proteins, and translation factors, are also present in the eB mRNA-protein complex; 5) knockdown of 14-3-3 β/α isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis-elements within eB of hSP-A2 mRNA in a sequence- and secondary structure-specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA. PMID:23525782

  1. The Pseudomonas syringae Effector HopQ1 Promotes Bacterial Virulence and Interacts with Tomato 14-3-3 Proteins in a Phosphorylation-Dependent Manner1[C][W][OA

    PubMed Central

    Li, Wei; Yadeta, Koste A.; Elmore, James Mitch; Coaker, Gitta

    2013-01-01

    A key virulence strategy of bacterial pathogens is the delivery of multiple pathogen effector proteins into host cells during infection. The Hrp outer protein Q (HopQ1) effector from Pseudomonas syringae pv tomato (Pto) strain DC3000 is conserved across multiple bacterial plant pathogens. Here, we investigated the virulence function and host targets of HopQ1 in tomato (Solanum lycopersicum). Transgenic tomato lines expressing dexamethasone-inducible HopQ1 exhibited enhanced disease susceptibility to virulent Pto DC3000, the Pto ΔhrcC mutant, and decreased expression of a pathogen-associated molecular pattern-triggered marker gene after bacterial inoculation. HopQ1-interacting proteins were coimmunoprecipitated and identified by mass spectrometry. HopQ1 can associate with multiple tomato 14-3-3 proteins, including TFT1 and TFT5. HopQ1 is phosphorylated in tomato, and four phosphorylated peptides were identified by mass spectrometry. HopQ1 possesses a conserved mode I 14-3-3 binding motif whose serine-51 residue is phosphorylated in tomato and regulates its association with TFT1 and TFT5. Confocal microscopy and fractionation reveal that HopQ1 exhibits nucleocytoplasmic localization, while HopQ1 dephosphorylation mimics exhibit more pronounced nuclear localization. HopQ1 delivered from Pto DC3000 was found to promote bacterial virulence in the tomato genotype Rio Grande 76R. However, the HopQ1(S51A) mutant delivered from Pto DC3000 was unable to promote pathogen virulence. Taken together, our data demonstrate that HopQ1 enhances bacterial virulence and associates with tomato 14-3-3 proteins in a phosphorylation-dependent manner that influences HopQ1’s subcellular localization and virulence-promoting activities in planta. PMID:23417089

  2. Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa.

    PubMed

    Chandna, Ruby; Augustine, Rehna; Kanchupati, Praveena; Kumar, Roshan; Kumar, Pawan; Arya, Gulab C; Bisht, Naveen C

    2016-01-01

    14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1-5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses. PMID:26858736

  3. Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa

    PubMed Central

    Chandna, Ruby; Augustine, Rehna; Kanchupati, Praveena; Kumar, Roshan; Kumar, Pawan; Arya, Gulab C.; Bisht, Naveen C.

    2016-01-01

    14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1–5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses. PMID:26858736

  4. Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis

    PubMed Central

    Das, Maitreyi; Nuñez, Illyce; Rodriguez, Marbelys; Wiley, David J.; Rodriguez, Juan; Sarkeshik, Ali; Yates, John R.; Buchwald, Peter; Verde, Fulvia

    2015-01-01

    Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24–Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence. PMID:26246599

  5. Identification of a functional splice variant of 14-3-3E1 in rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 14-3-3 proteins are a family of regulatory proteins involved in diverse cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. In this study, we report the identification and charact...

  6. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    PubMed

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-01

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization. PMID:25748451

  7. 14-3-3 sigma and 14-3-3 zeta plays an opposite role in cell growth inhibition mediated by transforming growth factor-beta 1.

    PubMed

    Hong, Hye-Young; Jeon, Woo-Kwang; Bae, Eun-Jin; Kim, Shin-Tae; Lee, Ho-Jae; Kim, Seong-Jin; Kim, Byung-Chul

    2010-03-01

    The expression of 14-3-3 proteins is dysregulated in various types of cancer. This study was undertaken to investigate the effects of 14-3-3 zeta and 14-3-3 sigma on cell growth inhibition mediated by transforming growth factor-beta 1 (TGF-beta1). Mouse mammary epithelial cells (Eph4) that are transformed with oncogenic c-H-Ras (EpRas) and no longer sensitive to TGF-beta1-mediated growth inhibition displayed increased expression of 14-3-3 zeta and decreased expression of 14-3-3 sigma compared with parental Eph4 cells. Using small interfering RNA-mediated knockdown and overexpression of 14-3-3 sigma or 14-3-3 zeta, we showed that 14-3-3 sigma is required for TGF-beta1-mediated growth inhibition whereas 14-3-3 zeta negatively modulates this growth inhibitory response. Notably, overexpression of 14-3-3 zeta increased the level of Smad3 protein that is phosphorylated at linker regions and cannot mediate the TGF-beta1 growth inhibitory response. Consistent with this finding, mutation of the 14-3-3 zeta phosphorylation sites in Smad3 markedly reduced the 14-3-3 zeta-mediated inhibition of TGF-beta1-induced p15 promoter-reporter activity and cell cycle arrest, suggesting that these residues are critical targets of 14-3-3 zeta in the suppression of TGF-beta1-mediated growth. Taken together, our findings indicate that dysregulation of 14-3-3 sigma or 14-3-3 zeta contributes to TGF-beta1 resistance in cancer cells. PMID:20082218

  8. Phosphate differentially regulates 14-3-3 family members and GRF9 plays a role in Pi-starvation induced responses.

    PubMed

    Cao, Aiqin; Jain, Ajay; Baldwin, James C; Raghothama, Kashchandra G

    2007-10-01

    The 14-3-3s are phosphoserine-binding proteins that act as key regulators of many metabolic pathways. Several biotic and abiotic stresses have been shown to modulate the expression of 14-3-3 genes. In Arabidopsis thaliana, 15 genes are known to code for 14-3-3 isoforms belonging to epsilon and non-epsilon groups. Since phosphorus is one of the essential macronutrients for plants, we examined its role in the regulation of the expression of 14-3-3 isoforms belonging to epsilon (GRF9, GRF10, GRF11, GRF13) and non-epsilon (GRF1, GRF3, GRF6, GRF8) groups. The effect of Pi deprivation was differential on the members of non-epsilon group ranging from a significant reduction in the transcripts of GRF3 to non-perceptible changes in the transcripts of other members. Suppressive effect of Pi-deficiency was more pronounced on some of the members of epsilon group with transcripts levels of GRF9 and GRF13 barely detectable. A concurrent increase in the transcript levels of GRF9 with an increase in the Pi concentration suggested a correlation between gene expression and Pi availability. However, neither Pi deficiency at low temperature nor Fe and K deficiency failed to suppress GRF9 expression. In planta role of GRF9 was elucidated by the analysis of the loss-of-function mutant under Pi-replete condition. The analyses revealed exaggerated Pi-starvation responses in the form of starch accumulation in the leaves and modulated root system architecture (RSA). An inverse relationship between the abundance of GRF9 transcripts and accumulation of starch in transgenic lines over-expressing this gene provided further evidence towards the role of GRF9 in modulation of metabolic pathways during Pi-starvation responses. PMID:17598127

  9. Tomato 14-3-3 protein 7 (TFT7) positively regulates immunity-associated programmed cell death by enhancing accumulation and signaling ability of MAPKKKalpha

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Programmed cell death (PCD) is triggered when Pto, a serine-threonine protein kinase recognizes either the AvrPto or AvrPtoB effector from Pseudomonas syringae pv. tomato. This PCD requires MAPKKKalpha as a positive regulator in tomato and Nicotiana benthamiana. To examine how PCD-eliciting activi...

  10. Genetic variations of 14-3-3E1 isoform in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly conserved family of 14-3-3 proteins functions in the regulation of a wide variety of cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. Several studies have observed diffe...

  11. 14-3-3 in Thoracic Aortic Aneurysms

    PubMed Central

    Chakravarti, Ritu; Gupta, Karishma; Swain, Mamuni; Willard, Belinda; Scholtz, Jaclyn; Svensson, Lars G.; Roselli, Eric E.; Pettersson, Gosta; Johnston, Douglas R.; Soltesz, Edward G.; Yamashita, Michifumi; Stuehr, Dennis; Daly, Thomas M.; Hoffman, Gary S.

    2015-01-01

    Objective Large vessel vasculitides (LVV) are a group of autoimmune diseases characterized by injury to and anatomic modifications of large vessels, including the aorta and its branch vessels. Disease etiology is unknown. This study was undertaken to identify antigen targets within affected vessel walls in aortic root, ascending aorta, and aortic arch surgical specimens from patients with LVV, including giant cell arteritis, Takayasu arteritis, and isolated focal aortitis. Methods Thoracic aortic aneurysm specimens and autologous blood were acquired from consenting patients who underwent aorta reconstruction procedures. Aorta proteins were extracted from both patients with LVV and age-, race-, and sex-matched disease controls with noninflammatory aneurysms. A total of 108 serum samples from patients with LVV, matched controls, and controls with antinuclear antibodies, different forms of vasculitis, or sepsis were tested. Results Evaluation of 108 serum samples and 22 aortic tissue specimens showed that 78% of patients with LVV produced antibodies to 14-3-3 proteins in the aortic wall (93.7% specificity), whereas controls were less likely to do so (6.7% produced antibodies). LVV patient sera contained autoantibody sufficient to immunoprecipitate 14-3-3 protein(s) from aortic lysates. Three of 7 isoforms of 14-3-3 were found to be up-regulated in aorta specimens from patients with LVV, and 2 isoforms (ε and ζ) were found to be antigenic in LVV. Conclusion This is the first study to use sterile, snap-frozen thoracic aorta biopsy specimens to identify autoantigens in LVV. Our findings indicate that 78% of patients with LVV have antibody reactivity to 14-3-3 protein(s). The precise role of these antibodies and 14-3-3 proteins in LVV pathogenesis deserves further study. PMID:25917817

  12. Phosphorylation and Interaction with the 14-3-3 Protein of the Plasma Membrane H+-ATPase are Involved in the Regulation of Magnesium-Mediated Increases in Aluminum-Induced Citrate Exudation in Broad Bean (Vicia faba. L).

    PubMed

    Chen, Qi; Kan, Qi; Wang, Ping; Yu, Wenqian; Yu, Yuzhen; Zhao, Yan; Yu, Yongxiong; Li, Kunzhi; Chen, Limei

    2015-06-01

    Several studies have shown that external application of micromolar magnesium (Mg) can increase the resistance of legumes to aluminum (Al) stress by enhancing Al-induced citrate exudation. However, the exact mechanism underlying this regulation remains unknown. In this study, the physiological and molecular mechanisms by which Mg enhances Al-induced citrate exudation to alleviate Al toxicity were investigated in broad bean. Micromolar concentrations of Mg that alleviated Al toxicity paralleled the stimulation of Al-induced citrate exudation and increased the activity of the plasma membrane (PM) H(+)-ATPase. Northern blot analysis shows that a putative MATE-like gene (multidrug and toxic compound extrusion) was induced after treatment with Al for 4, 8 and 12 h, whereas the mRNA abundance of the MATE-like gene showed no significant difference between Al plus Mg and Al-only treatments during the entire treatment period. Real-time reverse transcription-PCR (RT-PCR) and Western blot analyses suggest that the transcription and translation of the PM H(+)-ATPase were induced by Al but not by Mg. In contrast, immunoprecipitation suggests that Mg enhanced the phosphorylation levels of VHA2 and its interaction with the vf14-3-3b protein under Al stress. Taken together, our results suggest that micromolar concentrations of Mg can alleviate the Al rhizotoxicity by increasing PM H(+)-ATPase activity and Al-induced citrate exudation in YD roots. This enhancement is likely to be attributable to Al-induced increases in the expression of the MATE-like gene and vha2 and Mg-induced changes in the phosphorylation levels of VHA2, thus changing its interaction with the vf14-3-3b protein. PMID:25745032

  13. A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1

    PubMed Central

    Kilisch, Markus; Lytovchenko, Olga; Arakel, Eric C.; Bertinetti, Daniela; Schwappach, Blanche

    2016-01-01

    ABSTRACT The transport of the K+ channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic. PMID:26743085

  14. A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1.

    PubMed

    Kilisch, Markus; Lytovchenko, Olga; Arakel, Eric C; Bertinetti, Daniela; Schwappach, Blanche

    2016-02-15

    The transport of the K(+) channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic. PMID:26743085

  15. Dysregulated 14-3-3 Family in Peripheral Blood Leukocytes of Patients with Schizophrenia

    PubMed Central

    Qing, Ying; Sun, Liya; Yang, Chao; Jiang, Jie; Yang, Xuhan; Hu, Xiaowen; Cui, Donghong; Xu, Yifeng; He, Lin; Han, Dongmei; Wan, Chunling

    2016-01-01

    The 14-3-3 family, which is composed of seven distinct members in humans, plays important roles in the cell cycle, apoptosis, synaptic plasticity and neuronal differentiation and migration. Previous genetic and post-mortem gene expression studies have linked this family to schizophrenia. However, the direction of gene expression changes in these studies has been inconsistent, and reports of 14-3-3 gene expression in living schizophrenic patients are still lacking. Here, we assessed 14-3-3 gene and protein expression levels in peripheral blood leukocytes from drug-naïve first-episode schizophrenic patients and matched controls. mRNA and protein expression levels were quantified by qRT-PCR and UPLC-MRM/MS, respectively. Expression analysis revealed four downregulated and one upregulated mRNA transcripts as well as five downregulated protein levels of 14-3-3 isoforms in schizophrenia. Moreover, significant positive correlations between 14-3-3 mRNA and protein expression levels were found in schizophrenia, and we also identified negative correlations between ε, θ and ζ isoform expression levels and positive symptoms of schizophrenia. Our results suggest that gene and protein expression levels for the 14-3-3 family are dysregulated in schizophrenia, perhaps owing to specific regulatory mechanisms, and we also suggest that expression of the 14-3-3ε, θ and ζ isoform genes could be useful indicators of disease severity. PMID:27030512

  16. Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos

    NASA Astrophysics Data System (ADS)

    Lau, Jeffrey M. C.; Muslin, Anthony J.

    The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 τ exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 γ exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 ζ appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique.

  17. Locomotor hyperactivity in 14-3-3ζ KO mice is associated with dopamine transporter dysfunction

    PubMed Central

    Ramshaw, H; Xu, X; Jaehne, E J; McCarthy, P; Greenberg, Z; Saleh, E; McClure, B; Woodcock, J; Kabbara, S; Wiszniak, S; Wang, Ting-Yi; Parish, C; van den Buuse, M; Baune, B T; Lopez, A; Schwarz, Q

    2013-01-01

    Dopamine (DA) neurotransmission requires a complex series of enzymatic reactions that are tightly linked to catecholamine exocytosis and receptor interactions on pre- and postsynaptic neurons. Regulation of dopaminergic signalling is primarily achieved through reuptake of extracellular DA by the DA transporter (DAT) on presynaptic neurons. Aberrant regulation of DA signalling, and in particular hyperactivation, has been proposed as a key insult in the presentation of schizophrenia and related neuropsychiatric disorders. We recently identified 14-3-3ζ as an essential component of neurodevelopment and a central risk factor in the schizophrenia protein interaction network. Our analysis of 14-3-3ζ-deficient mice now shows that baseline hyperactivity of knockout (KO) mice is rescued by the antipsychotic drug clozapine. 14-3-3ζ KO mice displayed enhanced locomotor hyperactivity induced by the DA releaser amphetamine. Consistent with 14-3-3ζ having a role in DA signalling, we found increased levels of DA in the striatum of 14-3-3ζ KO mice. Although 14-3-3ζ is proposed to modulate activity of the rate-limiting DA biosynthesis enzyme, tyrosine hydroxylase (TH), we were unable to identify any differences in total TH levels, TH localization or TH activation in 14-3-3ζ KO mice. Rather, our analysis identified significantly reduced levels of DAT in the absence of notable differences in RNA or protein levels of DA receptors D1–D5. Providing insight into the mechanisms by which 14-3-3ζ controls DAT stability, we found a physical association between 14-3-3ζ and DAT by co-immunoprecipitation. Taken together, our results identify a novel role for 14-3-3ζ in DA neurotransmission and provide support to the hyperdopaminergic basis of pathologies associated with schizophrenia and related disorders. PMID:24301645

  18. 14-3-3ζ Interacts with Stat3 and Regulates Its Constitutive Activation in Multiple Myeloma Cells

    PubMed Central

    Li, Wenliang; Xiong, Qian; Yang, Mingkun; Zheng, Peng; Li, Chongyang; Pei, Jianfeng; Ge, Feng

    2012-01-01

    The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner and function as adapter or scaffold proteins in signal transduction pathways. One family member, 14-3-3ζ, is believed to function in cell signaling, cycle control, and apoptotic death. A systematic proteomic analysis done in our laboratory has identified signal transducers and activators of transcription 3 (Stat3) as a novel 14-3-3ζ interacting protein. Following our initial finding, in this study, we provide evidence that 14-3-3ζ interacts physically with Stat3. We further demonstrate that phosphorylation of Stat3 at Ser727 is vital for 14-3-3ζ interaction and mutation of Ser727 to Alanine abolished 14-3-3ζ/Stat3 association. Inhibition of 14-3-3ζ protein expression in U266 cells inhibited Stat3 Ser727 phosphorylation and nuclear translocation, and decreased both Stat3 DNA binding and transcriptional activity. Moreover, 14-3-3ζ is involved in the regulation of protein kinase C (PKC) activity and 14-3-3ζ binding to Stat3 protects Ser727 dephosphorylation from protein phosphatase 2A (PP2A). Taken together, our findings support the model that multiple signaling events impinge on Stat3 and that 14-3-3ζ serves as an essential coordinator for different pathways to regulate Stat3 activation and function in MM cells. PMID:22279540

  19. Differential expression of 14-3-3 isoforms in human alcoholic brain

    PubMed Central

    MacKay, Rachel K.; Colson, Natalie J.; Dodd, Peter R.; Lewohl, Joanne M.

    2011-01-01

    Background Neuropathological damage due to chronic alcohol abuse often results in impairment of cognitive function. The damage is particularly marked in the frontal cortex. The 14-3-3 protein family consists of 7 proteins, β, γ, ε, ζ, η, θ and σ, encoded by 7 distinct genes. They are highly conserved molecular chaperones with roles in regulation of metabolism, signal transduction, cell-cycle control, protein trafficking, and apoptosis. They may also play an important role in neurodegeneration in chronic alcoholism. Methods We used Real-Time PCR to measure the expression of 14-3-3 mRNA transcripts in both the dorsolateral prefrontal cortex and motor cortex of human brains obtained at autopsy. Results We found significantly lower 14-3-3β, γ and θ expression in both cortical areas of alcoholics; but no difference in 14-3-3η expression, and higher expression of 14-3-3σ, in both areas. Levels of 14-3-3ζ and ε transcripts were significantly lower only in alcoholic motor cortex. Conclusions Altered 14-3-3 expression could contribute to synaptic dysfunction and altered neurotransmission in chronic alcohol misuse by human subjects. PMID:21332526

  20. Ywhaz/14-3-3ζ Deletion Improves Glucose Tolerance Through a GLP-1-Dependent Mechanism.

    PubMed

    Lim, Gareth E; Piske, Micah; Lulo, James E; Ramshaw, Hayley S; Lopez, Angel F; Johnson, James D

    2016-07-01

    Multiple signaling pathways mediate the actions of metabolic hormones to control glucose homeostasis, but the proteins that coordinate such networks are poorly understood. We previously identified the molecular scaffold protein, 14-3-3ζ, as a critical regulator of in vitro β-cell survival and adipogenesis, but its metabolic roles in glucose homeostasis have not been studied in depth. Herein, we report that Ywhaz gene knockout mice (14-3-3ζKO) exhibited elevated fasting insulin levels while maintaining normal β-cell responsiveness to glucose when compared with wild-type littermate controls. In contrast with our observations after an ip glucose bolus, glucose tolerance was significantly improved in 14-3-3ζKO mice after an oral glucose gavage. This improvement in glucose tolerance was associated with significantly elevated fasting glucagon-like peptide-1 (GLP-1) levels. 14-3-3ζ knockdown in GLUTag L cells elevated GLP-1 synthesis and increased GLP-1 release. Systemic inhibition of the GLP-1 receptor attenuated the improvement in oral glucose tolerance that was seen in 14-3-3ζKO mice. When taken together these findings demonstrate novel roles of 14-3-3ζ in the regulation of glucose homeostasis and suggest that modulating 14-3-3ζ levels in intestinal L cells may have beneficial metabolic effects through GLP-1-dependent mechanisms. PMID:27167773

  1. FTICR-MS analysis of 14-3-3 isoform substrate selection.

    PubMed

    Cardasis, Helene L; Sehnke, Paul C; Laughner, Beth; Eyler, John R; Powell, David H; Ferl, Robert J

    2007-07-01

    The 14-3-3s are a ubiquitous class of eukaryotic proteins that participate in a second regulatory step in many phosphorylation-based signal transduction systems. The Arabidopsis family of 14-3-3 proteins represents a rather large 14-3-3 gene family. The biological motive for such diversity within a single protein family is not yet completely understood. The work presented here utilizes 14-3-3 micro-affinity chromatography in conjunction with Fourier transform ion cyclotron resonance mass spectrometry to survey the substrate sequence selectivity of two Arabidopsis 14-3-3 isoforms that represent the two major subclasses of this protein family. A method was developed to compare the relative binding of eight synthetic phosphopeptide sequences. The degree to which each phosphopeptide bound to either isoform was assigned a relative value, defined here as the binding ratio. The method provided a simple means for visualizing differences in substrate sequence selection among different 14-3-3 isoforms. A reproducible preference for specific phosphopeptide sequences was measured for both isoforms. This binding preference was consistent among the two classes of isoforms, suggesting that any pressure for isoform selectivity must reside outside the central core that interacts with the phosphopeptide sequence of the client. PMID:17569603

  2. 14-3-3ζ: A numbers game in adipocyte function?

    PubMed Central

    Lim, Gareth E.; Johnson, James D.

    2016-01-01

    ABSTRACT Molecular scaffolds are often viewed as passive signaling molecules that facilitate protein-protein interactions. However, new evidence gained from the use of loss-of-function or gain-of-function models is dispelling this notion. Our own recent discovery of 14-3-3ζ as an essential regulator of adipogenesis highlights the complex roles of this member of the 14-3-3 protein family. Depletion of the 14-3-3ζ isoform affected parallel pathways that drive adipocyte development, including pathways controlling the stability of key adipogenic transcription factors and cell cycle progression. Going beyond adipocyte differentiation, this study opens new avenues of research in the context of metabolism, as 14-3-3ζ binds to a variety of well-established metabolic proteins that harbor its canonical phosphorylation binding motifs. This suggests that 14-3-3ζ may contribute to key metabolic signaling pathways, such as those that facilitate glucose uptake and fatty acid metabolism. Herein, we discuss these novel areas of research, which will undoubtedly shed light onto novel roles of 14-3-3ζ, and perhaps its related family members, on glucose homeostasis. PMID:27386155

  3. Decreased expression of 14-3-3 in Paracoccidioides brasiliensis confirms its involvement in fungal pathogenesis.

    PubMed

    Marcos, Caroline Maria; Silva, Julhiany de Fátima ds; Oliveira, Haroldo Cesar de; Assato, Patrícia Akemi; Singulani, Junya de Lacorte; Lopez, Angela Maria; Tamayo, Diana Patricia; Hernandez-Ruiz, Orville; McEwen, Juan G; Mendes-Giannini, Maria José Soares; Fusco-Almeida, Ana Marisa

    2016-01-01

    The interaction between the fungal pathogen Paracoccidioides brasiliensis and host cells is usually mediated by specific binding events between adhesins on the fungal surface and receptors on the host extracellular matrix or cell surface. One molecule implicated in the P. brasiliensis-host interaction is the 14-3-3 protein. The 14-3-3 protein belongs to a family of conserved regulatory molecules that are expressed in all eukaryotic cells and are involved in diverse cellular functions. Here, we investigated the relevance of the 14-3-3 protein to the virulence of P. brasiliensis. Using antisense RNA technology and Agrobacterium tumefaciens-mediated transformation, we generated a 14-3-3-silenced strain (expression reduced by ˜55%). This strain allowed us to investigate the interaction between 14-3-3 and the host and to correlate the functions of P. brasiliensis 14-3-3 with cellular features, such as morphological characteristics and virulence, that are important for pathogenesis. PMID:26646480

  4. Expression of bactericidal/permeability-increasing protein requires C/EBP epsilon.

    PubMed

    Tanaka, Miyuki; Gombart, Adrian F; Koeffler, H Phillip; Shiohara, Masaaki

    2007-05-01

    Bactericidal/permeability-increasing protein (BPI) is a 55-kd cationic protein found mainly in neutrophil primary granules. BPI shows cytotoxicity against Gram-negative bacteria. In this study, we studied the role of a myeloid-specific transcription factor, CCAAT/enhancer binding protein epsilon (C/EBP epsilon), in the regulation of BPI gene expression. A patient with neutrophil-specific granule deficiency with a homozygous inactivating mutation in the CEBP epsilon gene showed severely impaired expression of both BPI messenger RNA (mRNA) and BPI protein. Both U937 and NB4 cells treated with 10-7 M all-trans retinoic acid (ATRA) for 6 days displayed increased levels of BPI protein and accompanying up-regulated C/EBP epsilon expression. Chromatin-immunoprecipitation analysis and electrophoretic mobility shift assays revealed binding of the C/EBP epsilon protein to the C/EBP-binding site in the BPI gene promoter. U937 cells stably transfected with a zinc-inducible C/EBP epsilon expression vector showed a 30-fold increase in BPI mRNA levels compared with cells transfected with control empty vector after culturing for 48 hours with 100 microM ZnSO4. BPI mRNA expression was severely reduced in the bone marrow of C/EBP epsilon-deficient mice compared with wild-type mice. Expression of BPI in human cord blood cells was increased by incubation with 10-7 MATRA for 48 hours. These results demonstrate the requirement for C/EBP epsilon in mediating BPI gene expression in myeloid cells in vitro and in vivo. PMID:17483073

  5. Identification and expression analysis of four 14-3-3 genes during fruit ripening in banana (Musa acuminata L. AAA group, cv. Brazilian).

    PubMed

    Li, Mei-Ying; Xu, Bi-Yu; Liu, Ju-Hua; Yang, Xiao-Liang; Zhang, Jian-Bin; Jia, Cai-Hong; Ren, Li-Cheng; Jin, Zhi-Qiang

    2012-02-01

    To investigate the regulation of 14-3-3 proteins in banana (Musa acuminata L. AAA group, cv. Brazilian) fruit postharvest ripening, four cDNAs encoding 14-3-3 proteins were isolated from banana and designated as Ma-14-3-3a, Ma-14-3-3c, Ma-14-3-3e, and Ma-14-3-3i, respectively. Amino acid sequence alignment showed that the four 14-3-3 proteins shared a highly conserved core structure and variable C-terminal as well as N-terminal regions with 14-3-3 proteins from other plant species. Phylogenetic analysis revealed that the four 14-3-3 genes belong to the non-ε groups. They were differentially and specifically expressed in various tissues. Real-time RT-PCR analysis indicated that these four genes function differentially during banana fruit postharvest ripening. Three genes, Ma-14-3-3a, Ma-14-3-3c, and Ma-14-3-3e, were significantly induced by exogenous ethylene treatment. However, gene function differed in naturally ripened fruits. Ethylene could induce Ma-14-3-3c expression during postharvest ripening, but expression patterns of Ma-14-3-3a and Ma-14-3-3e suggest that these two genes appear to be involved in regulating ethylene biosynthesis during fruit ripening. No obvious relationship emerged between Ma-14-3-3i expression in naturally ripened and 1-MCP (1-methylcyclopropene)-treated fruit groups during fruit ripening. These results indicate that the 14-3-3 proteins might be involved in various regulatory processes of banana fruit ripening. Further studies will mainly focus on revealing the detailed biological mechanisms of these four 14-3-3 genes in regulating banana fruit postharvest ripening. PMID:22009053

  6. Genome-Wide Identification and Expression Analysis of the 14-3-3 Family Genes in Medicago truncatula

    PubMed Central

    Qin, Cheng; Cheng, Linming; Shen, Jingqin; Zhang, Yunhong; Cao, Huimin; Lu, Dan; Shen, Chenjia

    2016-01-01

    The 14-3-3 gene family, which is conserved in eukaryotes, is involved in protein-protein interactions and mediates signal transduction. However, detailed investigations of the 14-3-3 gene family in Medicago truncatula are largely unknown. In this study, the identification and study of M. truncatula 14-3-3-family genes were performed based on the latest M. truncatula genome. In the M. truncatula genome, 10 14-3-3 family genes were identified, and they can be grouped into ε and non-ε groups. An exon-intron analysis showed that the gene structures are conserved in the same group. The protein structure analysis showed that 14-3-3 proteins in M. truncatula are composed of nine typical antiparallel α-helices. The expression patterns of Mt14-3-3 genes indicated that they are expressed in all tissues. Furthermore, the gene expression levels of Mt14-3-3 under hormone treatment and Sinorhizobium meliloti infection showed that the Mt14-3-3 genes were involve in nodule formation. Our findings lay a solid foundation for further functional studies of 14-3-3 in M. truncatula. PMID:27047505

  7. The pro-inflammatory cytokine 14-3-3ε is a ligand of CD13 in cartilage

    PubMed Central

    Nefla, Meriam; Sudre, Laure; Denat, Guillaume; Priam, Sabrina; Andre-Leroux, Gwenaëlle; Berenbaum, Francis; Jacques, Claire

    2015-01-01

    ABSTRACT Osteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13 (also known as aminopeptidase N, APN) in the chondrocyte response to 14-3-3ε. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3ε-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3ε signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3ε. Direct interaction between CD13 and 14-3-3ε was then demonstrated by surface plasmon resonance. Using labeled 14-3-3ε, we also found that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3ε might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3ε–CD13 interaction could be a new therapeutic target in osteoarthritis. PMID:26208633

  8. The pro-inflammatory cytokine 14-3-3ε is a ligand of CD13 in cartilage.

    PubMed

    Nefla, Meriam; Sudre, Laure; Denat, Guillaume; Priam, Sabrina; Andre-Leroux, Gwenaëlle; Berenbaum, Francis; Jacques, Claire

    2015-09-01

    Osteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13 (also known as aminopeptidase N, APN) in the chondrocyte response to 14-3-3ε. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3ε-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3ε signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3ε. Direct interaction between CD13 and 14-3-3ε was then demonstrated by surface plasmon resonance. Using labeled 14-3-3ε, we also found that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3ε might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3ε-CD13 interaction could be a new therapeutic target in osteoarthritis. PMID:26208633

  9. The 14-3-3 Gene Function of Cryptococcus neoformans Is Required for its Growth and Virulence.

    PubMed

    Li, Jingbo; Chang, Yun C; Wu, Chun-Hua; Liu, Jennifer; Kwon-Chung, Kyung J; Huang, Sheng-He; Shimada, Hiro; Fante, Rob; Fu, Xiaowei; Jong, Ambrose

    2016-05-28

    Cryptococcus neoformans is a life-threatening pathogenic yeast that causes devastating meningoencephalitis. The mechanism of cryptococcal brain invasion is largely unknown, and recent studies suggest that its extracellular microvesicles may be involved in the invasion process. The 14-3-3 protein is abundant in the extracellular microvesicles of C. neoformans, and the 14-3-3-GFP fusion has been used as the microvesicle's marker. However, the physiological role of 14-3-3 has not been explored. In this report, we have found that C. neoformans contains a single 14-3-3 gene that apparently is an essential gene. To explore the functions of 14-3-3, we substituted the promoter region of the 14-3-3 with the copper-controllable promoter CTR4. The CTR4 regulatory strain showed an enlarged cell size, drastic changes in morphology, and a decrease in the thickness of the capsule under copper-enriched conditions. Furthermore, the mutant cells produced a lower amount of total proteins in their extracellular microvesicles and reduced adhesion to human brain microvascular endothelial cells in vitro. Proteomic analyses of the protein components under 14-3-3-overexpressed and -suppressed conditions revealed that the 14-3-3 function(s) might be associated with the microvesicle biogenesis. Our results support that 14-3-3 has diverse pertinent roles in both physiology and pathogenesis in C. neoformans. Its gene functions are closely relevant to the pathogenesis of this fungus. PMID:26437944

  10. 14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization

    PubMed Central

    Sambandam, Sumitha A.T.; Kasetti, Ramesh Babu; Xue, Lei; Dean, Douglas C.; Lu, Qingxian; Li, Qiutang

    2015-01-01

    The homozygous repeated epilation (Er/Er) mouse mutant of the gene encoding 14-3-3σ displays an epidermal phenotype characterized by hyperproliferative keratinocytes and undifferentiated epidermis. Heterozygous Er/+ mice develop spontaneous skin tumors and are highly sensitive to tumor-promoting DMBA/TPA induction. The molecular mechanisms underlying 14-3-3σ regulation of epidermal proliferation, differentiation, and tumor formation have not been well elucidated. In the present study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro. In addition, enhanced Yap1 nuclear localization was also evident in DMBA/TPA-induced tumors from Er/+ skin. Furthermore, shRNA knockdown of Yap1 expression in Er/Er keratinocytes inhibited their proliferation, suggesting that YAP1 functions as a downstream effector of 14-3-3σ controlling epidermal proliferation. We then demonstrated that keratinocytes express all seven 14-3-3 protein isoforms, some of which form heterodimers with 14-3-3σ, either full-length WT or the mutant form found in Er/Er mice. However Er 14-3-3σ does not interact with Yap1, as demonstrated by co-immunoprecipitation. We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in Er/Er epidermis. PMID:25668240

  11. Dynamic interaction between 14-3-3zeta and bax during TNF-α-induced apoptosis in living cells

    NASA Astrophysics Data System (ADS)

    Gao, Xuejuan; Xing, Da; Chen, Tongsheng

    2006-09-01

    Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but redistributes to mitochondria and undergoes oligomerization to induce the release of apoptogenic factors such as cytochrome c in response to apoptotic stimuli. Cytoplasmic protein 14-3-3zeta binds to Bax and, upon apoptotic stimulation, releases Bax by a caspase-independent mechanism. However, the direct interaction of the cytoplasmic 14-3-3zeta and Bax in living cells has not been observed. In present study, to monitor the dynamic interaction between 14-3-3zeta and Bax in living cells in real time during apoptosis induced by tumor necrosis factor (TNF-α), DsRed-14-3-3zeta plasmid is constructed. By cotransfecting DsRed- 14-3-3zeta and GFP-Bax plasmids into human lung adenocarcinoma cells (ASTC-a-1), we observe the dynamic interaction between Bax and 14-3-3zeta using fluorescence resonance energy transfer (FRET) technique on laser scanning confocal microscope. The results show that 14-3-3zeta remains in the cytoplasm but GFP-Bax translocates to mitochondria completely after TNF-α stimulation. These results reveal that 14-3-3zeta binds directly to Bax in healthy cells, and that 14-3-3zeta negatively regulates Bax translocation to mitochondria during TNF-α-induced apoptosis.

  12. 14-3-3, an integrator of cell mechanics and cytokinesis.

    PubMed

    Robinson, Douglas N

    2010-11-01

    One of the goals of understanding cytokinesis is to uncover the molecular regulation of the cellular mechanical properties that drive cell shape change. Such regulatory pathways are likely to be used at multiple stages of a cell's life, but are highly featured during cell division. Recently, we demonstrated that 14-3-3 (encoded by a single gene in the social amoeba Dictyostelium discoideum) serves to integrate key cytoskeletal components-microtubules, Rac and myosin II-to control cell mechanics and cytokinesis. As 14-3-3 proteins are frequently altered in a variety of human tumors, we extend these observations to suggest possible additional roles for how 14-3-3 proteins may contribute to tumorigenesis. PMID:21686271

  13. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

    PubMed Central

    Li, Tong; Paudel, Hemant K.

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer’s disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445–6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  14. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells.

    PubMed

    Li, Tong; Paudel, Hemant K

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer's disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445-6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  15. 14-3-3θ is a binding partner of rat Eag1 potassium channels.

    PubMed

    Hsu, Po-Hao; Miaw, Shi-Chuen; Chuang, Chau-Ching; Chang, Pei-Yu; Fu, Ssu-Ju; Jow, Guey-Mei; Chiu, Mei-Miao; Jeng, Chung-Jiuan

    2012-01-01

    The ether-à-go-go (Eag) potassium (K(+)) channel belongs to the superfamily of voltage-gated K(+) channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and native hippocampal neurons. Electrophysiological studies showed that over-expression of 14-3-3θ led to a sizable suppression of rEag1 K(+) currents with no apparent alteration of the steady-state voltage dependence and gating kinetics. Furthermore, co-expression with 14-3-3θ failed to affect the total protein level, membrane trafficking, and single channel conductance of rEag1, implying that 14-3-3θ binding may render a fraction of the channel locked in a non-conducting state. Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons. PMID:22911758

  16. Ischemia preconditioning protects astrocytes from ischemic injury through 14-3-3γ.

    PubMed

    Pang, Ying; Chai, Chao Rui; Gao, Kai; Jia, Xi Hua; Kong, Jin Ge; Chen, Xiao Qian; Vatcher, Greg; Chen, Jian Guo; Yu, Albert Cheung Hoi

    2015-10-01

    Stroke is a leading cause of death and disability, and new strategies are required to reduce neuronal injury and improve prognosis. Ischemia preconditioning (IPC) is an intrinsic phenomenon that protects cells from subsequent ischemic injury and might provide promising mechanisms for clinical treatment. In this study, primary astrocytes exhibited significantly less cell death than control when exposed to different durations of IPC (15, 30, 60, or 120 min). A 15-min duration was the most effective IPC to protect astrocytes from 8-hr-ischemia injury. The protective mechanisms of IPC involve the upregulation of protective proteins, including 14-3-3γ, and attenuation of malondialdehyde (MDA) content and ATP depletion. 14-3-3γ is an antiapoptotic intracellular protein that was significantly upregulated for up to 84 hr after IPC. In addition, IPC promoted activation of the c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK)-1/2, p38, and protein kinase B (Akt) signaling pathways. When JNK was specifically inhibited with SP600125, the upregulation of 14-3-3γ induced by IPC was almost completely abolished; however, there was no effect on ATP or MDA levels. This suggests that, even though both energy preservation and 14-3-3γ up-regulation were turned on by IPC, they were controlled by different pathways. The ERK1/2, p38, and Akt signaling pathways were not involved in the 14-3-3γ upregulation and energy preservation. These results indicate that IPC could protect astrocytes from ischemia injury by inducing 14-3-3γ and by alleviating energy depletion through different pathways, suggesting multiple protection of IPC and providing new insights into potential stroke therapies. PMID:25711139

  17. Protein kinase C epsilon is localized to the Golgi via its zinc-finger domain and modulates Golgi function.

    PubMed Central

    Lehel, C; Olah, Z; Jakab, G; Anderson, W B

    1995-01-01

    Protein kinase C (PKC) is a multigene family of serine/threonine kinases that are central to many signal transduction pathways. Among the PKC isozymes, only PKC epsilon has been reported to exhibit full oncogenic potential. PKC epsilon also displays unique substrate specificity and intracellular localization. To examine the interrelationship between the biological effects and domain structure of PKC epsilon, NIH 3T3 cells were stably transfected to overexpress different epitope-tagged fragments of PKC epsilon. The overexpressed proteins each contain the epsilon-tag peptide at the C terminus to allow ready detection with an antibody specific for the tag. The holo-PKC epsilon was found to localize with the Golgi network and other compartments, whereas the zinc-finger domain localized exclusively at the Golgi. Golgi-specific glycosaminoglycan sulfation was strongly inhibited in cells overexpressing either holo-PKC epsilon or its zinc-finger domain, while the secretion of sulfated glycosaminoglycans into the medium was impaired in cells expressing the PKC epsilon zinc-finger domain. Thus, these results suggest that PKC epsilon may be involved in specifically regulating Golgi-related processes. Further, the results indicate that PKC epsilon domains other than the kinase domain may also have biological activity and that the zinc-finger domain may function as a subcellular localization signal. Images Fig. 1 Fig. 2 Fig. 3 PMID:7877991

  18. Possible additional roles in mating for Ustilago maydis Rho1 and 14-3-3 homologues

    PubMed Central

    Pham, Cau D

    2010-01-01

    Both the Rho GTPases and 14-3-3 proteins each belong to ubiquitous families of proteins involved in a variety of cellular processes, including cytokinesis, cell polarity, cellular differentiation and apoptosis. In fungi, these components of signaling pathways are involved in cell cycle regulation, cytokinesis and virulence. We study cellular differentiation and pathogenesis for Ustilago maydis, the dimorphic fungal pathogen of maize. We have reported on the interactions of Pdc1, a U. maydis homologue of human 14-3-3ɛ, with Rho1, a small GTP binding protein; these proteins participate in cell polarity and filamentation pathways that include another small G protein, Rac1, and its effector PAK kinase, Cla4. Here we describe additional experiments that explore possible relationships of Pdc1 and Rho1 with another PAK-like kinase pathway and with the a matingtype locus. PMID:20539785

  19. Modulation of 14-3-3 interaction with phosphorylated histone H3 by combinatorial modification patterns

    PubMed Central

    Winter, Stefan; Fischle, Wolfgang; Seiser, Christian

    2011-01-01

    Post-translational modifications of histones are determining factors in the global and local regulation of genome activity. Phosphorylation of histone H3 is globally associated with mitotic chromatin compaction but occurs in a much more restricted manner during interphase transcriptional regulation of a limited subset of genes. In the course of gene regulation, serine 10 phosphorylation at histone H3 is targeted to a very small fraction of nucleosomes that is highly susceptible to additional acetylation events. Recently, we and others have identified 14-3-3 as a binding protein that recognizes both phosphorylated serine 10 and phosphorylated serine 28 on histone H3. In vitro, the affinity of 14-3-3 for phosphoserine 10 is weak but becomes significantly increased by additional acetylation of either lysine 9 or lysine 14 on the same histone tail. In contrast, the histone H3S28 site matches elements of 14-3-3 high affinity consensus motifs. This region mediates an initial stronger interaction that is less susceptible to modulation by “auxiliary” modifications. Here we discuss the binding of 14-3-3 proteins to histone H3 in detail and putative biological implications of these interactions. PMID:18418070

  20. Sporadic Creutzfeldt-Jakob disease diagnostic accuracy is improved by a new CSF ELISA 14-3-3γ assay.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-05-13

    Protein 14-3-3 is a reliable marker of rapid neuronal damage, specifically increased in cerebrospinal fluid (CSF) of sporadic Creutzfeldt-Jakob disease (sCJD) patients. Its detection is usually performed by Western Blot (WB), prone to methodological issues. Our aim was to evaluate the diagnostic performance of a recently developed quantitative enzyme-linked immunosorbent (ELISA) assay for 14-3-3γ, in comparison with WB and other neurodegeneration markers. CSF samples from 145 patients with suspicion of prion disease, later classified as definite sCJD (n=72) or Non-prion diseases (Non-CJD; n=73) comprised our population. 14-3-3 protein was determined by WB and ELISA. Total Tau (t-Tau) and phosphorylated Tau (p-Tau) were also evaluated. Apolipoprotein E gene (ApoE) and prionic protein gene (PRNP) genotyping was assessed. ELISA 14-3-3γ levels were significantly increased in sCJD compared to Non-CJD patients (p<0.001), showing very good accuracy (AUC=0.982; sensitivity=97%; specificity=94%), and matching WB results in 81% of all cases. It strongly correlated with t-Tau and p-Tau (p<0.0001), showing slightly higher specificity (14-3-3 WB - 63%; Tau - 90%; p-Tau/t-Tau ratio - 88%). From WB inconclusive results (n=44), ELISA 14-3-3γ correctly classified 41 patients. Additionally, logistic regression analysis selected ELISA 14-3-3γ as the best single predictive marker for sCJD (overall accuracy=93%). ApoE and PRNP genotypes did not influence ELISA 14-3-3γ levels. Despite specificity for 14-3-3γ isoform, ELISA results not only match WB evaluation but also help discrimination of inconclusive results. Our results therefore reinforce this assay as a single screening test, allowing higher sample throughput and unequivocal results. PMID:26940479

  1. 14-3-3σ is an independent prognostic biomarker for gastric cancer and is associated with apoptosis and proliferation in gastric cancer.

    PubMed

    Li, Yi-Liang; Liu, Lihua; Xiao, Yang; Zeng, Tao; Zeng, Chao

    2015-01-01

    14-3-3 proteins participate in various cellular processes, including apoptosis, proliferation and malignant transformation. 14-3-3σ, a member of the 14-3-3 protein family, is important in several types of cancer; however, little is known about the clinical significance and biological roles of 14-3-3σ in gastric cancer. The present study analyzed the expression pattern of 14-3-3σ in gastric cancer and investigated its correlation with the prognosis of gastric cancer patients. Furthermore, the association of 14-3-3σ with Ki-67, Bcl-2 and Bax was evaluated. 14-3-3σ was expressed at higher level in gastric cancer tissue compared with healthy gastric tissue, and 14-3-3σ expression was significantly correlated with tumor size and tumor node metastasis stage (P<0.05). To the best of our knowledge, the present study data are the first to suggest that 14-3-3σ expression has been significantly associated with poor prognosis in gastric cancer. Additionally, 14-3-3σ overexpression was positively correlated with Ki-67 and Bcl-2 expression levels. Thus, 14-3-3σ is a potential prognostic marker for gastric cancer patients, and may be involved in regulating the apoptosis and proliferation of gastric cancer cells. PMID:25435977

  2. The 14-3-3σ/GSK3β/β-catenin/ZEB1 regulatory loop modulates chemo-sensitivity in human tongue cancer

    PubMed Central

    Xiong, Yan; Yin, Jiang; Li, Nan; Deng, Yingen; Luo, Kai; Zhang, Qiong; Wang, Chengkun; Zhang, Zhijie; Zheng, Guopei; He, Zhimin

    2015-01-01

    Here we demonstrated that chemotherapy induced 14-3-3σ expression in tongue cancer (TC) cells and overexpressed 14-3-3σ sensitized TC cells to chemotherapy especially in multidrug resistant TC (MDR-TC) cells. In agreement, 14-3-3σ knockdown enhanced resistance of TC cells to chemotherapy. Mechanically, we found 14-3-3σ physically bound to GSK3β in protein level and the binding inhibited β-catenin signaling. Coincidentally, chemotherapy as well as 14-3-3σ overexpression led to increase of GSK3β protein level. Increased GSK3β protein sensitized TC cells to chemotherapy. Moreover, deregulation of 14-3-3σ/GSK3β/β-catenin axis led to overexpressed ZEB1 in TC cells, especially in MDR-TC cells. As a negative feedback loop, ZEB1 bond to 14-3-3σ promoter to enhance promoter hypermethylation in TC cells. Promoter hypermethylation resulted into the decrease of 14-3-3σ expression. Importantly, a positive correlation was observed between 14-3-3σ and GSK3β protein expression in TC tissues from patients receiving chemotherapy. High levels of 14-3-3σ and GSK3β were associated with better prognosis in TC patients. PMID:26036631

  3. Loss of the 14-3-3σ is essential for LASP1-mediated colorectal cancer progression via activating PI3K/AKT signaling pathway

    PubMed Central

    Shao, Ziyun; Cai, Yanjun; Xu, Lijun; Yao, Xueqing; Shi, Jiaolong; Zhang, Feifei; Luo, Yuhao; Zheng, Kehong; Liu, Jian; Deng, Fengliu; Li, Rui; Zhang, Lanzhi; Wang, Hui; Li, Mingyi; Ding, Yanqing; Zhao, Liang

    2016-01-01

    LIM and SH3 protein 1 (LASP1) can promote colorectal cancer (CRC) progression and metastasis, but the direct evidence that elucidates the molecular mechanism remains unclear. Here, our proteomic data showed that LASP1 interacted with 14-3-3σ and decreased the expression of 14-3-3σ in CRC. Deletion of 14-3-3σ was required for LASP1-mediated CRC cell aggressiveness. In vitro gain- and loss-of-function assays showed that 14-3-3σ suppressed the ability of cell migration and decreased the phosphorylation of AKT in CRC cells. We further observed clearly co-localization between AKT and 14-3-3σ in CRC cells. Treatment of PI3K inhibitor LY294002 markedly prevented phosphorylation of AKT and subsequently counteract aggressive phenotype mediated by siRNA of 14-3-3σ. Clinically, 14-3-3σ is frequently down-regulated in CRC tissues. Down-regulation of 14-3-3σ is associated with tumor progression and poor prognosis of patients with CRC. Multivariate analysis confirmed low expression of 14-3-3σ as an independent prognostic factor for CRC. A combination of low 14-3-3σ and high LASP1 expression shows a worse trend with overall survival of CRC patients. Our research paves the path to future investigation of the LASP1-14-3-3σ axis as a target for novel anticancer therapies of advanced CRC. PMID:27156963

  4. 14-3-3ε and ζ Regulate Neurogenesis and Differentiation of Neuronal Progenitor Cells in the Developing Brain

    PubMed Central

    Wachi, Tomoka; Hunt, Robert F.; Baraban, Scott C.; Taya, Shinichiro; Ramshaw, Hayley; Kaibuchi, Kozo; Schwarz, Quenten P.; Lopez, Angel F.

    2014-01-01

    During brain development, neural progenitor cells proliferate and differentiate into neural precursors. These neural precursors migrate along the radial glial processes and localize at their final destination in the cortex. Numerous reports have revealed that 14-3-3 proteins are involved in many neuronal activities, although their functions in neurogenesis remain unclear. Here, using 14-3-3ε/ζ double knock-out mice, we found that 14-3-3 proteins are important for proliferation and differentiation of neural progenitor cells in the cortex, resulting in neuronal migration defects and seizures. 14-3-3 deficiency resulted in the increase of δ-catenin and the decrease of β-catenin and αN-catenin. 14-3-3 proteins regulated neuronal differentiation into neurons via direct interactions with phosphorylated δ-catenin to promote F-actin formation through a catenin/Rho GTPase/Limk1/cofilin signaling pathway. Conversely, neuronal migration defects seen in the double knock-out mice were restored by phosphomimic Ndel1 mutants, but not δ-catenin. Our findings provide new evidence that 14-3-3 proteins play important roles in neurogenesis and neuronal migration via the regulation of distinct signaling cascades. PMID:25186760

  5. 14-3-3ε and ζ regulate neurogenesis and differentiation of neuronal progenitor cells in the developing brain.

    PubMed

    Toyo-oka, Kazuhito; Wachi, Tomoka; Hunt, Robert F; Baraban, Scott C; Taya, Shinichiro; Ramshaw, Hayley; Kaibuchi, Kozo; Schwarz, Quenten P; Lopez, Angel F; Wynshaw-Boris, Anthony

    2014-09-01

    During brain development, neural progenitor cells proliferate and differentiate into neural precursors. These neural precursors migrate along the radial glial processes and localize at their final destination in the cortex. Numerous reports have revealed that 14-3-3 proteins are involved in many neuronal activities, although their functions in neurogenesis remain unclear. Here, using 14-3-3ε/ζ double knock-out mice, we found that 14-3-3 proteins are important for proliferation and differentiation of neural progenitor cells in the cortex, resulting in neuronal migration defects and seizures. 14-3-3 deficiency resulted in the increase of δ-catenin and the decrease of β-catenin and αN-catenin. 14-3-3 proteins regulated neuronal differentiation into neurons via direct interactions with phosphorylated δ-catenin to promote F-actin formation through a catenin/Rho GTPase/Limk1/cofilin signaling pathway. Conversely, neuronal migration defects seen in the double knock-out mice were restored by phosphomimic Ndel1 mutants, but not δ-catenin. Our findings provide new evidence that 14-3-3 proteins play important roles in neurogenesis and neuronal migration via the regulation of distinct signaling cascades. PMID:25186760

  6. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

    PubMed

    Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

    2016-01-01

    The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform. PMID:27556493

  7. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability

    PubMed Central

    Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

    2016-01-01

    The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform. PMID:27556493

  8. Design and synthesis of protein kinase C epsilon selective diacylglycerol lactones (DAG-lactones).

    PubMed

    Ann, Jihyae; Yoon, Suyoung; Baek, Jisoo; Kim, Da Hye; Lewin, Nancy E; Hill, Colin S; Blumberg, Peter M; Lee, Jeewoo

    2015-01-27

    DAG-lactones afford a synthetically accessible, high affinity platform for probing structure activity relationships at the C1 regulatory domain of protein kinase C (PKC). Given the central role of PKC isoforms in cellular signaling, along with their differential biological activities, a critical objective is the design of isoform selective ligands. Here, we report the synthesis of a series of DAG-lactones varying in their side chains, with a particular focus on linoleic acid derivatives. We evaluated their selectivity for PKC epsilon versus PKC alpha both under standard lipid conditions (100% phosphatidylserine, PS) as well as in the presence of a nuclear membrane mimetic lipid mixture (NML). We find that selectivity for PKC epsilon versus PKC alpha tended to be enhanced in the presence of the nuclear membrane mimetic lipid mixture and, for our lead compound, report a selectivity of 32-fold. PMID:25437619

  9. 14-3-3ζ promotes hepatocellular carcinoma venous metastasis by modulating hypoxia-inducible factor-1α

    PubMed Central

    Shi, Jie; Yu, Hongming; Zhang, Long; Wang, Kang; Liu, Shangrong; Cheng, Shuqun

    2016-01-01

    Portal vein tumor thrombus (PVTT) is a type of intrahepatic metastasis arising from hepatocellular carcinoma (HCC) and is highly correlated with a poor prognosis. Hypoxia is common in solider tumors, including HCC, where it alters the behavior of HCC cells. We asked whether and how hypoxia contributes to PVTT formation. We demonstrated that increased intratumoral hypoxia is strongly associated with PVTT formation in HCC. We also showed that 14-3-3ζ is induced by hypoxia in HCC cells and correlates with PVTT formation in clinical HCC samples. In addition, 14-3-3ζ up-regulates HIF-1α expression by recruiting HDAC4, which prevents HIF-1α acetylation, thereby stabilizing the protein. Under hypoxic conditions in vitro, 14-3-3ζ knockdown inhibits hypoxia-induced HCC invasion by the HIF-1α/EMT pathway. Blockade of 14-3-3ζ in HCC cells reduces PVTT formation and distant lung metastasis in vivo. Moreover, a combination of 14-3-3ζ and HIF-1α expression is more prognostic for HCC patients than either protein alone. These results suggest that the hypoxia/14-3-3ζ/HIF-1α pathway plays an important role in PVTT formation and HCC metastasis. PMID:26910835

  10. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    SciTech Connect

    Xin, Ying; Lu, Qingxian; Li, Qiutang

    2010-02-19

    14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  11. A Characterization of the expression of 14-3-3 isoforms in psoriasis, basal cell carcinoma, atopic dermatitis and contact dermatitis

    PubMed Central

    Raaby, Line; Otkjær, Kristian; Salvskov-Iversen, Maria Luise; Johansen, Claus; Iversen, Lars

    2010-01-01

    14-3-3 is a highly conserved protein involved in a number of cellular processes including cell signalling, cell cycle regulation and gene transcription. Seven isoforms of the protein have been identified; β, γ, ε, ζ η σ and τ. The expression profile of the various isoforms in skin diseases is unknown. To investigate the expression of the seven 14-3-3 isoforms in involved and uninvolved skin from psoriasis, basal cell carcinoma (BCC), atopic dermatitis and nickel induced allergic contact dermatitis. Punch biopsies from involved and uninvolved skin were analyzed with quantitative reverse transcription-polymerase chain reaction to determine the mRNA expression of the 14-3-3 isoforms. The protein level of 14-3-3 isoforms was measured by Western blot technique in keratome biopsies from patients with psoriasis. Evaluation of dermal and epidermal protein expression was performed by immunofluorescence staining. Increased 14-3-3τ mRNA levels were detected in involved skin from patients with psoriasis, contact dermatitis and BCC. 14-3-3σ mRNA expression was increased in psoriasis and contact dermatitis, but not in BCC. In atopic dermatitis no significant difference between involved and uninvolved skin was found. The expression of the 14-3-3 isoforms was also studied at the protein level in psoriasis. Only 14-3-3τ expression was significantly increased in involved psoriatic skin compared with uninvolved skin. Immunofluorescence staining with 14-3-3τ- and 14-3-3σ-specific antibodies showed localization of both isoforms to the cytoplasm of the keratinocytes in the various skin sections. These results demonstrate a disease specific expression profile of the 14-3-3τ and 14-3-3σ iso-forms. PMID:25386251

  12. Phosphoproteomic analysis identifies the tumor suppressor PDCD4 as a RSK substrate negatively regulated by 14-3-3

    PubMed Central

    Galan, Jacob A.; Geraghty, Kathryn M.; Lavoie, Geneviève; Kanshin, Evgeny; Tcherkezian, Joseph; Calabrese, Viviane; Jeschke, Grace R.; Turk, Benjamin E.; Ballif, Bryan A.; Blenis, John; Thibault, Pierre; Roux, Philippe P.

    2014-01-01

    The Ras/MAPK signaling cascade regulates various biological functions, including cell growth and proliferation. As such, this pathway is frequently deregulated in several types of cancer, including most cases of melanoma. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its exact function and the nature of its substrates. Herein, we used a quantitative phosphoproteomics approach to define the signaling networks regulated by RSK in melanoma. To more accurately predict direct phosphorylation substrates, we defined the RSK consensus phosphorylation motif and found significant overlap with the binding consensus of 14-3-3 proteins. We thus characterized the phospho-dependent 14-3-3 interactome in melanoma cells and found that a large proportion of 14-3-3 binding proteins are also potential RSK substrates. Our results show that RSK phosphorylates the tumor suppressor PDCD4 (programmed cell death protein 4) on two serine residues (Ser76 and Ser457) that regulate its subcellular localization and interaction with 14-3-3 proteins. We found that 14-3-3 binding promotes PDCD4 degradation, suggesting an important role for RSK in the inactivation of PDCD4 in melanoma. In addition to this tumor suppressor, our results suggest the involvement of RSK in a vast array of unexplored biological functions with relevance in oncogenesis. PMID:25002506

  13. miR-451 protects against erythroid oxidant stress by repressing 14-3-3zeta.

    PubMed

    Yu, Duonan; dos Santos, Camila O; Zhao, Guowei; Jiang, Jing; Amigo, Julio D; Khandros, Eugene; Dore, Louis C; Yao, Yu; D'Souza, Janine; Zhang, Zhe; Ghaffari, Saghi; Choi, John; Friend, Sherree; Tong, Wei; Orange, Jordan S; Paw, Barry H; Weiss, Mitchell J

    2010-08-01

    The bicistronic microRNA (miRNA) locus miR-144/451 is highly expressed during erythrocyte development, although its physiological roles are poorly understood. We show that miR-144/451 ablation in mice causes mild erythrocyte instability and increased susceptibility to damage after exposure to oxidant drugs. This phenotype is deeply conserved, as miR-451 depletion synergizes with oxidant stress to cause profound anemia in zebrafish embryos. At least some protective activities of miR-451 stem from its ability to directly suppress production of 14-3-3zeta, a phospho-serine/threonine-binding protein that inhibits nuclear accumulation of transcription factor FoxO3, a positive regulator of erythroid anti-oxidant genes. Thus, in miR-144/451(-/-) erythroblasts, 14-3-3zeta accumulates, causing partial relocalization of FoxO3 from nucleus to cytoplasm with dampening of its transcriptional program, including anti-oxidant-encoding genes Cat and Gpx1. Supporting this mechanism, overexpression of 14-3-3zeta in erythroid cells and fibroblasts inhibits nuclear localization and activity of FoxO3. Moreover, shRNA suppression of 14-3-3zeta protects miR-144/451(-/-) erythrocytes against peroxide-induced destruction, and restores catalase activity. Our findings define a novel miRNA-regulated pathway that protects erythrocytes against oxidant stress, and, more generally, illustrate how a miRNA can influence gene expression by altering the activity of a key transcription factor. PMID:20679398

  14. Inhibition of proteolytic processing of adenoviral proteins by epsilon-aminocaproic acid and ambenum in adenovirus-infected cells.

    PubMed

    Nosach, Lidiya; Dyachenko, Nataliya; Zhovnovataya, Valentina; Lozinskiy, Miron; Lozitsky, Victor

    2002-01-01

    Maturation of adenovirus particles is markedly affected by proteolytic processing. The possibility for blocking the conversion of precursor structural core protein (preVII) into mature structure protein VII by officinal drugs epsilon-aminocaproic acid and ambenum has been demonstrated in Hep-2 cells infected with adenovirus. Proteolytic processing may be regarded as one of the targets for inhibiting adenovirus reproduction. PMID:12545207

  15. The role of 14-3-3{beta} in transcriptional activation of estrogen receptor {alpha} and its involvement in proliferation of breast cancer cells

    SciTech Connect

    Kim, Yoonseo; Kim, Hyungjin; Jang, Sung-Wuk; Ko, Jesang

    2011-10-14

    Highlights: {yields} 14-3-3{beta} interacts with ER{alpha} and the interaction is Akt-dependent. {yields} 14-3-3{beta} regulates the transcriptional activity of ER{alpha} in a ligand-dependent manner. {yields} 14-3-3{beta} increases expressions of ER{alpha} target genes. {yields} 14-3-3{beta} increases breast cancer cell proliferation. -- Abstract: The estrogen receptor (ER) functions as a transcription factor that mediates the effects of estrogen. ER{alpha}, which plays a crucial role in the development and progression of breast cancer, is activated by estrogen binding, leading to receptor phosphorylation, dimerization, and recruitment of co-activators and chaperons to the estrogen-bound receptor complex. The 14-3-3 proteins bind to target proteins via phosphorylation and influence many cellular events by altering their subcellular localization or acting as a chaperone. However, regulation of ER{alpha} expression and transactivation by the 14-3-3 proteins has not been reported. We demonstrate that 14-3-3{beta} functions as a positive regulator of ER{alpha} through a direct protein-protein interaction in an estrogen-dependent manner. Ectopic expression of 14-3-3{beta} stimulated ER{alpha}-mediated transcriptional activity in MCF-7 breast cancer cells. Enhanced ER{alpha} transcriptional activity due to 14-3-3{beta} increased the expressions of the endogenous ER{alpha} target genes, leading to proliferation of breast cancer cells. We suggest that 14-3-3{beta} has oncogenic potential in breast cancer via binding to ER{alpha} and activation of the transcriptional activity of ER{alpha}.

  16. PRKCE gene encoding protein kinase C-epsilon-Dual roles at sarcomeres and mitochondria in cardiomyocytes.

    PubMed

    Scruggs, Sarah B; Wang, Ding; Ping, Peipei

    2016-09-15

    Protein kinase C-epsilon (PKCε) is an isoform of a large PKC family of enzymes that has a variety of functions in different cell types. Here we discuss two major roles of PKCε in cardiac muscle cells; specifically, its role in regulating cardiac muscle contraction via targeting the sarcomeric proteins, as well as modulating cardiac cell energy production and metabolism by targeting cardiac mitochondria. The importance of PKCε action is described within the context of intracellular localization, as substrate selectivity and specificity is achieved through spatiotemporal targeting of PKCε. Accordingly, the role of PKCε in regulating myocardial function in physiological and pathological states has been documented in both cardioprotection and cardiac hypertrophy. PMID:27312950

  17. Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ*

    PubMed Central

    Kleppe, Rune; Rosati, Sara; Jorge-Finnigan, Ana; Alvira, Sara; Ghorbani, Sadaf; Haavik, Jan; Valpuesta, José María; Heck, Albert J. R.; Martinez, Aurora

    2014-01-01

    Phosphorylated tyrosine hydroxylase (TH) can form complexes with 14-3-3 proteins, resulting in enzyme activation and stabilization. Although TH was among the first binding partners identified for these ubiquitous regulatory proteins, the binding stoichiometry and the activation mechanism remain unknown. To address this, we performed native mass spectrometry analyses of human TH (nonphosphorylated or phosphorylated on Ser19 (TH-pS19), Ser40 (TH-pS40), or Ser19 and Ser40 (TH-pS19pS40)) alone and together with 14-3-3γ. Tetrameric TH-pS19 (224 kDa) bound 14-3-3γ (58.3 kDa) with high affinity (Kd = 3.2 nM), generating complexes containing either one (282.4 kDa) or two (340.8 kDa) dimers of 14-3-3. Electron microscopy also revealed one major population of an asymmetric complex, consistent with one TH tetramer and one 14-3-3 dimer, and a minor population of a symmetric complex of one TH tetramer with two 14-3-3 dimers. Lower phosphorylation stoichiometries (0.15–0.54 phosphate/monomer) produced moderate changes in binding kinetics, but native MS detected much less of the symmetric TH:14-3-3γ complex. Interestingly, dephosphorylation of [32P]-TH-pS19 was mono-exponential for low phosphorylation stoichiometries (0.18–0.52), and addition of phosphatase accelerated the dissociation of the TH-pS19:14-3-3γ complex 3- to 4-fold. All together this is consistent with a model in which the pS19 residues in the TH tetramer contribute differently in the association to 14-3-3γ. Complex formation between TH-pS40 and 14-3-3γ was not detected via native MS, and surface plasmon resonance showed that the interaction was very weak. Furthermore, TH-pS19pS40 behaved similarly to TH-pS19 in terms of binding stoichiometry and affinity (Kd = 2.1 nM). However, we found that 14-3-3γ inhibited the phosphorylation rate of TH-pS19 by PKA (3.5-fold) on Ser40. We therefore conclude that Ser40 does not significantly contribute to the binding of 14-3-3γ, and rather has reduced accessibility in

  18. Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

    PubMed

    Stevers, Loes M; Lam, Chan V; Leysen, Seppe F R; Meijer, Femke A; van Scheppingen, Daphne S; de Vries, Rens M J M; Carlile, Graeme W; Milroy, Lech G; Thomas, David Y; Brunsveld, Luc; Ottmann, Christian

    2016-03-01

    Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics. PMID:26888287

  19. The 14-3-3 gene expression specificity in response to stress is promoter-dependent.

    PubMed

    Aksamit, Anna; Korobczak, Alina; Skala, Jacek; Lukaszewicz, Marcin; Szopa, Jan

    2005-10-01

    Genomic clone coding for the 16R isoform of 14-3-3 proteins from potato plants has recently been described. This paper reports on 20R-gene isolation and analysis, and compares two isoforms. The northern blot analysis of mRNA of the 20R 14-3-3 isoform suggests its similarity to 16R. Vascular tissue-specific expression and age-dependent synthesis in potato leaves has been detected in both promoters. Screening of the potato genomic library using 20R cDNA isoform resulted in identification and isolation of the corresponding gene. This gene contains four exons and three introns. Inspecting the promoter sequence of the 20R isoform revealed several boxes important for the regulation of gene expression. The strongest GUS expression in transgenic potato plants transformed with the uidA reporter gene under the 20R promoter has been found in young leaf and stem vascular tissue, root tips, pollen and ovules. Mature fragments exhibit a significant decrease in GUS staining, which suggests age-dependent promoter activity. The analysis of transgenic plants transformed with 20R-GUS in contrast to 16R-GUS has revealed strong activation of the 20R promoter by metal ions and NaCl. Instead the 16R promoter is strongly affected by virus and salicylic acid treatments. The only factor, which strongly induced both promoters, was abscisic acid. It is thus suggested that promoter domain composition is the main factor differentiating the appearance of 14-3-3 isoforms. PMID:16081528

  20. Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis

    PubMed Central

    Margolis, Seth S.; Perry, Jennifer A.; Forester, Craig M.; Nutt, Leta K.; Guo, Yanxiang; Jardim, Melanie J.; Thomenius, Michael J.; Freel, Christopher D.; Darbandi, Rashid; Ahn, Jung-Hyuck; Arroyo, Jason D.; Wang, Xiao-Fan; Shenolikar, Shirish; Nairn, Angus C.; Dunphy, William G.; Hahn, William C.; Virshup, David M.; Kornbluth, Sally

    2009-01-01

    Summary DNA-responsive checkpoints prevent cell cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. S287 phosphorylation is a major locus of G2/M checkpoint control, though several checkpoint-independent kinases can phosphorylate this site. We reported previously that mitotic entry requires 14-3-3 removal and S287 dephosphorylation. We show here that DNA-responsive checkpoints activate PP2A/B56δ phosphatase complexes to dephosphorylate Cdc25 at a site (T138) whose phosphorylation is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. Rather, our data suggest that creation of a 14-3-3 “sink”, consisting of phosphorylated 14-3-3-binding intermediate filament proteins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56δ as a central checkpoint effector, and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis. PMID:17110335

  1. Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose.

    PubMed

    Harthill, Jean E; Meek, Sarah E M; Morrice, Nick; Peggie, Mark W; Borch, Jonas; Wong, Barry H C; Mackintosh, Carol

    2006-07-01

    Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1. PMID:16771775

  2. Protein kinase C epsilon, which sensitizes skin to sun's UV radiation-induced cutaneous damage and development of squamous cell carcinomas, associates with Stat3.

    PubMed

    Aziz, Moammir H; Manoharan, Herbert T; Verma, Ajit K

    2007-02-01

    Chronic exposure to UV radiation (UVR) is the major etiologic factor in the development of human skin cancers including squamous cell carcinoma (SCC). We have shown that protein kinase C(epsilon) (PKC(epsilon)), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is an endogenous photosensitizer. PKC(epsilon) is among the six isoforms (alpha, delta, epsilon, eta, mu, and zeta) expressed in both mouse and human skin. PKC(epsilon) transgenic mice, which overexpress PKC(epsilon) in the basal epidermal cells and cells of the hair follicle, are highly sensitive to UVR-induced cutaneous damage and development of SCC. We now present that PKC(epsilon)-overexpressing, but not PKC(delta)-overexpressing, transgenic mice, when exposed to a single (4 kJ/m(2)) or repeated (four doses, 2 kJ/m(2)/dose, thrice weekly) UVR, emitted by Kodacel-filtered FS-40 sun lamps, elicit constitutive phosphorylation of signal transducers and activators of transcription 3 (Stat3) at both Tyr705 and Ser727 residues. UVR-induced phosphorylation of Stat3 accompanied increased expression of Stat3-regulated genes (c-myc, cyclin D1, cdc25A, and COX-2). In reciprocal immunoprecipitation/blotting experiments, phosphorylated Stat3 co-immunoprecipitated with PKC(epsilon). As observed in vivo using PKC(epsilon) knockout mice and in vitro in an immunocomplex kinase assay, PKC(epsilon) phosphorylated Stat3 at Ser727 residue. These results indicate for the first time that (a) PKC(epsilon) is a Stat3Ser727 kinase; (b) PKC(epsilon)-mediated phosphorylation of StatSer727 may be essential for transcriptional activity of Stat3; and (c) UVR-induced phosphorylation of Ser727 may be a key component of the mechanism by which PKC(epsilon) imparts sensitivity to UVR-induced development of SCC. PMID:17283176

  3. Whey protein concentrate doped electrospun poly(epsilon-caprolactone) fibers for antibiotic release improvement.

    PubMed

    Ahmed, Said Mahmoud; Ahmed, Hanaa; Tian, Chang; Tu, Qin; Guo, Yadan; Wang, Jinyi

    2016-07-01

    Design and fabrication of scaffolds using appropriate biomaterials are a key step for the creation of functionally engineered tissues and their clinical applications. Poly(epsilon-caprolactone) (PCL), a biodegradable and biocompatible material with negligible cytotoxicity, is widely used to fabricate nanofiber scaffolds by electrospinning for the applications of pharmaceutical products and wound dressings. However, the use of PCL as such in tissue engineering is limited due to its poor bioregulatory activity, high hydrophobicity, lack of functional groups and neutral charge. With the attempt to found nanofiber scaffolds with antibacterial activity for skin tissue engineering, in this study, whey protein concentrate (WPC) was used to modify the PCL nanofibers by doping it in the PCL electrospun solution. By adding proteins into PCL nanofibers, the degradability of the fibers may be increased, and this further allows an antibiotic incorporated in the fibers to be efficiently released. The morphology, wettability and degradation of the as-prepared PCL/WPC nanofibers were carefully characterized. The results showed that the PCL/WPC nanofibers possessed good morphology and wettability, as well as high degradation ability to compare with the pristine PCL fibers. Afterwords, tetracycline hydrochloride as a model antibiotic drug was doped in the PCL/WPC nanofibers. In vitro drug release assays demonstrated that PCL/WPC nanofibers had higher antibiotic release capability than the PCL nanofibers. Also, antibacterial activity evaluation against various bacteria showed that the drug-doped PCL/WPC fibers possessed more efficient antibacterial activity than the PCL nanofibers. PMID:27022878

  4. Systemic administration of an Fcgamma-Fc(epsilon)-fusion protein in house dust mite sensitive nonhuman primates.

    PubMed

    Van Scott, Michael R; Mertsching, Elisabeth; Negrou, Ella; Miles, Jeremy; Stallings, Howard W; Graff, Candace; Kehry, Marilyn R

    2008-09-01

    Crosslinking Fc(epsilon)RI and FcgammaRIIB receptors inhibits mast cell and basophil activation, decreasing mediator release. In this study, a fusion protein incorporating human Fcgamma and Fc(epsilon) domains, hGE2, was shown to inhibit degranulation of human mast cells and basophils, and to exhibit efficacy in a nonhuman primate model of allergic asthma. hGE2 increased the provocative concentration of dust mite aeroallergen that induced an early phase asthmatic response. The treatment effect lasted up to 4 weeks and was associated with reduction in the number of circulating basophils and decreased expression of Fc(epsilon)RI on repopulating basophils. Repeat hGE2 dosing induced production of serum antibodies against human Fcgamma and Fc(epsilon) domains and acute anaphylaxis-like reactions. Immune serum induced histamine release from human IgE or hGE2-treated cord blood-derived mast cells and basophils in vitro. These results indicate that repeat administration with hGE2 induced an antibody response to the human molecule that resulted in activation rather than inhibition of allergic responses. PMID:18583194

  5. Dissection of Binding between a Phosphorylated Tyrosine Hydroxylase Peptide and 14-3-3ζ: A Complex Story Elucidated by NMR

    PubMed Central

    Hritz, Jozef; Byeon, In-Ja L.; Krzysiak, Troy; Martinez, Aurora; Sklenar, Vladimir; Gronenborn, Angela M.

    2014-01-01

    Human tyrosine hydroxylase activity is regulated by phosphorylation of its N-terminus and by an interaction with the modulator 14-3-3 proteins. We investigated the binding of singly or doubly phosphorylated and thiophosphorylated peptides, comprising the first 50 amino acids of human tyrosine hydroxylase, isoform 1 (hTH1), that contain the critical interaction domain, to 14-3-3ζ, by 31P NMR. Single phosphorylation at S19 generates a high affinity 14-3-3ζ binding epitope, whereas singly S40-phosphorylated peptide interacts with 14-3-3ζ one order-of-magnitude weaker than the S19-phosphorylated peptide. Analysis of the binding data revealed that the 14-3-3ζ dimer and the S19- and S40-doubly phosphorylated peptide interact in multiple ways, with three major complexes formed: 1), a single peptide bound to a 14-3-3ζ dimer via the S19 phosphate with the S40 phosphate occupying the other binding site; 2), a single peptide bound to a 14-3-3ζ dimer via the S19 phosphorous with the S40 free in solution; or 3), a 14-3-3ζ dimer with two peptides bound via the S19 phosphorous to each binding site. Our system and data provide information as to the possible mechanisms by which 14-3-3 can engage binding partners that possess two phosphorylation sites on flexible tails. Whether these will be realized in any particular interacting pair will naturally depend on the details of each system. PMID:25418103

  6. Protein Kinase C Epsilon Promotes Cerebral Ischemic Tolerance Via Modulation of Mitochondrial Sirt5

    PubMed Central

    Morris-Blanco, Kahlilia C.; Dave, Kunjan R.; Saul, Isabel; Koronowski, Kevin B.; Stradecki, Holly M.; Perez-Pinzon, Miguel A.

    2016-01-01

    Sirtuin 5 (SIRT5) is a mitochondrial-localized NAD+-dependent lysine desuccinylase and a major regulator of the mitochondrial succinylome. We wanted to determine whether SIRT5 is activated by protein kinase C epsilon (PKCε)-mediated increases in mitochondrial Nampt and whether SIRT5 regulates mitochondrial bioenergetics and neuroprotection against cerebral ischemia. In isolated mitochondria from rat cortical cultures, PKCε activation increased SIRT5 levels and desuccinylation activity in a Nampt-dependent manner. PKCε activation did not lead to significant modifications in SIRT3 activity, the major mitochondrial lysine deacetylase. Assessments of mitochondrial bioenergetics in the cortex of wild type (WT) and SIRT5−/− mice revealed that SIRT5 regulates oxygen consumption in the presence of complex I, complex II, and complex IV substrates. To explore the potential role of SIRT5 in PKCε-mediated protection, we compared WT and SIRT5−/− mice by employing both in vitro and in vivo ischemia paradigms. PKCε-mediated decreases in cell death following oxygen-glucose deprivation were abolished in cortical cultures harvested from SIRT5−/− mice. Furthermore, PKCε failed to prevent cortical degeneration following MCAO in SIRT5−/− mice. Collectively this demonstrates that SIRT5 is an important mitochondrial enzyme for protection against metabolic and ischemic stress following PKCε activation in the brain. PMID:27435822

  7. Selective chemical genetic inhibition of protein kinase C epsilon reduces ethanol consumption in mice.

    PubMed

    Maiya, Rajani; McMahon, Thomas; Wang, Dan; Kanter, Benjamin; Gandhi, Dev; Chapman, Holly L; Miller, Jacklyn; Messing, Robert O

    2016-08-01

    Reducing expression or inhibiting translocation of protein kinase C epsilon (PKCε) prolongs ethanol intoxication and decreases ethanol consumption in mice. However, we do not know if this phenotype is due to reduced PKCε kinase activity or to impairment of kinase-independent functions. In this study, we used a chemical-genetic strategy to determine whether a potent and highly selective inhibitor of PKCε catalytic activity reduces ethanol consumption. We generated ATP analog-specific PKCε (AS-PKCε) knock-in mice harboring a point mutation in the ATP binding site of PKCε that renders the mutant kinase highly sensitive to inhibition by 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine (1-NA-PP1). Systemically administered 1-NA-PP1 readily crossed the blood brain barrier and inhibited PKCε-mediated phosphorylation. 1-NA-PP1 reversibly reduced ethanol consumption by AS-PKCε mice but not by wild type mice lacking the AS-PKCε mutation. These results support the development of inhibitors of PKCε catalytic activity as a strategy to reduce ethanol consumption, and they demonstrate that the AS- PKCε mouse is a useful tool to study the role of PKCε in behavior. PMID:26947945

  8. Characterization and subcellular localization of two 14-3-3 genes and their response to abiotic stress in wheat.

    PubMed

    Meng, Xiaodan; Chen, Xin; Wang, Yaying; Xiao, Ruixia; Liu, Hailun; Wang, Xinguo; Ren, Jiangping; Li, Yongchun; Niu, Hongbin; Wang, Xiang; Yin, Jun

    2014-02-01

    In order to investigate biological functions of the 14-3-3 genes and their response to abiotic stress, two cDNAs (designated as Ta14R1 and Ta14R2) encoding putative 14-3-3 proteins were isolated from wheat by PCR and rapid amplification of cDNA end (RACE) technique. The cDNA of Ta14R1 is 999bp and encodes a protein of 262 amino acids, while the cDNA of Ta14R2 is 897bp in length and encodes a protein of 261 amino acids. Transient expression assays using Ta14R1/Ta14R2-GFP fusion constructs indicated that Ta14R1 and Ta14R2 were located in cytoplasm and cell membrane but not in chloroplasts. Real-time quantitative (RT-PCR) analysis revealed that Ta14R1 and Ta14R2 were differentially expressed in wheat tissues and significantly up-regulated in roots and shoots 1d after germination, indicating they may play a role in process of seed germination. The expression of the two genes in roots and leaves were significantly induced by plant hormone ABA, as well as heat, cold and drought treatments, suggesting that the two 14-3-3 genes in wheat may be involved in ABA dependent stress-responding pathway and response to heat, cold and drought stress. PMID:24941745

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

    SciTech Connect

    Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki

    2015-08-21

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

  10. 14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression

    PubMed Central

    Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S.; Balyan, Renu; Borde, Lalit; Kundu, Tapas K.; Dalal, Sorab N.

    2016-01-01

    More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering. PMID:27253419

  11. 14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression.

    PubMed

    Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S; Balyan, Renu; Borde, Lalit; Kundu, Tapas K; Dalal, Sorab N

    2016-01-01

    More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering. PMID:27253419

  12. The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming

    PubMed Central

    Phan, Liem; Chou, Ping-Chieh; Velazquez-Torres, Guermarie; Samudio, Ismael; Parreno, Kenneth; Huang, Yaling; Tseng, Chieh; Vu, Thuy; Gully, Chris; Su, Chun-Hui; Wang, Edward; Chen, Jian; Choi, Hyun-Ho; Fuentes-Mattei, Enrique; Shin, Ji-Hyun; Shiang, Christine; Grabiner, Brian; Blonska, Marzenna; Skerl, Stephen; Shao, Yiping; Cody, Dianna; Delacerda, Jorge; Kingsley, Charles; Webb, Douglas; Carlock, Colin; Zhou, Zhongguo; Hsieh, Yun-Chih; Lee, Jaehyuk; Elliott, Andrew; Ramirez, Marc; Bankson, Jim; Hazle, John; Wang, Yongxing; Li, Lei; Weng, Shaofan; Rizk, Nibal; Wen, Yu Ye; Lin, Xin; Wang, Hua; Wang, Huamin; Zhang, Aijun; Xia, Xuefeng; Wu, Yun; Habra, Mouhammed; Yang, Wei; Pusztai, Lajos; Yeung, Sai-Ching; Lee, Mong-Hong

    2015-01-01

    Summary Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumourigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programs by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anti-cancer metabolism therapy development in future. PMID:26179207

  13. 14-3-3γ regulates cell viability and milk fat synthesis in lipopolysaccharide-induced dairy cow mammary epithelial cells

    PubMed Central

    LIU, LIXIN; ZHANG, LI; LIN, YE; BIAN, YANJIE; GAO, XUEJUN; QU, BO; LI, QINGZHANG

    2016-01-01

    Our previous study demonstrated that 14-3-3γ overexpression was able to inhibit the production of lipopolysaccharide (LPS)-induced cytokines in dairy cow mammary epithelial cells (DCMECs) by inhibiting the activation of nuclear factor-κB (NF-κB) signaling pathways. However, the association between 14-3-3γ overexpression and milk fat synthesis in LPS-induced DCMECs remains unclear. Therefore, the present study investigated the effect of 14-3-3γ on cell viability and milk fat synthesis in LPS-induced DCMECs. The results of the MTT assay and lactate dehydrogenase activity assay demonstrated that 14-3-3γ overexpression was able to attenuate LPS-induced cytotoxicity in DCMECs, and increase the viability of the cells. In addition, the results of reverse transcription-quantitative polymerase chain reaction suggested that mRNA expression levels of genes associated with milk fat synthesis, including sterol regulatory element binding protein (SREBP1), peroxisome proliferator-activated receptor-γ (PPARG), cluster of differentiation 36, acetyl-coA carboxylase (ACC), fatty acid synthase (FAS) and fatty acid binding protein-3, were significantly upregulated in cells overexpressing the 14-3-3γ protein. In addition, as compared with the LPS-treated group, the activities of FAS and ACC were significantly increased. Furthermore, western blotting demonstrated that 14-3-3γ overexpression enhanced the protein expression levels of phosphorylated SREBP1 and PPARG. These results suggested that high levels of 14-3-3γ protein were able to attenuate LPS-induced cell damage and promote milk fat synthesis in LPS-induced DCMECs by increasing the cell viability and upregulating the expression levels of transcription factors associated with milk fat synthesis. PMID:27073437

  14. 14-3-3 eta isoform colocalizes TDP-43 on the coarse granules in the anterior horn cells of patients with sporadic amyotrophic lateral sclerosis.

    PubMed

    Umahara, Takahiko; Uchihara, Toshiki; Shibata, Noriyuki; Nakamura, Ayako; Hanyu, Haruo

    2016-09-01

    The immunolocalization of the 14-3-3 eta isoform in the anterior horn cells (AHCs) of patients with sporadic amyotrophic lateral sclerosis (ALS) and controls was examined. Compared with the immunolocalization of other 14-3-3 isoforms, the immunolocalization of the 14-3-3 eta isoform was either synaptic at the periphery of AHCs, spindle-shaped in neurites, or granular in the cytoplasm. By double labeling with phosphorylated (p-)TDP-43, the transactivation response DNA binding protein of 43kDa (TDP-43) demonstrated frequent colocalization of the 14-3-3 eta isoform in granular structures (90%) and spindle-shaped structures (85.4%), but not in p-TDP-43-positive round inclusions. It is speculated that the 14-3-3 eta isoform is associated with not only a synaptic pathology of ALS but also TDP-positive small lesions in the cytoplasm and neurites. The absence of eta-like immunoreactivity in p-TDP-43-positive large inclusions suggests the restricted relevance of the 14-3-3 eta isoform during ALS pathogenesis to some phases of the p-TDP pathology. PMID:27256400

  15. Oxidative damage of 14-3-3 zeta and gamma isoforms in Alzheimer's disease and cerebral amyloid angiopathy.

    PubMed

    Santpere, G; Puig, B; Ferrer, I

    2007-06-01

    Previous studies have shown oxidative damage resulting from amyloid Abeta exposure to cultured cells and in murine models. A target of oxidation is 14-3-3 which comprises a group of proteins involved in kinase activation and chaperone activity. The present study shows glycoxidative damage, as revealed with mono and bi-dimensional gel electrophoresis and Western blotting, followed by in-gel digestion and mass spectrometry, in the frontal cortex in Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), a neurodegenerative disease with deposition of Abeta in cerebral blood vessels and in diffuse plaques unaccompanied by intraneuronal hyper-phosphorylated tau deposition. malondialdehyde-lysine (MDA-Lys)-, but not 4-hydroxy-2-nonenal (HNE)-immunoreactive adducts, and N-carboxyethyl-lysine (CEL), but not N-carboxymethyl-lysine (CML)-products, were present in 14-3-3 involving zeta and gamma isoforms in both AD and CAA. These findings demonstrate that 14-3-3 glyco- and lipoxidation occurs in AD and CAA, probably as a direct consequence of Abeta deposition. PMID:17445990

  16. Regulation of Molecular Chaperone Gene Transcription Involves the Serine Phosphorylation, 14-3-3ɛ Binding, and Cytoplasmic Sequestration of Heat Shock Factor 1

    PubMed Central

    Wang, XiaoZhe; Grammatikakis, Nicholas; Siganou, Aliki; Calderwood, Stuart K.

    2003-01-01

    Heat shock factor 1 (HSF1) regulates the transcription of molecular chaperone hsp genes. However, the cellular control mechanisms that regulate HSF1 activity are not well understood. In this study, we have demonstrated for the first time that human HSF1 binds to the essential cell signaling protein 14-3-3ɛ. Binding of HSF1 to 14-3-3ɛ occurs in cells in which extracellular signal regulated kinase (ERK) is activated and blockade of the ERK pathway by treatment with the specific ERK pathway inhibitor PD98059 in vivo strongly suppresses the binding. We previously showed that ERK1 phosphorylates HSF1 on serine 307 and leads to secondary phosphorylation by glycogen synthase kinase 3 (GSK3) on serine 303 within the regulatory domain and that these phosphorylation events repress HSF1. We show here that HSF1 binding to 14-3-3ɛ requires HSF1 phosphorylation on serines 303 and 307. Furthermore, the serine phosphorylation-dependent binding of HSF1 to 14-3-3ɛ results in the transcriptional repression of HSF1 and its sequestration in the cytoplasm. Leptomycin B, a specific inhibitor of nuclear export receptor CRM1, was found to reverse the cytoplasmic sequestration of HSF1 mediated by 14-3-3ɛ, suggesting that CRM1/14-3-3ɛ directed nuclear export plays a major role in repression of HSF1 by the ERK/GSK3/14-3-3ɛ pathway. Our experiments indicate a novel pathway for HSF1 regulation and suggest a mechanism for suppression of its activity during cellular proliferation. PMID:12917326

  17. Identification of 14-3-3β Gene as a Novel miR-152 Target Using a Proteome-based Approach*

    PubMed Central

    Jasinski-Bergner, Simon; Stehle, Franziska; Gonschorek, Evamaria; Kalich, Jana; Schulz, Kristin; Huettelmaier, Stefan; Braun, Juliane; Seliger, Barbara

    2014-01-01

    Recent studies demonstrated that miR-152 overexpression down-regulates the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G in human tumors thereby contributing to their immune surveillance. Using two-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry, the protein expression profile of HLA-G+, miR-152low cells, and their miR-152-overexpressing (miRhigh) counterparts was compared leading to the identification of 24 differentially expressed proteins. These were categorized according to their function and localization demonstrating for most of them an important role in the initiation and progression of tumors. The novel miR-152 target 14-3-3 protein β/α/YWHAB (14-3-3β) is down-regulated upon miR-152 overexpression, although its overexpression was often found in tumors of distinct origin. The miR-152-mediated reduction of the 14-3-3β expression was accompanied by an up-regulation of BAX protein expression resulting in a pro-apoptotic phenotype. In contrast, the reconstitution of 14-3-3β expression in miR-152high cells increased the expression of the anti-apoptotic BCL2 gene, enhances the proliferative activity in the presence of the cytostatic drug paclitaxel, and causes resistance to apoptosis induced by this drug. By correlating clinical microarray data with the patients' outcome, a link between 14-3-3β and HLA-G expression was found, which could be associated with poor prognosis and overall survival of patients with tumors. Because miR-152 controls both the expression of 14-3-3β and HLA-G, it exerts a dual role in tumor cells by both altering the immunogenicity and the tumorigenicity. PMID:25228695

  18. Effects of physical exercise on the P38MAPK/REDD1/14-3-3 pathways in the myocardium of diet-induced obesity rats.

    PubMed

    Pieri, B L S; Souza, D R; Luciano, T F; Marques, S O; Pauli, J R; Silva, A S R; Ropelle, E R; Pinho, R A; Lira, F S; De Souza, C T

    2014-08-01

    Obesity is associated with myocardial insulin resistance and impairment of the mammalian target of rapamycin (mTOR) signaling pathway. The activation of the mTOR cascade by exercise has been largely shown in skeletal muscle, but insufficiently analyzed in myocardial tissue. In addition, little is known regarding the mTOR upstream molecules in the hearts of obese animals and even less about the role of exercise in this process. Thus, the present study was aimed to evaluate the effects of physical exercise on P38 Mitogen-Activated Protein Kinase (P38MAPK) phosphorylation and the REDD1 (regulated in development and DNA damage responses 1) and 14-3-3 protein levels in the myocardium of diet-induced obesity (DIO) rats. After achievement of DIO and insulin resistance, Wistar rats were divided in 2 groups: sedentary obese rats and obese rats performed treadmill running (50-min/day, 5 days per week velocity of 1.0 km/h for 2 months). Forty-eight hours after the final physical exercise, the rats were killed, and the myocardial tissue was removed for Western blot analysis. DIO increased the REDD1 protein levels and reduced the 14-3-3 protein levels and P38MAPK, mTOR, P70S6k (p70 ribosomal S6 protein kinase), and 4EBP1 (4E-binding protein-1) phosphorylation. Interestingly, physical exercise reduced the REDD1 protein levels and increased the 14-3-3 protein levels and P38MAPK, mTOR, P70S6k, and 4EBP1 phosphorylation. Moreover, exercise increased the REDD1/14-3-3 association in the heart. Our results indicate that the phospho-P38MAPK, REDD1, and 14-3-3 protein levels were reduced in the myocardium of obese rats and that physical exercise increased the protein levels of these molecules. PMID:24691733

  19. Loss of ypk1 function causes rapamycin sensitivity, inhibition of translation initiation and synthetic lethality in 14-3-3-deficient yeast.

    PubMed Central

    Gelperin, Daniel; Horton, Lynn; DeChant, Anne; Hensold, Jack; Lemmon, Sandra K

    2002-01-01

    14-3-3 proteins bind to phosphorylated proteins and regulate a variety of cellular activities as effectors of serine/threonine phosphorylation. To define processes requiring 14-3-3 function in yeast, mutants with increased sensitivity to reduced 14-3-3 protein levels were identified by synthetic lethal screening. One mutation was found to be allelic to YPK1, which encodes a Ser/Thr protein kinase. Loss of Ypk function causes hypersensitivity to rapamycin, similar to 14-3-3 mutations and other mutations affecting the TOR signaling pathway in yeast. Similar to treatment with rapamycin, loss of Ypk function disrupted translation, at least in part by causing depletion of eIF4G, a central adaptor protein required for cap-dependent mRNA translation initiation. In addition, Ypk1 as well as eIF4G protein levels were rapidly depleted upon nitrogen starvation, but not during glucose starvation, even though both conditions inhibit translation initiation. These results suggest that Ypk regulates translation initiation in response to nutrient signals, either through the TOR pathway or in a functionally related pathway parallel to TOR. PMID:12196392

  20. N epsilon,N epsilon-dimethyl-lysine cytochrome c as an NMR probe for lysine involvement in protein-protein complex formation.

    PubMed Central

    Moore, G R; Cox, M C; Crowe, D; Osborne, M J; Rosell, F I; Bujons, J; Barker, P D; Mauk, M R; Mauk, A G

    1998-01-01

    The reductively dimethylated derivatives of horse and yeast iso-1-ferricytochromes c have been prepared and characterized for use as NMR probes of the complexes formed by cytochrome c with bovine liver cytochrome b5 and yeast cytochrome c peroxidase. The electrostatic properties and structures of the derivatized cytochromes are not significantly perturbed by the modifications; neither are the electrostatics of protein-protein complex formation or rates of interprotein electron transfer. Two-dimensional 1H-13C NMR spectroscopy of the complexes formed by the derivatized cytochromes with cytochrome b5 and cytochrome c peroxidase has been used to investigate the number and identity of lysine residues of cytochrome c that are involved in interprotein interactions of cytochrome c. The NMR data are incompatible with simple static models proposed previously for the complexes formed by these proteins, but are consistent with the presence of multiple, interconverting complexes of comparable stability, consistent with studies employing Brownian dynamics to model the complexes. The NMR characteristics of the Nepsilon,Nepsilon-dimethyl-lysine groups, their chemical shift dispersion, oxidation state and temperature dependences and the possibility of chemical exchange phenomena are discussed with relevance to the utility of Nepsilon, Nepsilon-dimethyl-lysine's being a generally useful derivative for characterizing protein-protein complexes. PMID:9601073

  1. Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein

    PubMed Central

    Kaushik, Himani; Deshmukh, Sachin; Mathur, Deepika Dayal; Tiwari, Archana; Garg, Lalit C

    2013-01-01

    Epsilon toxin secreted by Clostridium perfringens types B and D has been directly implicated as the causative agent of fatal enterotoxemia in domestic animals. The aim of the present study is to use in silico approach for identification of B-cell epitope(s) of epsilon toxin, and its expression in fusion with a carrier protein to analyze its potential as vaccine candidate(s). Using different computational analyses and bioinformatics tools, a number of antigenic determinant regions of epsilon toxin were identified. One of the B cell epitopes of epsilon toxin comprising the region (amino acids 40-62) was identified as a promising antigenic determinant. This Etx epitope (Etx40-62) was cloned and expressed as a translational fusion with B-subunit of heat labile enterotoxin (LTB) of E. coli in a secretory expression system. Similar to the native LTB, the recombinant fusion protein retained the ability to pentamerize and bind to GM1 ganglioside receptor of LTB. The rLTB.Etx40-62 could be detected both with anti-Etx and anti-LTB antisera. The rLTB.Etx40-62 fusion protein thus can be evaluated as a potential vaccine candidate against C. perfringens. Abbreviations aa - amino acid(s), Etx - epsilon toxin of Clostridium perfringens, LTB - B-subunit of heat labile enterotoxin of E. coli. PMID:23904738

  2. Klotho Regulates 14-3-3ζ Monomerization and Binding to the ASK1 Signaling Complex in Response to Oxidative Stress

    PubMed Central

    Brobey, Reynolds K.; Dheghani, Mehdi; Foster, Philip P.; Kuro-o, Makoto; Rosenblatt, Kevin P

    2015-01-01

    The reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1) signaling complex is a key regulator of p38 MAPK activity, a major modulator of stress-associated with aging disorders. We recently reported that the ratio of free ASK1 to the complex-bound ASK1 is significantly decreased in Klotho-responsive manner and that Klotho-deficient tissues have elevated levels of free ASK1 which coincides with increased oxidative stress. Here, we tested the hypothesis that: 1) covalent interactions exist among three identified proteins constituting the ASK1 signaling complex; 2) in normal unstressed cells the ASK1, 14-3-3ζ and thioredoxin (Trx) proteins simultaneously engage in a tripartite complex formation; 3) Klotho’s stabilizing effect on the complex relied solely on 14-3-3ζ expression and its apparent phosphorylation and dimerization changes. To verify the hypothesis, we performed 14-3-3ζ siRNA knock-down experiments in conjunction with cell-based assays to measure ASK1-client protein interactions in the presence and absence of Klotho, and with or without an oxidant such as rotenone. Our results show that Klotho activity induces posttranslational modifications in the complex targeting 14-3-3ζ monomer/dimer changes to effectively protect against ASK1 oxidation and dissociation. This is the first observation implicating all three proteins constituting the ASK1 signaling complex in close proximity. PMID:26517365

  3. 14-3-3-zeta participates in TLR3-mediated TICAM-1 signal-platform formation.

    PubMed

    Funami, Kenji; Matsumoto, Misako; Obuse, Chikashi; Seya, Tsukasa

    2016-05-01

    Recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) is important in innate immune signaling. Toll-like receptors (TLRs) are well-characterized PRRs and are pivotal in antiviral and antitumor host defense. TIR domain-containing adaptor molecule 1 (TICAM-1, also called TRIF) is an adapter molecule in TLR3- and TLR4-mediated IRF3 activation, late-phase NF-κB activation and MAPK-mediated AP-1 activation. When a TLR3 ligand is added to TLR3-positive cells, TICAM-1 transiently interacts with TLR3 and forms multimers in the cytosol. However, the precise mechanism of TICAM-1 multimer formation remains unknown. In this study, we identified 14-3-3-zeta as a molecule that functions in TLR3-mediated signaling. Knockdown of 14-3-3-zeta reduced production of type I interferon and inflammatory cytokines, nuclear translocation of IRF3 and phosphorylation of IκB via the TLR3-TICAM-1 pathway. Furthermore, TICAM-1 multimerization by ligand stimulation was prohibited by 14-3-3-zeta knockdown. These results suggest that 14-3-3-zeta is involved in the TLR3-TICAM-1 pathway in promoting multimerization of TICAM-1 for the formation of a TICAM-1 signalosome. PMID:27058640

  4. Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16

    PubMed Central

    Shehata, Saifeldin N.; Deak, Maria; Morrice, Nicholas A.; Ohta, Eriko; Hunter, Roger W.; Kalscheuer, Vera M.; Sakamoto, Kei

    2015-01-01

    PCTAIRE-1 [also known as cyclin-dependent kinase 16 (CDK16)] is implicated in various physiological processes such as neurite outgrowth and vesicle trafficking; however, its molecular regulation and downstream targets are largely unknown. Cyclin Y has recently been identified as a key interacting/activating cyclin for PCTAIRE-1; however, the molecular mechanism by which it activates PCTAIRE-1 is undefined. In the present study, we initially performed protein sequence analysis and identified two candidate phosphorylation sites (Ser12 and Ser336) on cyclin Y that might be catalysed by PCTAIRE-1. Although in vitro peptide analysis favoured Ser12 as the candidate phosphorylation site, immunoblot analysis of cell lysates that had been transfected with wild-type (WT) or kinase-inactive (KI) PCTAIRE-1 together with WT or phospho-deficient mutants of cyclin Y suggested Ser336, but not Ser12, as a PCTAIRE-1-dependent phosphorylation site. Monitoring phosphorylation of Ser336 may provide a useful read-out to assess cellular activity of PCTAIRE-1 in vivo; however, a phospho-deficient S336A mutant displayed normal interaction with PCTAIRE-1. Unbiased mass spectrometry and targeted mutagenesis analysis of cyclin Y identified key phosphorylation sites (Ser100 and Ser326) required for 14-3-3 binding. Recombinant WT cyclin Y, but not a S100A/S326A mutant, prepared in COS-1 cells co-purified with 14-3-3 and was able to activate bacterially expressed recombinant PCTAIRE-1 in cell-free assays. Finally, we observed that recently identified PCTAIRE-1 variants found in patients with intellectual disability were unable to interact with cyclin Y, and were inactive enzymes. Collectively, the present work has revealed a new mechanistic insight into activation of PCTAIRE-1, which is mediated through interaction with the phosphorylated form of cyclin Y in complex with 14-3-3. PMID:26205494

  5. Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16.

    PubMed

    Shehata, Saifeldin N; Deak, Maria; Morrice, Nicholas A; Ohta, Eriko; Hunter, Roger W; Kalscheuer, Vera M; Sakamoto, Kei

    2015-08-01

    PCTAIRE-1 [also known as cyclin-dependent kinase 16 (CDK16)] is implicated in various physiological processes such as neurite outgrowth and vesicle trafficking; however, its molecular regulation and downstream targets are largely unknown. Cyclin Y has recently been identified as a key interacting/activating cyclin for PCTAIRE-1; however, the molecular mechanism by which it activates PCTAIRE-1 is undefined. In the present study, we initially performed protein sequence analysis and identified two candidate phosphorylation sites (Ser(12) and Ser(336)) on cyclin Y that might be catalysed by PCTAIRE-1. Although in vitro peptide analysis favoured Ser(12) as the candidate phosphorylation site, immunoblot analysis of cell lysates that had been transfected with wild-type (WT) or kinase-inactive (KI) PCTAIRE-1 together with WT or phospho-deficient mutants of cyclin Y suggested Ser(336), but not Ser(12), as a PCTAIRE-1-dependent phosphorylation site. Monitoring phosphorylation of Ser(336) may provide a useful read-out to assess cellular activity of PCTAIRE-1 in vivo; however, a phospho-deficient S336A mutant displayed normal interaction with PCTAIRE-1. Unbiased mass spectrometry and targeted mutagenesis analysis of cyclin Y identified key phosphorylation sites (Ser(100) and Ser(326)) required for 14-3-3 binding. Recombinant WT cyclin Y, but not a S100A/S326A mutant, prepared in COS-1 cells co-purified with 14-3-3 and was able to activate bacterially expressed recombinant PCTAIRE-1 in cell-free assays. Finally, we observed that recently identified PCTAIRE-1 variants found in patients with intellectual disability were unable to interact with cyclin Y, and were inactive enzymes. Collectively, the present work has revealed a new mechanistic insight into activation of PCTAIRE-1, which is mediated through interaction with the phosphorylated form of cyclin Y in complex with 14-3-3. PMID:26205494

  6. Verteporfin inhibits YAP function through up-regulating 14-3-3σ sequestering YAP in the cytoplasm

    PubMed Central

    Wang, Chao; Zhu, Xiaoyong; Feng, Weiwei; Yu, Yinhua; Jeong, Kangjin; Guo, Wei; Lu, Yiling; Mills, Gordon B

    2016-01-01

    Yes-associated protein (YAP), the central mediator of Hippo pathway, not only regulates a diversity of cellular processes during development but also plays a pivotal role in tumorigenesis. YAP is overexpressed in many types of human cancers with its expression level being associated with patient outcomes. Thus, inhibiting YAP function could provide a novel therapeutic approach. Verteporfin, a photosensitizer, which has been used in photodynamic therapy (PDT), was recently identified as an inhibitor of the interaction of YAP with TEAD, which, in turn, blocks transcriptional activation of targets downstream of YAP. However, the mechanism by which Verteporfin inhibits YAP activity remains to be elucidated. We demonstrate that overexpression of YAP stimulates cell proliferation whereas knocking down YAP or treating cells with Verteporfin inhibited cell proliferation, even in the presence of growth factors. Protoporphyrin IX, another photosensitizer, did not have similar activity demonstrating specificity to Verteporfin. Verteporfin induced sequestration of YAP in cytoplasm through increasing levels of 14-3-3σ, a YAP chaperon protein that retains YAP in cytoplasm and targets it for degradation in the proteosome. Interestingly, while knockdown of YAP had no effect on the ability of Verteporfin to induce 14-3-3σ, p53 is required for this effect of Verteporfin. This provides potential approaches to select patients likely to benefit from Verteporfin. PMID:27073720

  7. Down-regulation of 14-3-3β exerts anti-cancer effects through inducing ER stress in human glioma U87 cells: Involvement of CHOP–Wnt pathway

    SciTech Connect

    Cao, Lei; Lei, Hui; Chang, Ming-Ze; Liu, Zhi-Qin; Bie, Xiao-Hua

    2015-07-10

    We previously identified 14-3-3β as a tumor-specific isoform of 14-3-3 protein in astrocytoma, but its functional role in glioma cells and underlying mechanisms are poorly understood. In the present study, we investigated the effects of 14-3-3β inhibition in human glioma U87 cells using specific targeted small interfering RNA (siRNA). The results showed that 14-3-3β is highly expressed in U87 cells but not in normal astrocyte SVGp12 cells. Knockdown of 14-3-3β by Si-14-3-3β transfection significantly decreased the cell viability but increased the LDH release in a time-dependent fashion in U87 cells, and these effects were accompanied with G0/G1 cell cycle arrest and apoptosis. In addition, 14-3-3β knockdown induced ER stress in U87 cells, as evidenced by ER calcium release, increased expression of XBP1S mRNA and induction of ER related pro-apoptotic factors. Down-regulation of 14-3-3β significantly decreased the nuclear localization of β-catenin and inhibited Topflash activity, which was shown to be reversely correlated with CHOP. Furthermore, Si-CHOP and sFRP were used to inhibit CHOP and Wnt, respectively. The results showed that the anti-cancer effects of 14-3-3β knockdown in U87 cells were mediated by increased expression of CHOP and followed inhibition of Wnt/β-catenin pathway. In summary, the remarkable efficiency of 14-3-3β knockdown to induce apoptotic cell death in U87 cells may find therapeutic application for the treatment of glioma patients. - Highlights: • Knockdown of 14-3-3β leads to cytotoxicity in human glioma U87 cells. • Knockdown of 14-3-3β induces cell cycle arrest and apoptosis in U87 cells. • Knockdown of 14-3-3β results in ER stress in U87 cells. • Knockdown of 14-3-3β inhibits Wnt/β-catenin pathway via CHOP activation.

  8. Upstream signaling of protein kinase C-epsilon in xenon-induced pharmacological preconditioning. Implication of mitochondrial adenosine triphosphate dependent potassium channels and phosphatidylinositol-dependent kinase-1.

    PubMed

    Weber, Nina C; Toma, Octavian; Damla, Halil; Wolter, Jessica I; Schlack, Wolfgang; Preckel, Benedikt

    2006-06-01

    Xenon elicits preconditioning of the myocardium via protein kinase C-epsilon. We determined the implication of (1) the mitochondrial adenosinetriphosphate dependent potassium (K(ATP)) channels and (2) the 3'phosphatidylinositol-dependent kinase-1 (PDK-1) in activating protein kinase C-epsilon. For infarct size measurements, anaesthetized rats were subjected to 25 min of coronary artery occlusion followed by 120 min of reperfusion. Rats received xenon 70% during three 5-min periods before ischaemia with or without the K(ATP) channel blocker 5-hydroxydecanoate or Wortmannin as PI3K/PDK-1 inhibitor. For Western blot, hearts were excised at five time points after xenon preconditioning (Control, 15, 25, 35, 45 min). Infarct size was reduced from 42+/-6% (mean+/-S.D.) to 27+/-8% after xenon preconditioning (P<0.05). Western blot revealed an increased activation of PKC-epsilon after 45 min and of PDK-1 after 25 min during xenon preconditioning. 5-hydroxydecanoate and Wortmannin blocked both effects. PKC-epsilon is activated downstream of mitochondrial K(ATP) channels and PDK-1. Both pathways are functionally involved in xenon preconditioning. PMID:16716295

  9. Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells.

    PubMed

    Manni, Marco M; Sot, Jesús; Goñi, Félix M

    2015-03-01

    Epsilon-toxin (ETX) is a powerful toxin produced by some strains of Clostridium perfringens (classified as types B and D) that is responsible for enterotoxemia in animals. ETX forms pores through the plasma membrane of eukaryotic cells, consisting of a β-barrel of 14 amphipathic β-strands. ETX shows a high specificity for certain cell lines, of which Madin-Darby canine kidney (MDCK) is the first sensitive cell line identified and the most studied one. The aim of this study was to establish the role of lipids in the toxicity caused by ETX and the correlation of its activity in model and biological membranes. In MDCK cells, using cell counting and confocal microscopy, we have observed that the toxin causes cell death mediated by toxin binding to plasma membrane. Moreover, ETX binds and permeabilizes the membranes of giant plasma membrane vesicles (GPMV). However, little effect is observed on protein-free vesicles. The data suggest the essential role of a protein receptor for the toxin in cell membranes. PMID:25485476

  10. Increased BDNF protein expression after ischemic or PKC epsilon preconditioning promotes electrophysiologic changes that lead to neuroprotection

    PubMed Central

    Neumann, Jake T; Thompson, John W; Raval, Ami P; Cohan, Charles H; Koronowski, Kevin B; Perez-Pinzon, Miguel A

    2015-01-01

    Ischemic preconditioning (IPC) via protein kinase C epsilon (PKCɛ) activation induces neuroprotection against lethal ischemia. Brain-derived neurotrophic factor (BDNF) is a pro-survival signaling molecule that modulates synaptic plasticity and neurogenesis. Interestingly, BDNF mRNA expression increases after IPC. In this study, we investigated whether IPC or pharmacological preconditioning (PKCɛ activation) promoted BDNF-induced neuroprotection, if neuroprotection by IPC or PKCɛ activation altered neuronal excitability, and whether these changes were BDNF-mediated. We used both in vitro (hippocampal organotypic cultures and cortical neuronal-glial cocultures) and in vivo (acute hippocampal slices 48 hours after preconditioning) models of IPC or PKCɛ activation. BDNF protein expression increased 24 to 48 hours after preconditioning, where inhibition of the BDNF Trk receptors abolished neuroprotection against oxygen and glucose deprivation (OGD) in vitro. In addition, there was a significant decrease in neuronal firing frequency and increase in threshold potential 48 hours after preconditioning in vivo, where this threshold modulation was dependent on BDNF activation of Trk receptors in excitatory cortical neurons. In addition, 48 hours after PKCɛ activation in vivo, the onset of anoxic depolarization during OGD was significantly delayed in hippocampal slices. Overall, these results suggest that after IPC or PKCɛ activation, there are BDNF-dependent electrophysiologic modifications that lead to neuroprotection. PMID:25370861

  11. 14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

    PubMed Central

    Liu, Lixin; Lin, Ye; Liu, Lili; Bian, Yanjie; Zhang, Li; Gao, Xuejun; Li, Qingzhang

    2015-01-01

    As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPAR

  12. Epsilon Aurigae

    NASA Astrophysics Data System (ADS)

    Turner, Rebecca; Price, A.; Henden, A.

    2009-05-01

    The IYA 2009 working group on Research Experiences for Students, Teachers, and Citizen-Scientists is planning a multi-year project involving the bright star Eps Aur. The project will go beyond simple observing and also include a major data analysis component. The goal is to introduce the participant to the full scientific process from background research to paper writing for a peer-reviewed journal. It begins with a 10 Star Training Program of several types of binary and transient variable stars that are easy to observe from suburban locations with the naked eye. Participants will be trained both in observing and also in basic data analysis of photometric datasets (light curve and period analysis). Eventually it will lead to a capstone project: monitoring the rare and mysterious 2009-2011 eclipse of Epsilon Aurigae. In the summer of IYA 2009, third-magnitude Eps Aur will experience its next eclipse, which occurs every 27.1 years and lasts 714 days, nearly two years. The star is bright enough to be seen with the naked eye from most urban areas. If fully funded, the project will also involve two public workshops on observing and data analysis in the summers of 2009 and 2010, respectively.

  13. Fatty acid represses insulin receptor gene expression by impairing HMGA1 through protein kinase C{epsilon}

    SciTech Connect

    Dey, Debleena; Bhattacharya, Anirban; Roy, SibSankar; Bhattacharya, Samir . E-mail: smrbhattacharya@gmail.com

    2007-06-01

    It is known that free fatty acid (FFA) contributes to the development of insulin resistance and type2 diabetes. However, the underlying mechanism in FFA-induced insulin resistance is still unclear. In the present investigation we have demonstrated that palmitate significantly (p < 0.001) inhibited insulin-stimulated phosphorylation of PDK1, the key insulin signaling molecule. Consequently, PDK1 phosphorylation of plasma membrane bound PKC{epsilon} was also inhibited. Surprisingly, phosphorylation of cytosolic PKC{epsilon} was greatly stimulated by palmitate; this was then translocated to the nuclear region and associated with the inhibition of insulin receptor (IR) gene transcription. A PKC{epsilon} translocation inhibitor peptide, {epsilon}V1, suppressed this inhibitory effect of palmitate, suggesting requirement of phospho-PKC{epsilon} migration to implement palmitate effect. Experimental evidences indicate that phospho-PKC{epsilon} adversely affected HMGA1. Since HMGA1 regulates IR promoter activity, expression of IR gene was impaired causing reduction of IR on cell surface and that compromises with insulin sensitivity.

  14. Fabrication of porous poly(epsilon-caprolactone) microparticles for protein release.

    PubMed

    Lin, W J; Huang, L I

    2001-01-01

    The particle morphology and in vitro release of protein from porous and non-porous PCL-F127 blended microparticles were evaluated. The BSA loaded PCL microparticles were prepared by the w/o/o/o emulsion-solvent evaporation method. Two types of homogenizer, a Polytron homogenizer and a probe ultrasonicator, were used to prepare the emulsion systems. The effects of solvent evaporation rate on the crystallinity and the performance of the microparticles were investigated. Both microparticles showed quite different shapes as well as surface morphology and release characteristics. The microparticles prepared with a Polytron homogenizer were quite porous in structure, which created channels for protein to continuously diffuse out, and resulted in sustained- and controlled-release characteristics. In addition, the initial burst release of protein from the microparticles was also reduced. Alteration of the evaporation rate of solvent did not change the crystallinity of the final microparticles. An influence of evaporation rate on the size of resulting microparticles was observed. The porous PCL microparticles were developed by choosing a proper homogenizer and fabrication conditions. Carefully controlling these variables resulted in microparticles with desirable release performance. PMID:11508763

  15. Tipping the balance between good and evil: aberrant 14-3-3ζ expression drives oncogenic TGF-β signaling in metastatic breast cancers.

    PubMed

    Morrison, Chevaun D; Schiemann, William P

    2015-01-01

    Transforming growth factor beta (TGF-β) readily suppresses the development of early-stage breast cancers, an activity that gives way to tumor promotion in their late-stage counterparts. The molecular mechanisms underlying this mysterious switch in TGF-β function remain murky. In addressing this conundrum, Xu et al. observed aberrant 14-3-3ζ expression to prevent the formation of tumor-suppressive Smad2/3:p53 complexes, while simultaneously driving the generation of oncogenic Smad2/3:Gli2 complexes. Once formed, Smad2/3:Gli2 complexes stimulate the expression of parathyroid hormone-related protein necessary for breast cancer metastasis to bone. This viewpoint highlights 14-3-3ζ as an essential driver of oncogenic signaling by Smad2/3 and TGF-β in metastatic breast cancers. PMID:26160166

  16. 14-3-3ζ up-regulates hypoxia-inducible factor-1α in hepatocellular carcinoma via activation of PI3K/Akt/NF-кB signal transduction pathway

    PubMed Central

    Tang, Yufu; Lv, Pengfei; Sun, Zhongyi; Han, Lei; Luo, Bichao; Zhou, Wenping

    2015-01-01

    14-3-3ζ protein, a member of 14-3-3 family, plays important roles in multiple cellular processes. Our previous study showed that 14-3-3ζ could bind to regulate the expression of hypoxia-inducible factor-1α (HIF-1α), which is induced by hypoxia and a crucial factor for induction of tumor metastasis. Moreover, we also have confirmed the response of 14-3-3ζ to hypoxia in our unpublished data as well. Thus, in the present study, we attempted to reveal that whether the regulation effect of 14-3-3ζ on HIF-1α functioned in a similar pattern as hypoxia. Stable regulation of 14-3-3ζ in human HCC cell line SMMC-772 and HCC-LM3 was achieved. The regulation of 14-3-3ζ on HIF-1α mRNA transcription was evaluated by luciferase activity assay and quantitative real-time PCR (qPCR). The effect of 14-3-3ζ on the production of HIF-1α and pathways determining HIF-1α’s response to hypoxia was assessed using western blotting assay. Our results showed that regulation of 14-3-3ζ expression influenced the activity of HIF-1α, phosphatidyl inositol 3-kinase (PI3K), Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), and nuclear factor kappa B (NF-кB). Blocking of these pathways using indicated inhibitors revealed that 14-3-3ζ enhanced the production of HIF-1α via the activation of PI3K/Akt/NF-кB pathway, which was identical to hypoxia induced HIF-1α expression. For the first time, our study described the key role of 14-3-3ζ in the HIF-1α production in HCC cells. And the molecule exerted its function on HIF-1α both by directly binding to it and via PI3K/Akt/NF-кB signal transduction pathway. PMID:26884855

  17. Inhibition of 5-lipoxygenase triggers apoptosis in prostate cancer cells via down-regulation of protein kinase C-epsilon

    PubMed Central

    Sarveswaran, Sivalokanathan; Thamilselvan, Vijayalakshmi; Brodie, Chaya; Ghosh, Jagadananda

    2012-01-01

    Previous studies have shown that human prostate cancer cells constitutively generate 5-lipoxygenase (5-LOX) metabolites from arachidonic acid, and inhibition of 5-LOX blocks production of 5-LOX metabolites and triggers apoptosis in prostate cancer cells. This apoptosis is prevented by exogenous metabolites of 5-LOX, suggesting an essential role of 5-LOX metabolites in the survival of prostate cancer cells. However, downstream signaling mechanisms which mediate the survival-promoting effects of 5-LOX metabolites in prostate cancer cells are still unknown. Recently, we reported that MK591, a specific inhibitor of 5-LOX activity, induces apoptosis in prostate cancer cells without inhibition of Akt, or ERK, two well-characterized regulators of pro-survival mechanisms, suggesting the existence of an Akt and ERK-independent survival mechanism in prostate cancer cells regulated by 5-LOX. Here, we report that 5-LOX inhibition-induced apoptosis in prostate cancer cells occurs via rapid inactivation of protein kinase C-epsilon (PKCε), and that exogenous 5-LOX metabolites prevent both 5-LOX inhibition-induced down-regulation of PKCε and induction of apoptosis. Interestingly, pre-treatment of prostate cancer cells with diazoxide (a chemical activator of PKCε), or KAE1-1 (a cell-permeable, octa-peptide specific activator of PKCε) prevents 5-LOX inhibition-induced apoptosis, which indicates that inhibition of 5-LOX triggers apoptosis in prostate cancer cells via down-regulation of PKCε. Altogether, these findings suggest that metabolism of arachidonic acid by 5-LOX activity promotes survival of prostate cancer cells via signaling through PKCε, a pro-survival serine/threonine kinase. PMID:21824498

  18. Arsenite Stress Down-regulates Phosphorylation and 14-3-3 Binding of Leucine-rich Repeat Kinase 2 (LRRK2), Promoting Self-association and Cellular Redistribution*

    PubMed Central

    Mamais, Adamantios; Chia, Ruth; Beilina, Alexandra; Hauser, David N.; Hall, Christine; Lewis, Patrick A.; Cookson, Mark R.; Bandopadhyay, Rina

    2014-01-01

    Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser910/Ser935 mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser910/Ser935 phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H2O2-induced stresses promote loss of Ser910/Ser935 phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function. PMID:24942733

  19. p38- and MK2-dependent signalling promotes stress-induced centriolar satellite remodelling via 14-3-3-dependent sequestration of CEP131/AZI1

    PubMed Central

    Tollenaere, Maxim A. X.; Villumsen, Bine H.; Blasius, Melanie; Nielsen, Julie C.; Wagner, Sebastian A.; Bartek, Jiri; Beli, Petra; Mailand, Niels; Bekker-Jensen, Simon

    2015-01-01

    Centriolar satellites (CS) are small granular structures that cluster in the vicinity of centrosomes. CS are highly susceptible to stress stimuli, triggering abrupt displacement of key CS factors. Here we discover a linear p38-MK2-14-3-3 signalling pathway that specifically targets CEP131 to trigger CS remodelling after cell stress. We identify CEP131 as a substrate of the p38 effector kinase MK2 and pinpoint S47 and S78 as critical MK2 phosphorylation sites in CEP131. Ultraviolet-induced phosphorylation of these residues generates direct binding sites for 14-3-3 proteins, which sequester CEP131 in the cytoplasm to block formation of new CS, thereby leading to rapid depletion of these structures. Mutating S47 and S78 in CEP131 is sufficient to abolish stress-induced CS reorganization, demonstrating that CEP131 is the key regulatory target of MK2 and 14-3-3 in these structures. Our findings reveal the molecular mechanism underlying dynamic CS remodelling to modulate centrosome functions on cell stress. PMID:26616734

  20. epsilon-Hexachlorocyclohexane (epsilon-HC)

    Integrated Risk Information System (IRIS)

    epsilon - Hexachlorocyclohexane ( epsilon - HC ) ; CASRN 6108 - 10 - 7 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard

  1. Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms.

    PubMed Central

    Zhao, Xiangshan; Gan, Lixia; Pan, Haiyun; Kan, Donghui; Majeski, Michael; Adam, Stephen A; Unterman, Terry G

    2004-01-01

    FOXO1, a Forkhead transcription factor, is an important target of insulin and growth factor action. Phosphorylation of Thr-24, Ser-256 and Ser-319 promotes nuclear exclusion of FOXO1, yet the mechanisms regulating nuclear/cytoplasmic shuttling of FOXO1 are poorly understood. Previous studies have identified an NLS (nuclear localization signal) in the C-terminal basic region of the DBD (DNA-binding domain), and a leucine-rich, leptomycin-B sensitive NES (nuclear export signal) located further downstream. Here, we find that other elements in the DBD also contribute to nuclear localization, and that multiple mechanisms contribute to nuclear exclusion of FOXO1. Phosphorylation of Ser-319 and a cluster of nearby residues (Ser-322, Ser-325 and Ser-329) functions co-operatively with the nearby NES to promote nuclear exclusion. The N-terminal region of FOXO1 (amino acids 1-149) also is sufficient to promote nuclear exclusion, and does so through multiple mechanisms. Amino acids 1-50 are sufficient to promote nuclear exclusion of green fluorescent protein fusion proteins, and the phosphorylation of Thr-24 is required for this effect. A leucine-rich, leptomycin B-sensitive export signal is also present nearby. Phosphorylated FOXO1 binds 14-3-3 proteins, and co-precipitation studies with tagged proteins indicate that 14-3-3 binding involves co-operative interactions with both Thr-24 and Ser-256. Ser-256 is located in the C-terminal region of the DBD, where 14-3-3 proteins may interfere both with DNA-binding and with nuclear-localization functions. Together, these studies demonstrate that multiple elements contribute to nuclear/cytoplasmic shuttling of FOXO1, and that phosphorylation and 14-3-3 binding regulate the cellular distribution and function of FOXO1 through multiple mechanisms. The presence of these redundant mechanisms supports the concept that the regulation of FOXO1 function plays a critical role in insulin and growth factor action. PMID:14664696

  2. 14-3-3σ confers cisplatin resistance in esophageal squamous cell carcinoma cells via regulating DNA repair molecules.

    PubMed

    Lai, Kenneth K Y; Chan, Kin Tak; Choi, Mei Yuk; Wang, Hector K; Fung, Eva Y M; Lam, Ho Yu; Tan, Winnie; Tung, Lai Nar; Tong, Daniel K H; Sun, Raymond W Y; Lee, Nikki P; Law, Simon

    2016-02-01

    Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer in Asia. Cisplatin is commonly used in chemoradiation for unresectable ESCC patients. However, the treatment efficacy is diminished in patients with established cisplatin resistance. To understand the mechanism leading to the development of cisplatin resistance in ESCC, we compared the proteomes from a cisplatin-resistant HKESC-2R cell line with its parental-sensitive counterpart HKESC-2 to identify key molecule involved in this process. Mass spectrometry analysis detected 14-3-3σ as the most abundant molecule expressed exclusively in HKESC-2R cells, while western blot result further validated it to be highly expressed in HKESC-2R cells when compared to HKESC-2 cells. Ectopic expression of 14-3-3σ increased cisplatin resistance in HKESC-2 cells, while its suppression sensitized SLMT-1 cells to cisplatin. Among the molecules involved in drug detoxification, drug transportation, and DNA repair, the examined DNA repair molecules HMGB1 and XPA were found to be highly expressed in HKESC-2R cells with high 14-3-3σ expression. Subsequent manipulation of 14-3-3σ by both overexpression and knockdown approaches concurrently altered the expression of HMGB1 and XPA. 14-3-3σ, HMGB1, and XPA were preferentially expressed in cisplatin-resistant SLMT-1 cells when compared to those more sensitive to cisplatin. In ESCC patients with poor response to cisplatin-based chemoradiation, their pre-treatment tumors expressed higher expression of HMGB1 than those with response to such treatment. In summary, our results demonstrate that 14-3-3σ induces cisplatin resistance in ESCC cells and that 14-3-3σ-mediated cisplatin resistance involves DNA repair molecules HMGB1 and XPA. Results from this study provide evidences for further work in researching the potential use of 14-3-3σ and DNA repair molecules HMGB1 and XPA as biomarkers and therapeutic targets for ESCC. PMID:26346170

  3. The nucleotide sequence of the mouse immunoglobulin epsilon gene: comparison with the human epsilon gene sequence.

    PubMed Central

    Ishida, N; Ueda, S; Hayashida, H; Miyata, T; Honjo, T

    1982-01-01

    We have determined the nucleotide sequence of the immunoglobulin epsilon gene cloned from newborn mouse DNA. The epsilon gene sequence allows prediction of the amino acid sequence of the constant region of the epsilon chain and comparison of it with sequences of the human epsilon and other mouse immunoglobulin genes. The epsilon gene was shown to be under the weakest selection pressure at the protein level among the immunoglobulin genes although the divergence at the synonymous position is similar. Our results suggest that the epsilon gene may be dispensable, which is in accord with the fact that IgE has only obscure roles in the immune defense system but has an undesirable role as a mediator of hypersensitivity. The sequence data suggest that the human and murine epsilon genes were derived from different ancestors duplicated a long time ago. The amino acid sequence of the epsilon chain is more homologous to those of the gamma chains than the other mouse heavy chains. Two membrane exons, separated by an 80-base intron, were identified 1.7 kb 3' to the CH4 domain of the epsilon gene and shown to conserve a hydrophobic portion similar to those of other heavy chain genes. RNA blot hybridization showed that the epsilon membrane exons are transcribed into two species of mRNA in an IgE hybridoma. Images Fig. 4. PMID:6329728

  4. Development of a polymerase chain reaction assay for the diagnosis of neosporosis using the Neospora caninum 14-3-3 gene.

    PubMed

    Lally, N C; Jenkins, M C; Dubey, J P

    1996-01-01

    Neospora caninum is a recently described apicomplexan parasite which causes neuromuscular disease in dogs, and abortion and neonatal morbidity in cattle, sheep and horses. Morphological similarities and serological cross-reactivity between N. caninum and the closely related parasite Toxoplasma gondii, have resulted in the frequent misdiagnosis of neosporosis as toxoplasmosis. This report describes the isolation and characterization of an N. caninum cDNA clone encoding a 14-3-3 protein homologue. The 14-3-3 proteins are a class of proteins which show a high degree of amino acid sequence conservation across several eukaryotic taxa. Using less conserved regions of the N. caninum cDNA clone, nested primers were designed for the amplification of a 614-bp N. caninum DNA fragment by the polymerase chain reaction (PCR). The DNA fragment was amplified from N. caninum genomic DNA, but not from T. gondii, Sarcocystis muris, Sarcocystis tenella, or Sarcocystis cruzi genomic DNA. Additionally, the fragment was amplified from DNA prepared from the brains of N. caninum-infected mice, but not from the brain of a mouse infected with T. gondii. These results suggest that this PCR assay may be useful for the diagnosis of neosporosis. PMID:8992315

  5. Suppression of 14-3-3γ-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells.

    PubMed

    Lee, Young-Sun; Lee, Jae Kwang; Bae, Yeonju; Lee, Bok-Soon; Kim, Eunju; Cho, Chang-Hoon; Ryoo, Kanghyun; Yoo, Jiyun; Kim, Chul-Ho; Yi, Gwan-Su; Lee, Seok-Geun; Lee, C Justin; Kang, Sang Soo; Hwang, Eun Mi; Park, Jae-Yong

    2016-01-01

    Anoctamin-1 (ANO1) acts as a Ca(2+)-activated Cl(-) channel in various normal tissues, and its expression is increased in several different types of cancer. Therefore, understanding the regulation of ANO1 surface expression is important for determining its physiological and pathophysiological functions. However, the trafficking mechanism of ANO1 remains elusive. Here, we report that segment a (N-terminal 116 amino acids) of ANO1 is crucial for its surface expression, and we identified 14-3-3γ as a binding partner for anterograde trafficking using yeast two-hybrid screening. The surface expression of ANO1 was enhanced by 14-3-3γ, and the Thr9 residue of ANO1 was critical for its interaction with 14-3-3γ. Gene silencing of 14-3-3γ and/or ANO1 demonstrated that suppression of ANO1 surface expression inhibited migration and invasion of glioblastoma cells. These findings provide novel therapeutic implications for glioblastomas, which are associated with poor prognosis. PMID:27212225

  6. Suppression of 14-3-3γ-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells

    PubMed Central

    Lee, Young-Sun; Lee, Jae Kwang; Bae, Yeonju; Lee, Bok-Soon; Kim, Eunju; Cho, Chang-Hoon; Ryoo, Kanghyun; Yoo, Jiyun; Kim, Chul-Ho; Yi, Gwan-Su; Lee, Seok-Geun; Lee, C. Justin; Kang, Sang Soo; Hwang, Eun Mi; Park, Jae-Yong

    2016-01-01

    Anoctamin-1 (ANO1) acts as a Ca2+-activated Cl− channel in various normal tissues, and its expression is increased in several different types of cancer. Therefore, understanding the regulation of ANO1 surface expression is important for determining its physiological and pathophysiological functions. However, the trafficking mechanism of ANO1 remains elusive. Here, we report that segment a (N-terminal 116 amino acids) of ANO1 is crucial for its surface expression, and we identified 14-3-3γ as a binding partner for anterograde trafficking using yeast two-hybrid screening. The surface expression of ANO1 was enhanced by 14-3-3γ, and the Thr9 residue of ANO1 was critical for its interaction with 14-3-3γ. Gene silencing of 14-3-3γ and/or ANO1 demonstrated that suppression of ANO1 surface expression inhibited migration and invasion of glioblastoma cells. These findings provide novel therapeutic implications for glioblastomas, which are associated with poor prognosis. PMID:27212225

  7. How to measure epsilon'/epsilon with lattice QCD

    SciTech Connect

    Sharpe, S.R.

    1987-04-01

    A pedagogical discussion is given of a lattice calculation of epsilon'. The method is outlined, and preliminary results are presented. They suggest that epsilon'/epsilon may be reduced from previous estimates by 60-70%.

  8. Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ

    PubMed Central

    Bersani, C; Xu, L-D; Vilborg, A; Lui, W-O; Wiman, K G

    2014-01-01

    Wig-1, also known as ZMAT3, is a p53 target gene that encodes an RNA-binding zinc-finger protein involved in the regulation of mRNA stability through binding to AU-rich elements (AREs). We have used microarray analysis to identify novel Wig-1 target mRNAs. We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells. Several p53 target genes were among the deregulated transcripts. We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53. We show that Wig-1 binds to FAS mRNA 3′-UTR and decreases its stability through an ARE in the 3′-UTR. Depletion of Wig-1 was associated with increased cell death and reduced cell cycle arrest upon DNA damage. Our results suggest a role of Wig-1 as a survival factor that directs the p53 stress response toward cell cycle arrest rather than apoptosis through the regulation of FAS and 14-3-3σ mRNA levels. PMID:24469038

  9. Decreased expression of 14-3-3σ is predictive of poor prognosis for patients with human uterine papillary serous carcinoma.

    PubMed

    Suzuki, Fumihiko; Nagase, Satoru; Suzuki, Kichiya; Oba, Etsuko; Hiroki, Eri; Matsuda, Yukika; Akahira, Jun-Ichi; Nishigori, Hidekazu; Sugiyama, Takashi; Otsuki, Takeo; Yoshinaga, Kousuke; Takano, Tadao; Niikura, Hitoshi; Ito, Kiyoshi; Sasano, Hironobu; Yaegashi, Nobuo

    2013-01-01

    Uterine papillary serous carcinoma (UPSC) morphologically resembles ovarian serous carcinoma and is categorized as a type II endometrial cancer. UPSC comprises about 10% of all types of endometrial cancer and has an aggressive clinical course and a poor prognosis. The 14-3-3σ gene was originally discovered as a p53-inducible gene; its expression is induced by DNA damage in a p53-dependent manner, which leads to G2 arrest and repair of damaged DNA. Moreover, it has been reported that expression of 14-3-3σ is frequently lost in various types of human cancer, including ovarian cancer. We therefore examined the association between 14-3-3σ expression determined by immunohistochemistry and clinical outcomes of 51 patients with UPSC. UPSC was considered positive for 14-3-3σ when > 30% of tumor cells were stained with a specific antibody. Of these patients, 29 (58.7%) showed positive immunoreactivity for 14-3-3σ and 22 (41.3%) had decreased 14-3-3σ staining. Decreased immunoreactivity for 14-3-3σ was associated with stage (P = 0.001) and lymphovascular space involvement (P = 0.005). Moreover, decreased 14-3-3σ expression was an independent risk factor for reduced overall survival (P = 0.0416) in multivariate analysis. Direct bisulfite sequencing was performed to evaluate the methylation status of the 27 CpG islands in the promoter region and first exon of the 14-3-3σ gene. These CpG islands were hypermethylated in 30% of 14-3-3σ-positive UPSC and 80% of 14-3-3σ-negative UPSC, although the difference was not statistically significant. These findings suggest that decreased expression of immunoreactive 14-3-3σ may be a predictor of poor prognosis in patients with UPSC. PMID:24201220

  10. The epsilon dataflow processor

    SciTech Connect

    Grafe, V.G.; Davidson, G.S.; Hoch, J.E.; Holmes, V.P.

    1988-01-01

    The epsilon dataflow architecture is designed for high speed uniprocessor execution as well as for parallel processing. The epsilon architecture directly matches ready operands, thus eliminating the need for associative matching stores. epsilon also supports low cost data fan out and critical sections. A 10 MFLOPS CMOS/TTL processor prototype is running and its performance has been measured with several benchmarks. The prototype processor has demonstrated sustained performance exceeding that of comparable control flow processors running at higher clock rates (three times faster than a 20 MHz transputer and 24 times faster than a Sun on a suite of arithmetic tests, for example). 22 refs., 16 figs.

  11. Proteomic analysis of differentially expressed proteins in Penaeus monodon hemocytes after Vibrio harveyi infection

    PubMed Central

    2010-01-01

    Background Viral and bacterial diseases can cause mass mortalities in commercial shrimp aquaculture. In contrast to studies on the antiviral response, the responses of shrimps to bacterial infections by high throughput techniques have been reported only at the transcriptional level and not at the translational level. In this study, a proteomic analysis of shrimp hemocytes to identify differentially expressed proteins in response to a luminous bacterium Vibrio harveyi was evaluated for its feasibility and is reported for the first time. Results The two-dimensional gel electrophoresis (2-DE) patterns of the hemocyte proteins from the unchallenged and V. harveyi challenged shrimp, Penaeus monodon, at 24 and 48 h post infection were compared. From this, 27 differentially expressed protein spots, and a further 12 weakly to non-differentially regulated control spots, were selected for further analyses by the LC-ESI-MS/MS. The 21 differentially expressed proteins that could be identified by homologous annotation were comprised of proteins that are directly involved in the host defense responses, such as hemocyanin, prophenoloxidase, serine proteinase-like protein, heat shock protein 90 and alpha-2-macroglobulin, and those involved in signal transduction, such as the14-3-3 protein epsilon and calmodulin. Western blot analysis confirmed the up-regulation of hemocyanin expression upon bacterial infection. The expression of the selected proteins which were the representatives of the down-regulated proteins (the 14-3-3 protein epsilon and alpha-2-macroglobulin) and of the up-regulated proteins (hemocyanin) was further assessed at the transcription level using real-time RT-PCR. Conclusions This work suggests the usefulness of a proteomic approach to the study of shrimp immunity and revealed hemocyte proteins whose expression were up regulated upon V. harveyi infection such as hemocyanin, arginine kinase and down regulated such as alpha-2-macroglobulin, calmodulin and 14-3-3

  12. Regulation of Aldo-keto-reductase family 1 B10 by 14-3-3ε and their prognostic impact of hepatocellular carcinoma

    PubMed Central

    Lu, Yi-Jhu; Liang, Shu-Man; Liu, Chia-Chia; Chen, Shyh-Chang; Wang, John; Shyue, Song-Kun; Liou, Jun-Yang

    2015-01-01

    14-3-3ε is overexpressed in hepatocellular carcinoma (HCC) and its expression significantly associates with a poor prognostic outcome. To uncover how 14-3-3ε contributes to the tumor progression of HCC, we investigated the potential downstream targets regulated by 14-3-3ε. We found that 14-3-3ε increases expression and nuclear translocation of β-catenin and that 14-3-3ε-induced cell proliferation is attenuated by β-catenin silencing in HCC cells. Moreover, 14-3-3ε induces aldo-keto reductase family 1 member B10 (AKR1B10) expression through the activation of β-catenin signaling. Knockdown of AKR1B10 by siRNAs abolished 14-3-3ε-induced in vitro cell proliferation, anchorage-independent growth as well as in vivo tumor growth. Furthermore, AKR1B10 silencing increased retinoic acid (RA) levels in the serum of tumor-bearing mice and RA treatment attenuated 14-3-3ε-induced HCC cell proliferation. We further examined 14-3-3ε and AKR1B10 expression and clinicopathological characteristics of HCC tumors. Although the expression of AKR1B10 was significantly correlated with 14-3-3ε, an increase of AKR1B10 expression in 14-3-3ε positive patients paradoxically had better overall survival and disease-free survival rates as well as lower metastatic incidence than those without an AKR1B10 increase. Finally, we found a loss of AKR1B10 expression in cells exhibiting a high capacity of invasiveness. Silencing of AKR1B10 resulted in inducing snail and vimentin expression in HCC cells. These results indicate that AKR1B10 may play a dual role during HCC tumor progression. Our results also indicate that 14-3-3ε regulates AKR1B10 expression by activating β-catenin signaling. A combination of 14-3-3ε with AKR1B10 is a potential therapeutic target and novel prognostic biomarker of HCC. PMID:26516929

  13. 14-3-3ζ deficient mice in the BALB/c background display behavioural and anatomical defects associated with neurodevelopmental disorders

    PubMed Central

    Xu, Xiangjun; Jaehne, Emily J.; Greenberg, Zarina; McCarthy, Peter; Saleh, Eiman; Parish, Clare L.; Camera, Daria; Heng, Julian; Haas, Matilda; Baune, Bernhard T.; Ratnayake, Udani; Buuse, Maarten van den; Lopez, Angel F.; Ramshaw, Hayley S.; Schwarz, Quenten

    2015-01-01

    Sequencing and expression analyses implicate 14-3-3ζ as a genetic risk factor for neurodevelopmental disorders such as schizophrenia and autism. In support of this notion, we recently found that 14-3-3ζ−/− mice in the Sv/129 background display schizophrenia-like defects. As epistatic interactions play a significant role in disease pathogenesis we generated a new congenic strain in the BALB/c background to determine the impact of genetic interactions on the 14-3-3ζ−/− phenotype. In addition to replicating defects such as aberrant mossy fibre connectivity and impaired spatial memory, our analysis of 14-3-3ζ−/− BALB/c mice identified enlarged lateral ventricles, reduced synaptic density and ectopically positioned pyramidal neurons in all subfields of the hippocampus. In contrast to our previous analyses, 14-3-3ζ−/− BALB/c mice lacked locomotor hyperactivity that was underscored by normal levels of the dopamine transporter (DAT) and dopamine signalling. Taken together, our results demonstrate that dysfunction of 14-3-3ζ gives rise to many of the pathological hallmarks associated with the human condition. 14-3-3ζ-deficient BALB/c mice therefore provide a novel model to address the underlying biology of structural defects affecting the hippocampus and ventricle, and cognitive defects such as hippocampal-dependent learning and memory. PMID:26207352

  14. A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

    PubMed

    Kular, Jasreen; Scheer, Kaitlin G; Pyne, Natasha T; Allam, Amr H; Pollard, Anthony N; Magenau, Astrid; Wright, Rebecca L; Kolesnikoff, Natasha; Moretti, Paul A; Wullkopf, Lena; Stomski, Frank C; Cowin, Allison J; Woodcock, Joanna M; Grimbaldeston, Michele A; Pitson, Stuart M; Timpson, Paul; Ramshaw, Hayley S; Lopez, Angel F; Samuel, Michael S

    2015-12-21

    ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities. PMID:26702834

  15. Decreased expression of 14-3-3 σ, an early event of malignant transformation of respiratory epithelium, also facilitates progression of squamous cell lung cancer

    PubMed Central

    Sun, Nan; Wu, Yongkai; Huang, Bo; Liu, Qian; Dong, Yinan; Ding, Jianqiao; Liu, Yongyu

    2015-01-01

    Background It has been shown that 14-3-3 σ serves as a tumor suppressor gene, and is downregulated in various tumor tissues. However, the role of 14-3-3 σ during the initiation and progression of lung squamous cell carcinoma (SqCC) is not well understood. Methods The expression status of 14-3-3 σ in archival tissue samples from 40 lung SqCC patients (36 with normal bronchia, 19 squamous metaplasia, and 17 dysplasia/carcinoma in situ, in their tissue samples) was examined by immunohistochemical analysis. The proliferation rate and tumor formation ability of the H520 cell transfected with 14-3-3 σ was tested with methyl thiazolyl tetrazolium assay and nude mice subcutaneous injection, respectively. Results In the normal bronchial epithelia, 14-3-3 σ was highly expressed, whereas it was significantly decreased in precancerous and cancerous tissues. Compared with matched invasive cancer tissues, the expression level of 14-3-3 σ in squamous metaplasia was significantly higher (P = 0.049), while that in dysplasia/carcinoma in situ showed no significant changes (P = 0.135). Statistical analysis showed that the expression level of 14-3-3 σ in tumor tissue was associated with the differentiation grade of the tumor (P = 0.001) and the prognosis of the patient (P = 0.003). The overexpression of 14-3-3 σ significantly suppressed the proliferation of H520 cells in vitro and in vivo. Conclusion The inactivation of 14-3-3 σ may be a very early event in tumorigenesis and could facilitate the initiation and progression of lung SqCC in a sustainable way. PMID:26557909

  16. PI 3-kinase-dependent phosphorylation of Plk1–Ser99 promotes association with 14-3-3γ and is required for metaphase–anaphase transition

    PubMed Central

    Kasahara, Kousuke; Goto, Hidemasa; Izawa, Ichiro; Kiyono, Tohru; Watanabe, Nobumoto; Elowe, Sabine; Nigg, Erich A; Inagaki, Masaki

    2013-01-01

    Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1–Thr210 phosphorylation. Plk1–Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1–Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1–Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1–Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1–Ser99 phosphorylation downstream of the PI3K–Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase. PMID:23695676

  17. Mapping Protein-Protein Interactions of the Resistance-Related Bacterial Zeta Toxin-Epsilon Antitoxin Complex (ε₂ζ₂) with High Affinity Peptide Ligands Using Fluorescence Polarization.

    PubMed

    Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg

    2016-01-01

    Toxin-antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta-Epsilon toxin-antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε₂ζ₂ complex. Three α helices of Zeta forming the protein-protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε₂ζ₂ complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay. PMID:27438853

  18. 14-3-3σ regulation of and interaction with YAP1 in acquired gemcitabine resistance via promoting ribonucleotide reductase expression

    PubMed Central

    Qin, Li; Dong, Zizheng; Zhang, Jian-Ting

    2016-01-01

    Gemcitabine is an important anticancer therapeutics approved for treatment of several human cancers including locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). Its clinical effectiveness, however, is hindered by existence of intrinsic and development of acquired resistances. Previously, it was found that 14-3-3σ expression associates with poor clinical outcome of PDAC patients. It was also found that 14-3-3σ expression is up-regulated in gemcitabine resistant PDAC cells and contributes to the acquired gemcitabine resistance. In this study, we investigated the molecular mechanism of 14-3-3σ function in gemcitabine resistance and found that 14-3-3σ up-regulates YAP1 expression and then binds to YAP1 to inhibit gemcitabine-induced caspase 8 activation and apoptosis. 14-3-3σ association with YAP1 up-regulates the expression of ribonucleotide reductase M1 and M2, which may mediate 14-3-3σ/YAP1 function in the acquired gemcitabine resistance. These findings suggest a possible role of YAP1 signaling in gemcitabine resistance. PMID:26894857

  19. Genetic and physical interaction of Ssp1 CaMKK and Rad24 14-3-3 during low pH and osmotic stress in fission yeast

    PubMed Central

    Freitag, Silja I.; Wong, Jimson; Young, Paul G.

    2014-01-01

    The Ssp1 calmodulin kinase kinase (CaMKK) is necessary for stress-induced re-organization of the actin cytoskeleton and initiation of growth at the new cell end following division in Schizosaccharomyces pombe. In addition, it regulates AMP-activated kinase and functions in low glucose tolerance. ssp1− cells undergo mitotic delay at elevated temperatures and G2 arrest in the presence of additional stressors. Following hyperosmotic stress, Ssp1-GFP forms transient foci which accumulate at the cell membrane and form a band around the cell circumference, but not co-localizing with actin patches. Hyperosmolarity-induced localization to the cell membrane occurs concomitantly with a reduction of its interaction with the 14-3-3 protein Rad24, but not Rad25 which remains bound to Ssp1. The loss of rad24 in ssp1− cells reduces the severity of hyperosmotic stress response and relieves mitotic delay. Conversely, overexpression of rad24 exacerbates stress response and concomitant cell elongation. rad24− does not impair stress-induced localization of Ssp1 to the cell membrane, however this response is almost completely absent in cells overexpressing rad24. PMID:24451546

  20. An autoantibody against N{sup {epsilon}}-(carboxyethyl)lysine (CEL): Possible involvement in the removal of CEL-modified proteins by macrophages

    SciTech Connect

    Mera, Katsumi; Nagai, Ryoji; Takeo, Kazuhiro; Izumi, Miyoko; Maruyama, Toru; Otagiri, Masaki

    2011-04-08

    Highlights: {yields} A higher amount of autoantibody against CEL than that of other AGEs was observed in human plasma. {yields} The purified human anti-CEL autoantibody specifically reacted with CEL. {yields} Anti-CEL antibody accelerated the uptake of {sup 125}I-CEL-HSA by macrophage in vitro. {yields} Endocytic uptake of {sup 125}I-CEL-HSA by mice liver was accelerated in the presence of anti-CEL antibody. -- Abstract: Advanced glycation end products (AGEs) are believed to play a significant role in the development of diabetic complications. In this study, we measured the levels of autoantibodies against several AGE structures in healthy human plasma and investigated the physiological role of the autoantibodies. A high titer of the autoantibody against N{sup {epsilon}}-(carboxyethyl)lysine (CEL) was detected in human plasma compared with other AGE structures such as CML and pentosidine. The purified human anti-CEL autoantibody reacted with CEL-modified human serum albumin (CEL-HSA), but not CML-HSA. A rabbit polyclonal anti-CEL antibody, used as a model autoantibody against CEL, accelerated the uptake of CEL-HSA by macrophages, but did not enhance the uptake of native HSA. Furthermore, when {sup 125}I-labeled CEL-HSA was injected into the tail vein of mice, accumulation of {sup 125}I-CEL-HSA in the liver was accelerated by co-injection of the rabbit anti-CEL antibody. These results demonstrate that the autoantibody against CEL in plasma may play a role in the macrophage uptake of CEL-modified proteins.

  1. Bone Tissue Engineering Using High Permeability Poly-epsilon-caprolactone Scaffolds Conjugated with Bone Morphogenetic Protein-2

    NASA Astrophysics Data System (ADS)

    Mitsak, Anna Guyer

    Bone is the second most commonly transplanted tissue in the United States. Limitations of current bone defect treatment options include morbidity at the autograft harvest site, mechanical failure, and poorly controlled growth factor delivery. Combining synthetic scaffolds with biologics may address these issues and reduce dependency on autografts. The ideal scaffolding system should promote tissue in-growth and nutrient diffusion, control delivery of biologics and maintain mechanical integrity during bone formation. This dissertation evaluates how scaffold permeability, conjugated bone morphogenetic protein-2 (BMP-2) and differentiation medium affect osteogenesis in vitro and bone growth in vivo.. "High" and "low" permeability polycaprolactone (PCL) scaffolds with regular architectures were manufactured using solid free form fabrication. Bone growth in vivo was evaluated in an ectopic mouse model. High permeability scaffolds promoted better 8 week bone growth, supported tissue penetration into the scaffold core, and demonstrated increased mechanical properties due to newly formed bone. Next, the effects of differentiation medium and conjugated BMP-2 on osteogenesis were compared. Conjugation may improve BMP-2 loading efficiency, help localize bone growth and control release. High permeability scaffolds were conjugated with BMP-2 using the crosslinker, sulfo-SMCC. When adipose-derived and bone marrow stromal cells were seeded onto constructs (with or without BMP-2), BMSC expressed more differentiation markers, and differentiation medium affected differentiation more than BMP-2. In vivo, scaffolds with ADSC pre-differentiated in osteogenic medium (with and without BMP-2) and scaffolds with only BMP-2 grew the most bone. Bone volume did not differ among these groups, but constructs with ADSC had evenly distributed, scaffold-guided bone growth. Analysis of two additional BMP-2 attachment methods (heparin and adsorption) showed highest conjugation efficiency for the

  2. A role for protein kinase C subtypes alpha and epsilon in phorbol-ester-enhanced K(+)- and carbachol-evoked noradrenaline release from the human neuroblastoma SH-SY5Y.

    PubMed Central

    Turner, N A; Rumsby, M G; Walker, J H; McMorris, F A; Ball, S G; Vaughan, P F

    1994-01-01

    Protein kinase C (PKC) consists of a family of closely related subtypes which differ in their localization and activation properties. Our previous studies have suggested a role for PKC in the regulation of noradrenaline (NA) release from the human neuroblastoma SH-SY5Y. Here we have used two approaches to characterize the PKC subtypes present in SH-SY5Y cells. Firstly, the PCR was used to show that SH-SY5Y cells contain mRNA encoding PKC subtypes alpha, beta, gamma, delta, epsilon and zeta. Secondly, immunoblotting showed that SH-SY5Y cells express PKC subtypes alpha, epsilon and zeta at the protein level. Prolonged (48 h) exposure of cells to the phorbol ester phorbol 12-myristate 13-acetate (PMA; 100 nM) resulted in a marked decrease in the amounts of PKC-alpha and PKC-epsilon, with no change in levels of PKC-zeta. Prolonged PMA treatment had no significant effect on K(+)-evoked NA release from SH-SY5Y cells, whereas carbachol-evoked release was increased 2.2-fold. However, prolonged exposure to PMA completely inhibited the ability of acute (12 min) PMA treatment to enhance both K(+)- and carbachol-evoked NA release. The specific PKC inhibitor RO 31-7459 (10 microM) was found to inhibit K(+)- and carbachol-evoked release by 27% and 68% respectively. RO 31-7549 also completely inhibited the ability of acute PMA treatment to enhance release. These data suggest that PKC-alpha and/or PKC-epsilon play an essential role in the regulation of PMA-enhanced K(+)- and carbachol-evoked NA release in SH-SY5Y cells. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8297348

  3. Role of protein kinase C epsilon (PKCε) in the reduction of ethanol reinforcement due to mGluR5 antagonism in the nucleus accumbens shell

    PubMed Central

    Gass, Justin T.; Olive, M. Foster

    2009-01-01

    Rationale The type 5 metabotropic glutamate receptor (mGluR5) and the epsilon isoform of protein kinase C (PKCε) regulate ethanol intake, and we have previously demonstrated that mGluR5 receptor antagonism reduces ethanol consumption via a PKCε-dependent mechanism. Objectives We explored the neuroanatomical substrates of the regulation of ethanol reinforcement by this mGluR5-PKCε signaling pathway by infusing selective inhibitors of these proteins into the shell or core region of the nucleus accumbens (NAc). Methods Male Wistar rats were trained to self-administer ethanol intravenously and received intra-NAc infusions of vehicle or the selective mGluR5 antagonist 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP) alone and in combination with a PKCε translocation inhibitor (εV1-2) or a scrambled control peptide (sεV1-2). The effects of intra-NAc MTEP on food-reinforced responding and open-field locomotor activity were also determined. Results MTEP (1 μg/μl) had no effect on ethanol or food reinforcement or locomotor activity when infused into the NAc core. MTEP (3 μg/μl) reduced ethanol reinforcement when infused into the NAc shell but not the core, and this effect was reversed by εV1-2 (1 μg/μl) but not sεV1-2 (1 μg/μl). In both regions, this concentration of MTEP did not alter food-reinforced responding or locomotor activity, and infusion of εV1-2 alone did not alter ethanol reinforcement. MTEP (10 μg/μl) reduced locomotor activity when infused into the shell, and therefore this concentration was not further tested on responding for ethanol or food. Conclusions Blockade of mGluR5 receptors in the NAc shell reduces ethanol reinforcement via a PKCε-dependent mechanism. PMID:19225761

  4. 14-3-3ɛ/ζ Affects the stability of δ-catenin and regulates δ-catenin-induced dendrogenesis.

    PubMed

    He, Yongfeng; Han, Jeong Ran; Chang, Ockyoung; Oh, Minsoo; James, Sarah E; Lu, Qun; Seo, Young-Woo; Kim, Hangun; Kim, Kwonseop

    2013-01-01

    Accumulated evidence suggests that aberrant regulation of δ-catenin leads to pathological consequences such as mental retardation and cognitive dysfunction. This study revealed that 14-3-3ɛ/ζ stabilizes δ-catenin, with different binding regions involved in the interaction. Furthermore, the specific inhibition of the interaction of 14-3-3 with δ-catenin reduced levels of δ-catenin and significantly impaired the capacity of δ-catenin to induce dendritic branching in both NIH3T3 fibroblasts and primary hippocampal neurons. However, the S1094A δ-catenin mutant, which cannot interact with 14-3-3ζ, still retained the capability of inducing dendrogenesis. Taken together, these results elucidate the underlying events that regulate the stability of δ-catenin and δ-catenin-induced dendrogenesis. PMID:23772369

  5. The EPSILON-2 multiprocessor system

    SciTech Connect

    Grafe, V.G.; Hoch, J.E. )

    1990-12-01

    This paper reports on Epsilon-2 which is a general purpose parallel computer architecture that addresses the twin goals of high performance and programmability. Epsilon-2 implements a hybrid computation model that combines the fine-grain parallelism of dataflow computing with the sequential efficiency characteristic of von Neumann computing. It provides instruction-level synchronization, single-cycle context switches, and RISC-like sequential execution. The general parallel computing model and the architecture of Epsilon-2 are described. A sample code is also presented to illustrate Epsilon-2's operation.

  6. An unusual arrangement of two 14-3-3-like domains in the SMG5–SMG7 heterodimer is required for efficient nonsense-mediated mRNA decay

    PubMed Central

    Jonas, Stefanie; Weichenrieder, Oliver; Izaurralde, Elisa

    2013-01-01

    The nonsense-mediated mRNA decay (NMD) pathway triggers the rapid degradation of aberrant mRNAs containing premature translation termination codons (PTCs). In metazoans, NMD requires three 14-3-3-like proteins: SMG5, SMG6, and SMG7. These proteins are recruited to PTC-containing mRNAs through the interaction of their 14-3-3-like domains with phosphorylated UPF1, the central NMD effector. Recruitment of SMG5, SMG6, and SMG7 causes NMD target degradation. In this study, we report the crystal structure of the Caenorhabditis elegans SMG5–SMG7 complex. The 14-3-3-like phosphopeptide recognition domains of SMG5 and SMG7 heterodimerize in an unusual perpendicular back-to-back orientation in which the peptide-binding sites face opposite directions. Structure-based mutants and functional assays indicate that the SMG5–SMG7 interaction is conserved and is crucial for efficient NMD in human cells. Notably, we demonstrate that heterodimerization increases the affinity of the SMG5–SMG7 complex for UPF1. Furthermore, we show that the degradative activity of the SMG5–SMG7 complex resides in SMG7 and that the SMG5–SMG7 complex and SMG6 play partially redundant roles in the degradation of aberrant mRNAs. We propose that the SMG5–SMG7 complex binds to phosphorylated UPF1 with high affinity and recruits decay factors to the mRNA target through SMG7, thus promoting target degradation. PMID:23348841

  7. Quark masses, B-parameters, and CP violation parameters {epsilon} and {epsilon}{prime}/{epsilon}

    SciTech Connect

    Gupta, R.

    1998-01-20

    After a brief introduction to lattice QCD, the author summarizes the results for the light quark masses and the bag parameters B{sub K}, B{sub 6}{sup 1/2}, and B{sub 8}{sup 3/2}. The implications of these results for the standard model estimates of CP violation parameters {epsilon} and {epsilon}{prime}/{epsilon} are also discussed.

  8. N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins.

    PubMed Central

    Ahmed, M U; Brinkmann Frye, E; Degenhardt, T P; Thorpe, S R; Baynes, J W

    1997-01-01

    Advanced glycation end-products and glycoxidation products, such as Nepsilon-(carboxymethyl)lysine (CML) and pentosidine, accumulate in long-lived tissue proteins with age and are implicated in the aging of tissue proteins and in the development of pathology in diabetes, atherosclerosis and other diseases. In this paper we describe a new advanced glycation end-product, Nepsilon-(carboxyethyl)lysine (CEL), which is formed during the reaction of methylglyoxal with lysine residues in model compounds and in the proteins RNase and collagen. CEL was also detected in human lens proteins at a concentration similar to that of CML, and increased with age in parallel with the concentration of CML. Although CEL was formed in highest yields during the reaction of methylglyoxal and triose phosphates with lysine and protein, it was also formed in reactions of pentoses, ascorbate and other sugars with lysine and RNase. We propose that levels of CML and CEL and their ratio to one another in tissue proteins and in urine will provide an index of glyoxal and methylglyoxal concentrations in tissues, alterations in glutathione homoeostasis and dicarbonyl metabolism in disease, and sources of advanced glycation end-products in tissue proteins in aging and disease. PMID:9182719

  9. Induction of activation-induced cytidine deaminase-targeting adaptor 14-3-3γ is mediated by NF-κB-dependent recruitment of CFP1 to the 5'-CpG-3'-rich 14-3-3γ promoter and is sustained by E2A.

    PubMed

    Mai, Thach; Pone, Egest J; Li, Guideng; Lam, Tonika S; Moehlman, J'aime; Xu, Zhenming; Casali, Paolo

    2013-08-15

    Class switch DNA recombination (CSR) crucially diversifies Ab biologic effector functions. 14-3-3γ specifically binds to the 5'-AGCT-3' repeats in the IgH locus switch (S) regions. By interacting directly with the C-terminal region of activation-induced cytidine deaminase (AID), 14-3-3γ targets this enzyme to S regions to mediate CSR. In this study, we showed that 14-3-3γ was expressed in germinal center B cells in vivo and induced in B cells by T-dependent and T-independent primary CSR-inducing stimuli in vitro in humans and mice. Induction of 14-3-3γ was rapid, peaking within 3 h of stimulation by LPSs, and sustained over the course of AID and CSR induction. It was dependent on recruitment of NF-κB to the 14-3-3γ gene promoter. The NF-κB recruitment enhanced the occupancy of the CpG island within the 14-3-3γ promoter by CFP1, a component of the COMPASS histone methyltransferase complex, and promoter-specific enrichment of histone 3 lysine 4 trimethylation (H3K4me3), which is indicative of open chromatin state and marks transcription-competent promoters. NF-κB also potentiated the binding of B cell lineage-specific factor E2A to an E-box motif located immediately downstream of the two closely-spaced transcription start sites for sustained 14-3-3γ expression and CSR induction. Thus, 14-3-3γ induction in CSR is enabled by the CFP1-mediated H3K4me3 enrichment in the promoter, dependent on NF-κB and sustained by E2A. PMID:23851690

  10. Amifostine alleviates radiation-induced lethal small bowel damage via promotion of 14-3-3σ-mediated nuclear p53 accumulation.

    PubMed

    Huang, Eng-Yen; Wang, Feng-Sheng; Chen, Yu-Min; Chen, Yi-Fan; Wang, Chung-Chi; Lin, I-Hui; Huang, Yu-Jie; Yang, Kuender D

    2014-10-30

    Amifostine (AM) is a radioprotector that scavenges free radicals and is used in patients undergoing radiotherapy. p53 has long been implicated in cell cycle arrest for cellular repair after radiation exposure. We therefore investigated the protective p53-dependent mechanism of AM on small bowel damage after lethal whole-abdominal irradiation (WAI). AM increased both the survival rate of rats and crypt survival following lethal 18 Gy WAI. The p53 inhibitor PFT-α compromised AM-mediated effects when administered prior to AM administration. AM significantly increased clonogenic survival in IEC-6 cells expressing wild type p53 but not in p53 knockdown cells. AM significantly increased p53 nuclear accumulation and p53 tetramer expression before irradiation through the inhibition of p53 degradation. AM inhibited p53 interactions with MDM2 but enhanced p53 interactions with 14-3-3σ. Knockdown of 14-3-3σ also compromised the effect of AM on clonogenic survival and p53 nuclear accumulation in IEC-6 cells. For the first time, our data reveal that AM alleviates lethal small bowel damage through the induction of 14-3-3σ and subsequent accumulation of p53. Enhancement of the p53/14-3-3σ interaction results in p53 tetramerization in the nucleus that rescues lethal small bowel damage. PMID:25230151

  11. Eclipse of epsilon Aurigae

    NASA Astrophysics Data System (ADS)

    Templeton, Matthew R.

    2009-07-01

    The bright, long-period, eclipsing binary star epsilon Aurigae is predicted to begin its next eclipse late July or early August of 2009. Epsilon Aurigae is now past solar conjunction and has reappeared as a morning object. All observers -- both visual and instrumental -- are encouraged to contribute observations of the eclipse during the next two years, beginning immediately for morning observers. Observations are urgently requested right now because it is less likely to be observed in the morning, and the eclipse will begin within the next month. The AAVSO is participating in a global campaign to record this eclipse as part of the International Year of Astronomy 2009 celebrations, organized by the Citizen Sky project (http://www.citizensky.org). For experienced visual observers, please observe this star on a weekly basis, using charts available via VSP from the AAVSO website. For novice visual observers, we recommend participating in this observing program by following the Citizen Sky 10-Star tutorial program, which provides a simple training experience in variable star observing. Photoelectric observers belonging to the AAVSO PEP-V program may submit data as usual via the WebObs feature of the AAVSO website Blue&Gold section. Photoelectric observers may also contribute reduced observations in all filters (including infrared J- and H-bands) directly to the AAVSO via WebObs. Observers using wide-field CCD and DSLR systems are also encouraged to participate; avoid saturating the star. For those with narrower-field systems (D < 2 degrees), we recommend taking a large number (10-100) of very short exposures and then stacking the resulting images. Observations should be submitted to the AAVSO International Database. Aaron Price is coordinating Citizen Sky for the AAVSO, and Dr. Robert Stencel and Jeffrey Hopkins are co-leading the precision photometry efforts.

  12. Molecular Characterization of the 14-3-3 Gene Family in Brachypodium distachyon L. Reveals High Evolutionary Conservation and Diverse Responses to Abiotic Stresses

    PubMed Central

    Cao, Hui; Xu, Yuxing; Yuan, Linlin; Bian, Yanwei; Wang, Lihui; Zhen, Shoumin; Hu, Yingkao; Yan, Yueming

    2016-01-01

    The 14-3-3 gene family identified in all eukaryotic organisms is involved in a wide range of biological processes, particularly in resistance to various abiotic stresses. Here, we performed the first comprehensive study on the molecular characterization, phylogenetics, and responses to various abiotic stresses of the 14-3-3 gene family in Brachypodium distachyon L. A total of seven 14-3-3 genes from B. distachyon and 120 from five main lineages among 12 species were identified, which were divided into five well-conserved subfamilies. The molecular structure analysis showed that the plant 14-3-3 gene family is highly evolutionarily conserved, although certain divergence had occurred in different subfamilies. The duplication event investigation revealed that segmental duplication seemed to be the predominant form by which the 14-3-3 gene family had expanded. Moreover, seven critical amino acids were detected, which may contribute to functional divergence. Expression profiling analysis showed that BdGF14 genes were abundantly expressed in the roots, but showed low expression in the meristems. All seven BdGF14 genes showed significant expression changes under various abiotic stresses, including heavy metal, phytohormone, osmotic, and temperature stresses, which might play important roles in responses to multiple abiotic stresses mainly through participating in ABA-dependent signaling and reactive oxygen species-mediated MAPK cascade signaling pathways. In particular, BdGF14 genes generally showed upregulated expression in response to multiple stresses of high temperature, heavy metal, abscisic acid (ABA), and salicylic acid (SA), but downregulated expression under H2O2, NaCl, and polyethylene glycol (PEG) stresses. Meanwhile, dynamic transcriptional expression analysis of BdGF14 genes under longer treatments with heavy metals (Cd2+, Cr3+, Cu2+, and Zn2+) and phytohormone (ABA) and recovery revealed two main expression trends in both roots and leaves: up-down and up

  13. Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo.

    PubMed

    Soler-Jover, Alex; Blasi, Juan; Gómez de Aranda, Inma; Navarro, Piedad; Gibert, Maryse; Popoff, Michel R; Martín-Satué, Mireia

    2004-07-01

    Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock. Recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-prototoxin-GFP were successfully expressed in Escherichia coli. MTT assays on MDCK cells confirmed that recombinant epsilon-toxin-GFP retained the cytotoxicity of the native toxin. Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent. epsilon-Toxin-GFP and epsilon-prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain. However, fluorescence mainly accumulated in kidneys. Mice injected with epsilon-toxin-GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules. Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species. We demonstrate with new recombinant fluorescence tools that epsilon-toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect. Our binding experiments indicate that an epsilon-toxin receptor is expressed on renal distal tubules of mammalian species, including human. PMID:15208360

  14. Mapping Protein–Protein Interactions of the Resistance-Related Bacterial Zeta Toxin–Epsilon Antitoxin Complex (ε2ζ2) with High Affinity Peptide Ligands Using Fluorescence Polarization

    PubMed Central

    Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg

    2016-01-01

    Toxin–antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta–Epsilon toxin–antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein–protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay. PMID:27438853

  15. The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX

    PubMed Central

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Finelli, Renata; Sferra, Gabriella; Müller, Joachim; Ricci, Giorgio; Pozio, Edoardo

    2015-01-01

    The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions. PMID:26082764

  16. The usefulness of S100P, mesothelin, fascin, prostate stem cell antigen, and 14-3-3 sigma in diagnosing pancreatic adenocarcinoma in cytological specimens obtained by endoscopic ultrasound guided fine-needle aspiration.

    PubMed

    Dim, Daniel C; Jiang, Feng; Qiu, Qi; Li, Ting; Darwin, Peter; Rodgers, William H; Peng, Hong Qi

    2014-03-01

    Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) of the pancreas is an efficient and minimally invasive procedure for the diagnosis and staging of pancreatic adenocarcinoma. Because of some limitations of EUS-FNA in diagnosis of well-differentiated or early stage cancers, the purpose of this study is to assess the added benefit of immunohistochemistry. We studied five proteins overexpressed in pancreatic adenocarcinoma, namely, prostate stem cell antigen, fascin, 14-3-3 sigma, mesothelin and S100P utilizing immunohistochemistry on paraffin sections from cellblocks obtained by EUS-FNA. Sixty-two cases of EUS-FNA of the pancreas that had follow-up histological and/or clinical diagnosis and sufficient material in cell blocks were included. Using histological diagnosis and/or clinical outcome as the reference standard, EUS-FNA shows the highest sensitivity (95%) and specificity (91%) and is superior to any marker in this study. Among five antibodies, S100P reveals the best diagnostic characters showing 90% of sensitivity and 67% of specificity. Fascin shows high specificity (92%) but low sensitivity (38%). Mesothelin has a moderate sensitivity (74%) and low specificity (33%), PSCA and 14-3-3 show high sensitivity but zero specificity. S100P and mesothelin were useful in nine indeterminate cases. S100P correctly predicted six of seven cancers and one of one without cancer and mesothelin correctly diagnosed five of seven cancers and one of two noncancers in this group. EUS-FNA cytomorphology is superior to any of the immunohistochemical markers used in this study. Use of S100P and mesothelin in cytologically borderline cases can increase the diagnostic accuracy in this group. PMID:21538952

  17. Aqueous Extract from Hibiscus sabdariffa Linnaeus Ameliorate Diabetic Nephropathy via Regulating Oxidative Status and Akt/Bad/14-3-3γ in an Experimental Animal Model

    PubMed Central

    Wang, Shou-Chieh; Lee, Shiow-Fen; Wang, Chau-Jong; Lee, Chao-Hsin; Lee, Wen-Chin; Lee, Huei-Jane

    2011-01-01

    Several studies point out that oxidative stress maybe a major culprit in diabetic nephropathy. Aqueous extract of Hibiscus sabdariffa L. (HSE) has been demonstrated as having beneficial effects on anti-oxidation and lipid-lowering in experimental studies. This study aimed at investigating the effects of Hibiscus sabdariffa L. on diabetic nephropathy in streptozotocin induced type 1 diabetic rats. Our results show that HSE is capable of reducing lipid peroxidation, increasing catalase and glutathione activities significantly in diabetic kidney, and decreasing the plasma levels of triglyceride, low-density lipoprotein (LDL) and increasing high-density lipoprotein (HDL) value. In histological examination, HSE improves hyperglycemia-caused osmotic diuresis in renal proximal convoluted tubules (defined as hydropic change) in diabetic rats. The study also reveals that up-regulation of Akt/Bad/14-3-3γ and NF-κB-mediated transcription might be involved. In conclusion, our results show that HSE possesses the potential effects to ameliorate diabetic nephropathy via improving oxidative status and regulating Akt/Bad/14-3-3γ signaling. PMID:19965962

  18. Akt and 14-3-3 control a PACS-2 homeostatic switch that integrates membrane traffic with TRAIL-induced apoptosis

    PubMed Central

    Aslan, Joseph E.; You, Huihong; Williamson, Danielle M.; Endig, Jessica; Youker, Robert T.; Thomas, Laurel; Shu, Hongjun; Du, Yuhong; Milewski, Robert L.; Brush, Matthew H.; Possemato, Anthony; Sprott, Kam; Fu, Haian; Greis, Kenneth D.; Runckel, Douglas N.; Vogel, Arndt; Thomas, Gary

    2009-01-01

    Summary TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis. PMID:19481529

  19. Akt and 14-3-3 control a PACS-2 homeostatic switch that integrates membrane traffic with TRAIL-induced apoptosis.

    PubMed

    Aslan, Joseph E; You, Huihong; Williamson, Danielle M; Endig, Jessica; Youker, Robert T; Thomas, Laurel; Shu, Hongjun; Du, Yuhong; Milewski, Robert L; Brush, Matthew H; Possemato, Anthony; Sprott, Kam; Fu, Haian; Greis, Kenneth D; Runckel, Douglas N; Vogel, Arndt; Thomas, Gary

    2009-05-14

    TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo, and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis. PMID:19481529

  20. Alzheimer amyloid-beta peptide forms denaturant-resistant complex with type epsilon 3 but not type epsilon 4 isoform of native apolipoprotein E.

    PubMed Central

    Zhou, Z.; Smith, J. D.; Greengard, P.; Gandy, S.

    1996-01-01

    BACKGROUND: The apolipoprotein E (apoE) type epsilon 4 isoform specifies increased cerebral and cerebrovascular accumulation of amyloid-beta protein (A beta) and contributes to the genetic susceptibility underlying a large proportion (approximately 60%) of typical, sporadic Alzheimer disease. Unfortunately, in vitro biochemical studies of direct apoE isoform-specific interactions with A beta have been inconsistent, perhaps due to the use by different research groups of apoE isoform preparations in different conformational states (purified denatured versus native). MATERIALS AND METHODS: In the current study, we have investigated the possibility that synthetic A beta(1-40) preferentially associates with native apoE of either the type epsilon 3 or the type epsilon 4 isoform. RESULTS: Here, we demonstrate the preferential association of synthetic A beta(1-40) with native apoE epsilon 3. The complex between apoE epsilon 3 and A beta(1-40) could not be disrupted by sodium dodecyl sulfate. In a parallel assay, no denaturant-resistant association of A beta(1-40) with apoE epsilon 4 was detectable. CONCLUSIONS: These results support the notion that the apoE epsilon 4 isoform may foster beta-amyloidogenesis because apoE epsilon 4 is inefficient in forming complexes with A beta. Images FIG. 1 FIG. 2 PMID:8726460

  1. epsilon-N-trimethyllysine availability regulates the rate of carnitine biosynthesis in the growing rat

    SciTech Connect

    Rebouche, C.J.; Lehman, L.J.; Olson, L.

    1986-05-01

    Rates of carnitine biosynthesis in mammals depend on the availability of substrates and the activity of enzymes subserving the pathway. This study was undertaken to test the hypothesis that the availability of epsilon-N-trimethyllysine is rate-limiting for synthesis of carnitine in the growing rat and to evaluate diet as a source of this precursor for carnitine biosynthesis. Rats apparently absorbed greater than 90% of a tracer dose of (methyl-/sup 3/H)epsilon-N-trimethyllysine, and approximately 30% of that was incorporated into tissues as (/sup 3/H)carnitine. Rats given oral supplements of epsilon-N-trimethyllysine (0.5-20 mg/d), but no dietary carnitine, excreted more carnitine than control animals receiving no dietary epsilon-N-trimethyllysine or carnitine. Rates of carnitine excretion increased in a dose-dependent manner. Tissue and serum levels of carnitine also increased with dietary epsilon-N-trimethyllysine supplementation. There was no evidence that the capacity for carnitine biosynthesis was saturated even at the highest level of oral epsilon-N-trimethyllysine supplementation. Common dietary proteins (casein, soy protein and wheat gluten) were found to be poor sources of epsilon-N-trimethyllysine for carnitine biosynthesis. The results of this study indicate that the availability of epsilon-N-trimethyllysine limits the rate of carnitine biosynthesis in the growing rat.

  2. Differential regulation of alternative 3{prime} splicing of {epsilon} messenger RNA variants

    SciTech Connect

    Diaz-Sanchez, D.; Zhang, K.; Saxon, A.

    1995-08-15

    Alternative 3{prime} splicing of the one active human {epsilon} heavy chain gene results in variants of {epsilon} mRNA encoding distinct IgE proteins. The same relative amounts of these {epsilon} mRNA variants were produced by non-atopic donor B cells when driven in a variety of T-dependent or T-independent systems. The most abundant variants were those for classic secreted {epsilon} and a novel secreted form (CH4-M2{double_prime}). In contrast, cells from subjects with high levels of serum IgE secondary to parasitic infection or atopy spontaneously produced higher relative levels of the CH4-M2{prime} {epsilon} mRNA variant, lower relative amounts of both the membrane and CH4-M2{double_prime} secreted variants, and very low levels of the CH4{prime}-CH5 variant. The existence of and corresponding changes in levels of the CH4-M2{prime}-enclosed secreted protein were demonstrated. IL-10 induced this same differential expression of {epsilon} splice variants in vitro when used to costimulate IL-4 plus CD40-driven B cells and could differentially enhance the production of CH4-M2{prime} protein by established IgE-secreting cell lines. Inhibition of IgE by cross-linking the low affinity IgE receptor (CD23) decreased the levels of {epsilon} mRNA and resulted in a distinct pattern of {epsilon} mRNA characterized by a dramatic decrease in CH4-M2{prime} splice variant. IL-6, IL-2, or IFN-{gamma} did not change the {epsilon} mRNA pattern. Overall, the absolute and relative amounts of the different {epsilon} mRNA splice variants produced appear to be controlled in a differentiation-related fashion.

  3. Echinococcus multilocularis proliferation in mice and respective parasite 14-3-3 gene expression is mainly controlled by an αβ+ CD4+ T-cell-mediated immune response

    PubMed Central

    Dai, Wen Juan; Waldvogel, Andreas; Siles-Lucas, Mar; Gottstein, Bruno

    2004-01-01

    The role of specific B lymphocytes and T-cell populations in the control of experimental Echinococus multilocularis infection was studied in µMT, nude, T-cell receptor (TCR)-β–/–, major histocompatibility complex (MHC)-I–/– and MHC-II–/– mice. At 2 months postinfection, the parasite mass was more than 10 times higher in nude, TCR-β–/– and MHC-II–/– mice than in infected C57BL/6 wild-type (WT) mice, and these T-cell-deficient mice started to die of the high parasite load at this time-point. In contrast, MHC-I–/– and µMT mice exhibited parasite growth rates similar to those found in WT controls. These findings clearly point to the major role that CD4+ αβ+ T cells play in limiting the E. multilocularis proliferation, while CD8+ T and B cells appeared to play a minor role in the control of parasite growth. In the absence of T cells, especially CD4+ or αβ+ T cells, the cellular immune response to infection was impaired, as documented by the lack of hepatic granuloma formation around the parasite and by a decreased splenocyte responsiveness to concanavalin A (Con A) and parasite antigen stimulation. Surprisingly, in T-cell-deficient mice, the ex vivo expression of interferon-γ (IFN-γ) and other inflammatory cytokines (except for interleukin-6) were increased in association with a high parasite load. Thus, the relative protection mediated by CD4+ αβ+ T cells against E. multilocularis infection seemed not be IFN-γ dependent, but rather to rely on the effector's function of CD4+ αβ+ T cells. The local restriction of parasite germinal cell proliferation was reflected by a regulatory effect on the expression of 14-3-3 protein within the parasite tissue in T-cell-deficient mice. These results provide a strong indication that the CD4+ αβ+ T-cell-mediated immune response contributes to the control of the parasite growth and to the regulation of production of the parasite 14-3-3 protein in metacestode tissues. PMID:15196217

  4. ErbB2, FoxM1 and 14-3-3ζ prime breast cancer cells for invasion in response to ionizing radiation

    PubMed Central

    Kambach, DM; Sodi, VL; Lelkes, PI; Azizkhan-Clifford, J; Reginato, MJ

    2014-01-01

    ErbB2 is frequently highly expressed in premalignant breast cancers, including ductal carcinoma in situ (DCIS); however, little is known about the signals or pathways it contributes to progression into the invasive/malignant state. Radiotherapy is often used to treat early premalignant lesions regardless of ErbB2 status. Here, we show that clinically relevant doses of ionizing radiation (IR)-induce cellular invasion of ErbB2-expressing breast cancer cells, as well as MCF10A cells overexpressing ErbB2. ErbB2-negative breast cancer cells, such as MCF7 and T47D, do not invade following treatment with IR nor do MCF10A cells overexpressing epidermal growth factor receptor. ErbB2 becomes phosphorylated at tyrosine 877 in a dose- and time- dependent manner following exposure to X-rays, and activates downstream signaling cascades including PI3K/Akt. Inhibition of these pathways, as well as inhibition of reactive oxygen species (ROS) with antioxidants, prevents IR-induced invasion. Activation of ErbB2-dependent signaling results in upregulation of the forkhead family transcription factor, FoxM1, and its transcriptional targets, including matrix metalloproteinase 2 (MMP2). Inhibition of FoxM1 by RNA interference prevented induction of invasion by IR, and overexpression of FoxM1 in MCF10A cells was sufficient to promote IR-induced invasion. Moreover, we found that 14-3-3ζ is also upregulated by IR in cancer cells in a ROS-dependent manner, is required for IR-induced invasion in ErbB2-positive breast cancer cells and together with FoxM1 is sufficient for invasion in ErbB2-negative breast cancer cells. Thus, our data show that IR-mediated activation of ErbB2 and induction of 14-3-3ζ collaborate to regulate FoxM1 and promote invasion of breast cancer cells and furthermore, may serve as therapeutic targets to enhance radiosensitivity of breast cancers. PMID:23318431

  5. Binding of epsilon-toxin from Clostridium perfringens in the nervous system.

    PubMed

    Dorca-Arévalo, Jonatan; Soler-Jover, Alex; Gibert, Maryse; Popoff, Michel R; Martín-Satué, Mireia; Blasi, Juan

    2008-09-18

    Epsilon-toxin (epsilon-toxin), produced by Clostridium perfringens type D, is the main agent responsible for enterotoxaemia in livestock. Neurological disorders are a characteristic of the onset of toxin poisoning. Epsilon-Toxin accumulates specifically in the central nervous system, where it produces a glutamatergic-mediated excitotoxic effect. However, no detailed study of putative binding structures in the nervous tissue has been carried out to date. Here we attempt to identify specific acceptor moieties and cell targets for epsilon-toxin, not only in the mouse nervous system but also in the brains of sheep and cattle. An epsilon-toxin-GFP fusion protein was produced and used to incubate brain sections, which were then analyzed by confocal microscopy. The results clearly show specific binding of epsilon-toxin to myelin structures. epsilon-Prototoxin-GFP and epsilon-toxin-GFP, the inactive and active forms of the toxin, respectively, showed identical results. By means of pronase E treatment, we found that the binding was mainly associated to a protein component of the myelin. Myelinated peripheral nerve fibres were also stained by epsilon-toxin. Moreover, the binding to myelin was not only restricted to rodents, but was also found in humans, sheep and cattle. Curiously, in the brains of both sheep and cattle, the toxin strongly stained the vascular endothelium, a result that may explain the differences in potency and effect between species. Although the binding of epsilon-toxin to myelin does not directly explain its neurotoxic effect, this feature opens up a new line of enquiry into its mechanism of toxicity and establishes the usefulness of this toxin for the study of the mammalian nervous system. PMID:18406080

  6. The EPSILON-2 hybrid dataflow architecture

    SciTech Connect

    Grafe, V.G.; Hoch, J.E.

    1989-11-08

    EPSILON-2 is a general parallel computer architecture that combines the fine grain parallelism of dataflow computing with the sequential efficiency common to von Neumann computing. Instruction level synchronization, single cycle context switches, and RISC-like sequential efficiency are all supported in EPSILON-2. The general parallel computing model of EPSILON-2 is described, followed by a description of the processing element architecture. A sample code is presented in detail, and the progress of the physical implementation discussed. 11 refs., 14 figs.

  7. Antibacterial functionalization of wool via mTGase-catalyzed grafting of epsilon-poly-L-lysine.

    PubMed

    Wang, Qiang; Jin, Guibiao; Fan, Xuerong; Zhao, Xianfei; Cui, Li; Wang, Ping

    2010-04-01

    epsilon-Poly-L-lysine (epsilon-PL), a natural biomacromolecule having a broad spectrum of antibacterial activity, was grafted on the wool fiber via the acyl transfer reaction catalyzed by microbial transglutaminase (mTGase) to develop a new strategy for antibacterial functionalization of proteinous materials. The effects of the concentrations of epsilon-PLs and mTGases on the graft yields were investigated. A coating of epsilon-PL that almost completely covered the scale profile on the wool surface was visualized by scanning electron microscopy (SEM) and further demonstrated in terms of Allwörden's reaction characteristic of wool. Identifiable differences in lysine content and color depth among the stained wool samples reveal the changes in the surface composition and polarity caused by the incorporation of epsilon-PL onto the wool substrate, respectively. The ratio of bacteriostasis to Escherichia coli of the wool fabric grafting epsilon-PL reached 96.6 %, indicating an excellent antibacterial effect. The application of epsilon-PL and corresponding mTGase-catalyzed grafting reaction would provide a worthwhile reference for antibacterial functionalization of proteinous materials in various forms. PMID:19649747

  8. Motility in the epsilon-proteobacteria.

    PubMed

    Beeby, Morgan

    2015-12-01

    The epsilon-proteobacteria are a widespread group of flagellated bacteria frequently associated with either animal digestive tracts or hydrothermal vents, with well-studied examples in the human pathogens of Helicobacter and Campylobacter genera. Flagellated motility is important to both pathogens and hydrothermal vent members, and a number of curious differences between the epsilon-proteobacterial and enteric bacterial motility paradigms make them worthy of further study. The epsilon-proteobacteria have evolved to swim at high speed and through viscous media that immobilize enterics, a phenotype that may be accounted for by the molecular architecture of the unusually large epsilon-proteobacterial flagellar motor. This review summarizes what is known about epsilon-proteobacterial motility and focuses on a number of recent discoveries that rationalize the differences with enteric flagellar motility. PMID:26590774

  9. The search for companions to Epsilon Eridani.

    PubMed

    Lawton, A T; Wright, P

    1990-12-01

    The authors review efforts to examine the star Epsilon Eridani and determine the possibility for the existence of an Earth-like planet. Early data indicated that there must be a habitable ecosphere about 82.5 million Km from the primary. Research into the existence of another planetary system determined that Epsilon Eridani was a binary star with an Oort cloud system, indicating the possibility of planet formation. A review of the evidence suggests that the presence of the small red Dwarf companion star precludes the existence of a planetary system surrounding Epsilon Eridani. It is suggested that observations continue to provide further data about the formation of binary systems. PMID:11540498

  10. Systematic effects of the quenched approximation on the strong penguin contribution to epsilon-prime / epsilon

    SciTech Connect

    Aubin, C.; Christ, N.H.; Dawson, C.; Laiho, J.W.; Noaki, J.; Li, S.; Soni, A.; /Brookhaven

    2006-03-01

    We discuss the implementation and properties of the quenched approximation in the calculation of the left-right, strong penguin contributions (i.e. Q{sub 6}) to {epsilon}{prime}/{epsilon}. The coefficient of the new chiral logarithm, discovered by Golterman and Pallante, which appears at leading order in quenched chiral perturbation theory is evaluated using both the method proposed by those authors and by an improved approach which is free of power divergent corrections. The result implies a large quenching artifact in the contribution of Q{sub 6} to {epsilon}{prime}/{epsilon}. This failure of the quenched approximation affects only the strong penguin operators and so does not affect the Q8 contribution to {epsilon}{prime}/{epsilon} nor ReA{sub 0}, ReAP{sub 2} and thus, the {Delta}I = 1/2 rule at tree level in chiral perturbation theory.

  11. Systematic effects of the quenched approximation on the strong penguin contribution to {epsilon}{sup '}/{epsilon}

    SciTech Connect

    Aubin, C.; Christ, N. H.; Li, S.; Dawson, C.; Noaki, J.; Laiho, J. W.; Soni, A.

    2006-08-01

    We discuss the implementation and properties of the quenched approximation in the calculation of the left-right, strong penguin contributions (i.e. Q{sub 6}) to {epsilon}{sup '}/{epsilon}. The coefficient of the new chiral logarithm, discovered by Golterman and Pallante, which appears at leading order in quenched chiral perturbation theory is evaluated using both the method proposed by those authors and by an improved approach which is free of power divergent corrections. The result implies a large quenching artifact in the contribution of Q{sub 6} to {epsilon}{sup '}/{epsilon}. This failure of the quenched approximation affects only the strong penguin operators and so does not affect the Q{sub 8} contribution to {epsilon}{sup '}/{epsilon} nor ReA{sub 0}, ReA{sub 2} and thus, the {delta}I=1/2 rule at tree level in chiral perturbation theory.

  12. Epsilon Aurigae Eclipse 2009 - Ingress

    NASA Astrophysics Data System (ADS)

    Hopkins, Jeffrey L.; Stencel, Robert E.; Leadbeater, Robin; Beckmann, Paul J.; Buil, Christian; Collins, Donald; Colombo, Tiziano; Garrel, Thierry; Gorodenski, Stanley; Gudmundsson, Snaevarr; Karlsson, Mukund Kurtadikar; Lindberg, Hans-Goran; Loughney, Des; Mauclaire, Benji; McCandless, Brian E.; Melillo, Frank J.; Miles, Richard; Pearson, Robert T.; Samolyk, Gerard; Schanne, Lothar; Strikis, Iakovos Marios; Teyssier, François; Thizy, Olivier

    The mysterious star system epsilon Aurigae undergoes an eclipse every 27.1 years that lasts nearly two years. The most recent eclipse started during the late summer of 2009. An international campaign for observing this eclipse was created in 2006, with a web site for information and, to-date, 17 periodic newsletters for details, as well as a Yahoo forum List for immediate announcements and comments. Photometric data in the UBVRIJH bands have been submitted. Ingress occurred with first contact in the V band estimated at the second week of 2009 August and second contact estimated at 2010 mid-January. Spectroscopic data were also obtained during ingress. Spectroscopic data have been provided in the potassium I region, hydrogen alpha and beta regions and sodium D line region of the star system's spectrum. In this paper we describe details of observations and preliminary analysis during ingress and second contact. We introduce the observers and discuss plans for observing throughout totality and the end of the eclipse in 2011.

  13. Combinatorial H3K9acS10ph histone modification in IgH locus S regions targets 14-3-3 adaptors and AID to specify antibody class-switch DNA recombination.

    PubMed

    Li, Guideng; White, Clayton A; Lam, Tonika; Pone, Egest J; Tran, Daniel C; Hayama, Ken L; Zan, Hong; Xu, Zhenming; Casali, Paolo

    2013-11-14

    Class-switch DNA recombination (CSR) is central to the antibody response, in that it changes the immunoglobulin heavy chain (IgH) constant region, thereby diversifying biological effector functions of antibodies. The activation-induced cytidine deaminase (AID)-centered CSR machinery excises and rejoins DNA between an upstream (donor) and a downstream (acceptor) S region, which precede the respective constant region DNA. AID is stabilized on S regions by 14-3-3 adaptors. These adaptors display a high affinity for 5'-AGCT-3' repeats, which recur in all S regions. However, how 14-3-3, AID, and the CSR machinery target exclusively the donor and acceptor S regions is poorly understood. Here, we show that histone methyltransferases and acetyltransferases are induced by CD40 or Toll-like receptor signaling and catalyze H3K4me3 and H3K9ac/K14ac histone modifications, which are enriched in S regions but do not specify the S region targets of CSR. By contrast, the combinatorial H3K9acS10ph modification specifically marks the S regions set to recombine and directly recruits 14-3-3 adaptors for AID stabilization there. Inhibition of the enzymatic activity of GCN5 and PCAF histone acetyltransferases reduces H3K9acS10ph in S regions, 14-3-3 and AID stabilization, and CSR. Thus, H3K9acS10ph is a histone code that is "written" specifically in S regions and is "read" by 14-3-3 adaptors to target AID for CSR as an important biological outcome. PMID:24209747

  14. Regulation of the expression of human C[epsilon] germline transcript

    SciTech Connect

    Ichiki, T.; Takahashi, W.; Watanabe, T. )

    1993-06-15

    Transcriptional regulation for Ig H chain germline transcripts induced by cytokines is a topic of recent interest for the understanding of the mechanism of class switch recombination. Among human B cell lines examined, the authors have found that a human IgM-producing B cell line, DND39 (EBV negative) expressed germ-line transcripts of [epsilon] constant gene (C[epsilon]) when stimulated with lL-4. In this study, the regulatory element responsible for the expression of lL-4-induced human C[epsilon] germ-line transcript was determined using DND39 cells. To identify the lL-4 responsive promotor/enhancer element, deletion analysis of the upstream region of the germ-line exon (l[epsilon]) of the C[epsilon] germ-line transcript which is located 5' to the switch region, was performed by using a luciferase gene as a reporter. Deletion analysis showed that a DNA fragment which lies between [minus]215 and [minus]154 bp upstream from the most 3' transcriptional initiation site of human l[epsilon] gene is fully responsible for the induction of germ-line transcripts by IL-4. According to a mutational analysis, the DNA fragment between [minus]163 and [minus]152 bp was identified to be a novel IL-4 responsive element in a human C[epsilon] gene. Electrophoretic gel mobility shift assay showed the presence of IL-4-induced nuclear factor that specifically bound to this IL-4 responsive element. This novel IL-4 responsive element and an IL-4-induced DNA binding protein may play an important role for the induction of C[epsilon] germ-line transcript as well as class switching to IgE. 54 refs., 7 figs.

  15. A novel, myeloid transcription factor, C/EBP epsilon, is upregulated during granulocytic, but not monocytic, differentiation.

    PubMed

    Morosetti, R; Park, D J; Chumakov, A M; Grillier, I; Shiohara, M; Gombart, A F; Nakamaki, T; Weinberg, K; Koeffler, H P

    1997-10-01

    Human C/EBP epsilon is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBP epsilon, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBP epsilon mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBP epsilon was the only C/EBP family member that was easily detected by RT-PCR. No C/EBP epsilon mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBP epsilon. Northern blot and RT-PCR analyses showed that C/EBP epsilon mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBP epsilon protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBP epsilon protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38-), purified from humans had very weak expression of C/EBP epsilon mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher

  16. Potency against enterotoxemia of a recombinant Clostridium perfringens type D epsilon toxoid in ruminants.

    PubMed

    Lobato, Francisco C F; Lima, Catarina G R D; Assis, Ronnie A; Pires, Prhiscylla S; Silva, Rodrigo O S; Salvarani, Felipe M; Carmo, Anderson O; Contigli, Christiane; Kalapothakis, Evanguedes

    2010-08-31

    Enterotoxemia, a disease that affects domestic ruminants, is caused mainly by the epsilon toxin from Clostridium perfringens type D. Its eradication is virtually impossible, control and prophylaxis are based on systematic vaccination of herds with epsilon toxoids that are efficient in inducing protective antibody production. The use of recombinant toxins is one of the most promising of these strategies. This work evaluates the potency of a Cl. perfringens type D epsilon toxoid expressed by Escherichia coli administered to goats, sheep, and cattle. The etx gene was cloned into the pET-11a plasmid of E. coli strain BL21 to produce the recombinant toxin. Rabbits (n=8), goats, sheep, and cattle (n=5 for each species) were immunized with 0.2mg of the insoluble recombinant protein fraction to evaluate vaccine potency of the epsilon toxoid studied. Antibody titers were 40, 14.3, 26, and 13.1 IU/mL in the rabbit, goat, sheep, and cattle serum pools, respectively. The epsilon toxoid produced and tested in this work is adequate for immunization of ruminants against enterotoxemia. PMID:20670910

  17. Cloning of the novel human myeloid-cell-specific C/EBP-epsilon transcription factor.

    PubMed Central

    Chumakov, A M; Grillier, I; Chumakova, E; Chih, D; Slater, J; Koeffler, H P

    1997-01-01

    Chicken NF-M transcription factor, in cooperation with either c-Myb or v-Myb, is active in the combinatorial activation of myeloid-cell-specific genes in heterologous cell types, such as embryonic fibroblasts. In humans, similar effects were observed with homologous members of the CCAAT/enhancer-binding protein (C/EBP) family of transcriptional regulators, especially the human homolog of chicken NF-M, C/EBP-beta (NF-IL6). However, the NF-IL6 gene is expressed in a variety of nonmyeloid cell types and is strongly inducible in response to inflammatory stimuli, making it an unlikely candidate to have an exclusive role as a combinatorial differentiation switch during myelopoiesis in human cells. By using a reverse transcription-PCR-based approach and a set of primers specific for the DNA-binding domains of highly homologous members of the C/EBP family of transcriptional regulators, we have cloned a novel human gene encoding a member of the C/EBP gene family, identified as the human homolog of CRP1, C/EBP-epsilon. A 1.2-kb cDNA encoding full-length human C/EBP-epsilon was cloned from a promyelocyte-late myeloblast-derived lambda gt11 library. Molecular analysis of the cDNA and genomic clones indicated the presence of two exons encoding a protein with an apparent molecular mass of 32 kDa and a pI of 9.5. Primer extension analysis of C/EBP-epsilon mRNA detected a single major transcription start site approximately 200 bp upstream of the start codon. The putative promoter area is similar to those of several other myeloid-cell-specific genes in that it contains no TATAAA box but has a number of purine-rich stretches with multiple sites for the factors of the Ets family of transcriptional regulators. Northern blot analyses indicated a highly restricted mRNA expression pattern, with the strongest expression occurring in promyelocyte and late-myeloblast-like cell lines. Western blot and immunoprecipitation studies using rabbit anti-C/EBP-epsilon antibodies raised against the N

  18. Interaction of 5-aza-2'-deoxycytidine and depsipeptide on antineoplastic activity and activation of 14-3-3sigma, E-cadherin and tissue inhibitor of metalloproteinase 3 expression in human breast carcinoma cells.

    PubMed

    Gagnon, Jacynthe; Shaker, Sepideh; Primeau, Mélanie; Hurtubise, Annie; Momparler, Richard L

    2003-03-01

    Genes that suppress tumorigenesis can be silenced by epigenetic events, such as aberrant DNA methylation and modification of chromatin structure. Inhibitors of DNA methylase and histone deacetylase (HDAC) can potentially reverse these events. The aim of this study was to determine the in vitro antineoplastic activity of 5-aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylase, in combination with depsipeptide (depsi), an inhibitor of HDAC, on human breast carcinoma cells. We observed a synergistic antineoplastic interaction between 5-AZA-CdR and depsi in their capacity to inhibit colony formation of Hs578T and MCF-7 breast carcinoma cells. In order to understand the molecular mechanism of this interaction, we investigated the effect of these drugs on the activation of the 14-3-3sigma, E-cadherin and tissue inhibitor of metalloproteinase 3 (TIMP3) cancer-related genes, which were reported to be silenced by aberrant methylation in many breast tumor cell lines. 14-3-3sigma was reported to produce G cell cycle arrest following DNA damage. E-cadherin and TIMP3 function as suppressors of tumor metastasis. Semi-quantitative RT-PCR was used to determine the effect of the co-administration of 5-AZA-CdR and depsi on four breast carcinoma cell lines for the reactivation of these genes. We observed a synergistic activation of E-cadherin by the combination in Hs578T, MDA-MB-231 and MDA-MB-435 tumor cells. For 14-3-3sigma, we demonstrated an additive to synergistic activation by the combination for Hs578T and MDA-MB-435 tumor cells, respectively. In the MCF-7 tumor cells, the drug combination produced a synergistic activation of TIMP3. The association between the synergistic antineoplastic activity and the synergistic activation of the target genes in this study suggests that the mechanism of anticancer activity of 5-AZA-CdR, in combination with depsi, is probably related to their enhanced activation of different types of tumor suppressor genes that have been

  19. epsilon3epsilon4 genotype as risk factor of myocardial infarction in middle-aged people in Spain.

    PubMed

    Garcés, Carmen; Maicas, Carolina; Grande, Rosario; Benavente, Mercedes; Viturro, Enrique; Cano, Beatriz; López, Dolores; de Oya, Manuel

    2005-01-01

    Apolipoprotein E (apoE) plays an important role in lipid metabolism. Its epsilon4 allele has been consistently associated with lipoprotein disorders but its connection to myocardial infarction (MI) is controversial. Because epsilon4 frequency decreases with age we thought that the contradictory results in different studies could be due to the wide age range of the subjects included. To test our hypothesis, ApoE genotyping was performed in 474 MI cases and an analysis was performed by percentiles of age. The frequencies of epsilon3epsilon4 genotype and epsilon4 allele in the MI group as a whole (subjects aged 31 to 92) were not significantly different from those in our area general population. However, significant differences were observed when comparing by group of age. The frequencies decreased as age increased. The epsilon3epsilon4 and epsilon4 frequencies were significantly higher in MI subjects aged 31 to 56 than in subjects over 74. The epsilon3epsilon4 genotype prevalence in an age and sex matched control group of subjects aged 31 to 56 was significantly lower than in the 31-56 year-old MI group. In conclusion, our data shows different epsilon3epsilon4 and epsilon4 frequencies depending on the age range of the subjects with MI, being significantly higher in the middle-aged group. This finding may help explain the discrepancies between studies analyzing association between apoE genotype and MI, and emphasizes the idea of considering apoE genotype for prevention at early age. PMID:16276010

  20. Clostridium perfringens Epsilon Toxin: A Malevolent Molecule for Animals and Man?

    PubMed Central

    Stiles, Bradley G.; Barth, Gillian; Barth, Holger; Popoff, Michel R.

    2013-01-01

    Clostridium perfringens is a prolific, toxin-producing anaerobe causing multiple diseases in humans and animals. One of these toxins is epsilon, a 33 kDa protein produced by Clostridium perfringens (types B and D) that induces fatal enteric disease of goats, sheep and cattle. Epsilon toxin (Etx) belongs to the aerolysin-like toxin family. It contains three distinct domains, is proteolytically-activated and forms oligomeric pores on cell surfaces via a lipid raft-associated protein(s). Vaccination controls Etx-induced disease in the field. However, therapeutic measures are currently lacking. This review initially introduces C. perfringens toxins, subsequently focusing upon the Etx and its biochemistry, disease characteristics in various animals that include laboratory models (in vitro and in vivo), and finally control mechanisms (vaccines and therapeutics). PMID:24284826

  1. Epsilon Aur monitoring during predicted pulsation phase

    NASA Astrophysics Data System (ADS)

    Waagen, Elizabeth O.; Templeton, Matthew R.

    2014-09-01

    Dr. Robert Stencel (University of Denver Astronomy Program) has requested that AAVSO observers monitor epsilon Aurigae from now through the end of the observing season. "Studies of the long-term, out-of-eclipse photometry of this enigmatic binary suggest that intervals of coherent pulsation occur at roughly 1/3 of the 27.1-year orbital period. Kloppenborg, et al. noted that stable variation patterns develop at 3,200-day intervals' implying that 'the next span of dates when such events might happen are circa JD ~2457000 (2014 December)'. "These out-of-eclipse light variations often have amplitudes of ~0.1 magnitude in U, and ~0.05 in V, with characteristic timescales of 60-100 days. The AAVSO light curve data to the present may indicate that this coherent phenomenon has begun, but we encourage renewed efforts by observers...to help deduce whether these events are internal to the F star, or externally-driven by tidal interaction with the companion star." Nightly observations or one observation every few days (CCD/PEP/DSLR, VUBR (amplitude too small for visual)) are requested. Finder charts with sequence may be created using the AAVSO Variable Star Plotter (http://www.aavso.org/vsp). Observations should be submitted to the AAVSO International Database. Epsilon Aur was the subject of major international campaigns and the AAVSO's Citizen Sky project as it went through its 27.1-year eclipse in 2009-2011. Over 700 observers worldwide submitted over 20,000 multicolor observations to the AAVSO International Database for this project. Much information on eps Aur is available from the AAVSO, including material on the Citizen Sky website (http://www.aavso.org/epsilon-aurigae and http://www.citizensky.org/content/star-our-project). The Journal of the AAVSO, Volume 40, No. 2 (2012) was devoted to discussion of and research results from this event. See full Alert Notice for more details and observations.

  2. Classical closure theory and Lam's interpretation of epsilon-RNG

    NASA Technical Reports Server (NTRS)

    Zhou, YE

    1995-01-01

    Lam's phenomenological epsilon-renormalization group (RNG) model is quite different from the other members of that group. It does not make use of the correspondence principle and the epsilon-expansion procedure. We demonstrate that Lam's epsilon-RNG model is essentially the physical space version of the classical closure theory in spectral space and consider the corresponding treatment of the eddy viscosity and energy backscatter.

  3. New atmospheric model of Epsilon Eridani

    NASA Astrophysics Data System (ADS)

    Vieytes, Mariela; Fontenla, Juan; Buccino, Andrea; Mauas, Pablo

    2016-05-01

    We present a new semi-empirical model of the atmosphere of the widely studied K-dwarf Epsilon Eridani (HD 22049). The model is build to reproduce the visible spectral observations from 3800 to 6800 Angstrom and the h and k Mg II lines profiles. The computations were carried out using the Solar-Stellar Radiation Physical Modeling (SSRPM) tools, which calculate non-LTE population for the most important species in the stellar atmosphere. We show a comparison between the synthetic and observed spectrum, obtaining a good agreement in all the studied spectral range.

  4. Revealing the Hot Side of Epsilon Aurigae

    NASA Astrophysics Data System (ADS)

    Hoard, Donald; Stencel, Robert; Howell, Steve

    2012-12-01

    We request a small investment of 24 minutes of Spitzer time, to obtain four IRAC observations of epsilon Aurigae. A naked eye object located near Capella, epsilon Aurigae is the eclipsing binary star with the longest known orbital period, showing a single long duration (~2 yr) eclipse every 27.1 yr. For much of the last 200 years, the nature of the eclipsing object defied explanation. We recently demonstrated that epsilon Aurigae consists of a high luminosity F0 post-AGB star in orbit with a B5 V star surrounded by a solar system sized (~8 AU diameter) disk of cool, dust-dominated material. The eclipse of epsilon Aurigae is a rare event; moreover, it is a unique astrophysical opportunity, since the backlighting of the disk by the high luminosity eclipsed star reveals details that cannot be detected in similar dusty disks around single stars. The current eclipse started in August 2009 and ended in July 2011; we are now in the post-eclipse phase, when the irradiation-heated side of the disk will begin rotating into view. The goals for these observations include: (1) extend our ongoing IRAC monitoring campaign covering the current eclipse to post-eclipse visits; (2) provide a consistent, well-calibrated space-based set of IR photometry for comparison with ongoing ground-based work; and (3) use the composite results to constrain the thermal profile of the disk. A key expectation of these particular observations is to reveal the irradiation-heated portion of the disk, which will be visible on its trailing side following eclipse. Observations of this side of the disk will be crucial to test and constrain new models of disk structure. As part of our overall monitoring campaign with Spitzer, Hubble, Herschel, and numerous ground-based facilities, these proposed observations will make an important contribution to the understanding of stellar evolution in binary stars, including mass transfer and evolution studies, along with new insights into astrophysical disks and post

  5. Epsilon Aurigae at the End of Eclipse

    NASA Astrophysics Data System (ADS)

    Hoard, Donald; Stencel, R.; Howell, S.

    2011-05-01

    We request a small investment of 24 minutes of Spitzer time, to obtain four IRAC observations of epsilon Aurigae. A naked eye object located near Capella, epsilon Aurigae is the eclipsing binary star with the longest known orbital period, showing a single long duration (~2 yr) eclipse every 27.1 yr. For much of the last 150 years, the nature of the eclipsing object defied explanation. We recently demonstrated that epsilon Aurigae consists of a high luminosity F0 post-AGB star in orbit with a B5 V star surrounded by a solar system sized (~8 AU diameter) disk of cool, dust-dominated material. The eclipse of epsilon Aurigae is a rare event; moreover, it is a unique astrophysical opportunity, since the backlighting of the disk by the high luminosity eclipsed star reveals details that cannot be detected in similar dusty disks around single stars. The current eclipse started in August 2009 and is expected to reach its photometric conclusion in May 2011 (with the spectroscopic conclusion as late as December 2011). The goals for these observations include: (1) extend our ongoing IRAC monitoring campaign covering the current eclipse to late-phase and post-eclipse visits; (2) provide a consistent, well-calibrated space-based set of IR photometry for comparison with ongoing ground-based work; and (3) use the composite results to constrain the thermal profile of the disk. A key expectation of these particular observations is to reveal the irradiation-heated portion of the disk, which will be visible on its trailing side following eclipse. Observations of this side of the disk will be crucial to test and constrain new models of disk structure. As part of our overall monitoring campaign with Spitzer, Hubble, Herschel, and numerous ground-based facilities, these proposed observations will make an important contribution to the understanding of stellar evolution in binary stars, including mass transfer and evolution studies, along with new insights into astrophysical disks and post

  6. The Final Measurement of Epsilon'/Epsilon from KTeV

    SciTech Connect

    Worcester, E.T.

    2009-10-01

    The authors present precise measurements of CP and CPT symmetry based on the full dataset of K {yields} {pi}{pi} decays collected by the KTeV experiment at Fermi National Accelerator Laboratory during 1996, 1997, and 1999. This dataset contains about 15 million K {yields} {pi}{sup 0}{pi}{sup 0} and 70 million K {yields} {pi}{sup +}{pi}{sup -} decays. They measure the direct CP violation parameter Re({epsilon}'/{epsilon}) = (19.2 {+-} 2.1) x 10{sup -4}. they find the K{sub L}-K{sub S} mass difference {Delta}m = (5265 {+-} 10) x 10{sup 6} {bar h}s{sup -1} and the K{sub S} lifetime {tau}{sub S} = (89.62 {+-} 0.05) x 10{sup -12} s. They test CPT symmetry by finding the phase of the indirect CP violation parameter {epsilon}, {phi}{sub {epsilon}} = (44.09 {+-} 1.00){sup o}, and the difference of the relative phases between the CP violating and CP conserving decay amplitudes for K {yields} {pi}{sup +}{pi}{sup -} ({phi}{sub +-}) and for K {yields} {pi}{sup 0}{pi}{sup 0} ({phi}{sub 00}), {Delta}{phi} = (0.29 {+-} 0.31){sup o}. these results are consistent with other experimental results and with CPT symmetry.

  7. Distribution of type I Fc epsilon-receptors on the surface of mast cells probed by fluorescence resonance energy transfer.

    PubMed Central

    Kubitscheck, U; Schweitzer-Stenner, R; Arndt-Jovin, D J; Jovin, T M; Pecht, I

    1993-01-01

    The aggregation state of type I Fc epsilon-receptors (Fc epsilon RI) on the surface of single living mast cells was investigated by resonance fluorescence energy transfer. Derivatization of Fc epsilon RI specific ligands, i.e., immunoglobulin E or Fab fragments of a Fc epsilon RI specific monoclonal antibody, with donor and acceptor fluorophores provided a means for measuring receptor clustering through energy transfer between the receptor probes. The efficiency of energy transfer between the ligands carrying distinct fluorophores was determined on single cells in a microscope by analyzing the photobleaching kinetics of the donor fluorophore in the presence and absence of receptor ligands labeled with acceptor fluorophores. To rationalize the energy transfer data, we developed a theoretical model describing the dependence of the energy transfer efficiency on the geometry of the fluorescently labeled macromolecular ligands and their aggregation state on the cell surface. To this end, the transfer process was numerically calculated first for one pair and then for an ensemble of Fc epsilon RI bound ligands on the cell surface. The model stipulates that the aggregation state of the Fc epsilon RI is governed by an attractive lipid-protein mediated interaction potential. The corresponding pair-distribution function characterizes the spatial distribution of the ensemble. Using this approach, the energy transfer efficiency of the ensemble was calculated for different degrees of receptor aggregation. Comparison of the theoretical modeling results with the experimental energy transfer data clearly suggests that the Fc epsilon RI are monovalent, randomly distributed plasma membrane proteins. The method provides a novel approach for determining the aggregation state of cell surface components. PMID:8431535

  8. Epsilon Metal Summary Report FY 2011

    SciTech Connect

    Strachan, Denis M.; Crum, Jarrod V.; Zumhoff, Mac R.; Bovaird, Chase C.; Windisch, Charles F.; Riley, Brian J.

    2011-09-30

    The Epsilon-metal ({var_epsilon}-metal) phase was selected in FY 2009 as a potential waste form to for immobilizing the noble metals found in the undissolved solids + aqueous stream, and the soluble Tc from ion-exchange process, each resulting from proposed aqueous reprocessing. {var_epsilon}-metal phase is observed in used nuclear fuel and the natural reactors of Oklobono in Gabon, where the long-term corrosion behavior was demonstrated. This makes {var_epsilon}-metal a very attractive waste form. Last fiscal year, {var_epsilon}-metal was successfully fabricated by combining the five-metals, Mo, Ru, Rh, Pd and Re (surrogate for Tc), into pellets followed by consolidation with an arc melter. The arc melter produced fully dense samples with the epsilon structure. However, some chemistry differences were observed in the microstructure that resulted in regions rich in Re and Mo, and others rich in Pd, while Ru and Rh remained fairly constant throughout. This year, thermal stability (air), and corrosion testing of the samples fabricated by arc melting were the main focus for experimental work. Thermal stability was measured with a differential scanning calorimeter - thermogravimetric analyzer, by both ramp heating as well as step heating. There is clear evidence during the ramp heating experiment of an exothermic event + a weight loss peak both beginning at {approx}700 C. Step heating showed an oxidation event at {approx}690 C with minimal weight gain that occurs just before the weight loss event at 700 C. The conclusion being that the e-metal begins to oxidize and then become volatile. These findings are useful for considering the effects of voloxidation process. Three different pellets were subjected to electrochemical testing to study the corrosion behavior of the epsilon-metal phase in various conditions, namely acidic, basic, saline, and inert. Test was done according to an interim procedure developed for the alloy metal waste form. First an open circuit potential

  9. Nesprin-2 epsilon: A novel nesprin isoform expressed in human ovary and Ntera-2 cells

    SciTech Connect

    Lam, Le Thanh; Boehm, Sabrina V.; Roberts, Roland G.; Morris, Glenn E.

    2011-08-26

    Highlights: {yields} A novel epsilon isoform of nesprin-2 has been discovered. {yields} This 120 kDa protein was predicted by bioinformatic analysis, but has not previously been observed. {yields} It is the main isoform expressed in a teratocarcinoma cell line and is also found in ovary. {yields} Like other nesprins, it is located at the nuclear envelope. {yields} We suggest it may have a role in very early development or in some ovary-specific function. -- Abstract: The nuclear envelope-associated cytoskeletal protein, nesprin-2, is encoded by a large gene containing several internal promoters that produce shorter isoforms. In a study of Ntera-2 teratocarcinoma cells, a novel isoform, nesprin-2-epsilon, was found to be the major mRNA and protein product of the nesprin-2 gene. Its existence was predicted by bioinformatic analysis, but this is the first direct demonstration of both the mRNA and the 120 kDa protein which is located at the nuclear envelope. In a panel of 21 adult and foetal human tissues, the nesprin-2-epsilon mRNA was strongly expressed in ovary but was a minor isoform elsewhere. The expression pattern suggests a possible link with very early development and a likely physiological role in ovary.

  10. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site.

    PubMed

    Wongsantichon, Jantana; Robinson, Robert C; Ketterman, Albert J

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  11. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site

    PubMed Central

    Wongsantichon, Jantana; Robinson, Robert C.; Ketterman, Albert J.

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  12. Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice.

    PubMed

    Losada-Eaton, D M; Fernandez-Miyakawa, M E

    2010-12-01

    Epsilon toxin produced by Clostridium perfringens type B and D is a potent toxin that is responsible for a highly fatal enterotoxemia in sheep and goats. In vitro, epsilon toxin produces contraction of the rat ileum as the result of an indirect action, presumably mediated through the autonomic nervous system. To examine the impact of epsilon toxin in the intestinal transit, gastric emptying (GE) and gastrointestinal transit (GIT) were evaluated after intravenous and oral administration of epsilon toxin in mice. Orally administered epsilon toxin produced a delay on the GIT. Inhibition of the small intestinal transit was observed as early as 1 h after the toxin was administered orally but the effects were not observed after 1 week. Epsilon toxin also produced an inhibition in GE and a delay on the GIT when relatively high toxin concentrations were given intravenously. These results indicate that epsilon toxin administered orally or intravenously to mice transitorily inhibits the GIT. The delay in the GIT induced by epsilon toxin could be relevant in the pathogenesis of C. perfringens type B and D enterotoxemia. PMID:20434186

  13. Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1

    PubMed Central

    Ivie, Susan E.; McClain, Mark S.

    2012-01-01

    Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1. PMID:22938730

  14. Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1.

    PubMed

    Ivie, Susan E; McClain, Mark S

    2012-09-25

    Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1. PMID:22938730

  15. T*{sub {epsilon}} integral analysis of fracture specimens

    SciTech Connect

    Omori, Y.; Ma, L.; Kobayashi, A.S.

    1996-12-31

    T*{sub {epsilon}} integral values associated with stable crack growth in thin 2024-T3 aluminum compact (CT) specimens and A606 HSLA steel single edge notched (SEN) specimens were determined directly from the crack tip displacement field obtained by moire interferometry. Stable crack growth in the SEN specimen was also simulated by an elastic-plastic finite element (FE) model which was driven by the experimentally determined boundary conditions. T*{sub {epsilon}} obtained experimentally and by FE were in reasonable agreements with each other. Unlike the vanishing J integrals with crack extension, T*{sub {epsilon}} reached steady state values with stable crack growth. Thus, for a given integration contour, {Gamma}{sub {epsilon}}, near the crack tip, T*{sub {epsilon}} can be used as a stable crack growth as well as a ductile fracture criteria.

  16. Fyn kinase controls Fc{epsilon}RI receptor-operated calcium entry necessary for full degranulation in mast cells

    SciTech Connect

    Sanchez-Miranda, Elizabeth; Ibarra-Sanchez, Alfredo; Gonzalez-Espinosa, Claudia

    2010-01-22

    IgE-antigen-dependent crosslinking of the high affinity IgE receptor (Fc{epsilon}RI) on mast cells leads to degranulation, leukotriene synthesis and cytokine production. Calcium (Ca{sup 2+}) mobilization is a sine qua non requisite for degranulation, allowing the rapid secretion of stored pro-inflammatory mediators responsible for allergy symptoms. Fyn is a Src-family kinase that positively controls Fc{epsilon}RI-induced mast cell degranulation. However, our understanding of the mechanism connecting Fyn activation to secretion of pre-synthesized mediators is very limited. We analyzed Fc{epsilon}RI-dependent Ca{sup 2+} mobilization in bone marrow-derived mast cells (BMMCs) differentiated from WT and Fyn -/- knock out mice. Fyn -/- BMMCs showed a marked defect in extracellular Ca{sup 2+} influx after Fc{epsilon}RI crosslinking but not after thapsigargin addition. High concentrations of Gadolinium (Gd{sup 3+}) partially blocked Fc{epsilon}RI-induced Ca{sup 2+} influx in WT cells but, in contrast, completely inhibited Ca{sup 2+} mobilization in Fyn -/- cells. Low concentrations of an inhibitor of the canonical transient receptor potential (TRPC) Ca{sup 2+} channels (2-aminoethoxyphenyl-borane, 2-APB) blocked Fc{epsilon}RI-induced maximal Ca{sup 2+} rise in WT but not in Fyn -/- cells. Ca{sup 2+} entry through Fyn-controlled, 2-APB sensitive channels was found to be important for full degranulation and IL-2 mRNA accumulation in WT cells. Immunoprecipitation assays showed that Fyn kinase interacts with TRPC 3/6/7 channels after IgE-antigen stimulation, but its association is not related to protein tyrosine phosphorylation. Results indicate Fyn kinase mediates the receptor-dependent activation of TRPC channels that contribute to degranulation in Fc{epsilon}RI-stimulated mast cells.

  17. The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site.

    PubMed

    Kanno, Takeshi; Yamamoto, Hideyuki; Yaguchi, Takahiro; Hi, Rika; Mukasa, Takeshi; Fujikawa, Hirokazu; Nagata, Tetsu; Yamamoto, Satoshi; Tanaka, Akito; Nishizaki, Tomoyuki

    2006-06-01

    This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation. PMID:16520488

  18. Modelling Epsilon Aurigae without solid particles

    NASA Technical Reports Server (NTRS)

    Cheng, A. Y. S.; Woolf, N. J.

    1985-01-01

    Three components can be expected to contribute to the emission of epsilon Aurigae. There is a primary F star. There is an opaque disk which occults it, and there is a gas stream which is observed to produce absorption lines. Evidence that the disk is not responsible for the gas stream lines comes both from the radial velocities, which are too small, and from the IR energy distribution out of eclipse, which shows free-free emission that would produce inadequate optical depth in electron scattering. The color temperature of the IR excess can give misleading indications of low temperature material. Free-free emission at 10,000 K between 10 and 20 microns has a color temperature of 350 K. Attempts to mold the system are discussed.

  19. Modeling the Variable Polarization of Epsilon Aurigae

    NASA Astrophysics Data System (ADS)

    Ignace, Richard; Henson, Gary D.; Asbury, William

    2016-06-01

    The nature of the edge-on eclipsing binary Epsilon Aurigae remains perplexing, despite notable progress since the recent 2009-2011 eclipse. The binary involves an early F supergiant with a still unknown companion enshrouded in a disk. Although the eclipse geometry produces a significant broad band polarization signature, semiregular pulsations of the F supergiant are also a source of variable polarization, with an amplitude that is commensurate with the effect of the eclipse. This fact makes use of the polarization for studying the disk of the companion far more challenging. In an effort to better understand the pulsation nature of the supergiant, we explore a simple model for the stellar contribution to the polarization signal. The model does reasonably well in characterizing the gross properties of the time-variable polarization.

  20. Perturbative matching of the staggered four-fermion operators for {epsilon}'/{epsilon}

    SciTech Connect

    Lee, Weonjong

    2001-09-01

    Using staggered fermions, we calculate the perturbative corrections to the bilinear and four-fermion operators that are used in the numerical study of weak matrix elements for {epsilon}'/{epsilon}. We present results for one-loop matching coefficients between continuum operators, calculated in the naive dimensional regularization (NDR) scheme, and gauge invariant staggered fermion operators. In particular, we concentrate on Feynman diagrams of the current-current insertion type. We also present results for the tadpole improved operators. These results, combined with existing results for penguin diagrams, provide a complete one-loop renormalization of the staggered four-fermion operators. Therefore, using our results, it is possible to match a lattice calculation of K{sup 0}-{bar K}{sup 0} mixing and K{yields}{pi}{pi} decays to the continuum NDR results with all corrections of O(g{sup 2}) included.

  1. Brain lesions associated with clostridium perfringens type D epsilon toxin in a Holstein heifer calf.

    PubMed

    Mete, A; Garcia, J; Ortega, J; Lane, M; Scholes, S; Uzal, F A

    2013-09-01

    A 6-month-old dairy heifer calf with no premonitory signs was acutely down after the morning feeding and could not rise. On presentation, the heifer was in right lateral recumbency and moribund with opisthotonus and left hind limb paddling. Following euthanasia, gross examination of the brain revealed multifocal loss of gray-white matter distinction and extensive petechiae throughout the brainstem. On histopathological examination, there was striking white matter edema and marked perivascular proteinaceous edema surrounding many arterioles and venules (microangiopathy), mainly in the white matter of the internal capsule, thalamus, midbrain, cerebellum, and cerebellar peduncles. The perivascular neuropil was strongly positive for Alzheimer precursor protein A4. Clostridium perfringens epsilon toxin was detected in the intestinal contents. This is the first report of microangiopathy in postneonatal cattle associated with the detection of epsilon toxin in the intestinal contents. PMID:23381925

  2. Assays for the measurement of tissue transglutaminase (type II) mediated protein crosslinking via epsilon-(gamma-glutamyl) lysine and N',N'-bis (gamma-glutamyl) polyamine linkages using biotin labelled casein.

    PubMed

    Lilley, G R; Griffin, M; Bonner, P L

    1997-02-01

    Two colorimetric assays for tissue transglutaminase (type II) activity involving the crosslinking of proteins have been developed. In one assay, biotin labelled casein is crosslinked into chemically modified casein bound to a microtiter plate by tissue transglutaminase and the biotin labelled reaction product is detected by conjugation to Extravidin peroxidase. The assay can detect activity in 10 ng of commercially available purified guinea pig liver transglutaminase and in the crude homogenate derived from 400 human endothelial cells (cell line ECV 304). A correlation (r2 = 0.977) was shown between this assay and the radiolabeled putrescine incorporation assay for the detection of transglutaminase activity. This assay measures the protein crosslinking activity of tissue transglutaminase as opposed to polyamine incorporation and offers a rapid, non-radiometric method for screening large sample numbers. Typical inter-assay variability is 13.9 +/- 1.5% (n = 8). In a second assay, the ability of tissue transglutaminase to catalyze the formation of N',N'-bis (gamma-glutamyl) polyamine bridges is measured. N',N'-dimethylcasein is bound to a microtiter plate and modified enzymatically using commercially available purified guinea pig liver transglutaminase to incorporate polyamines into glutamine residues. Biotin labelled casein is then crosslinked into the immobilized polyamines by tissue transglutaminase resulting in the formation of N',N'-bis (gamma-glutamyl) polyamine linkages. PMID:9089382

  3. VARIABILITY IN OPTICAL SPECTRA OF {epsilon} ORIONIS

    SciTech Connect

    Thompson, Gregory B.; Morrison, Nancy D. E-mail: nmorris@utnet.utoledo.edu

    2013-04-15

    We present the results of a time series analysis of 130 echelle spectra of {epsilon} Ori (B0 Ia), acquired over seven observing seasons between 1998 and 2006 at Ritter Observatory. The equivalent widths of H{alpha} (net) and He I {lambda}5876 were measured and radial velocities were obtained from the central absorption of He I {lambda}5876. Temporal variance spectra (TVS) revealed significant wind variability in both H{alpha} and He I {lambda}5876. The He I TVS have a double-peaked profile consistent with radial velocity oscillations. A periodicity search was carried out on the equivalent width and radial velocity data, as well as on wavelength-binned spectra. This analysis has revealed several periods in the variability with timescales of two to seven days. Many of these periods exhibit sinusoidal modulation in the associated phase diagrams. Several of these periods were present in both H{alpha} and He I, indicating a possible connection between the wind and the photosphere. Due to the harmonic nature of these periods, stellar pulsations may be the origin of some of the observed variability. Periods on the order of the rotational period were also detected in the He I line in the 1998-1999 season and in both lines during the 2004-2005 season. These periods may indicate rotational modulation due to structure in the wind.

  4. EPSILON AURIGAE: AN IMPROVED SPECTROSCOPIC ORBITAL SOLUTION

    SciTech Connect

    Stefanik, Robert P.; Torres, Guillermo; Lovegrove, Justin; Latham, David W.; Zajac, Joseph; Pera, Vivian E.; Mazeh, Tsevi

    2010-03-15

    A rare eclipse of the mysterious object {epsilon} Aurigae will occur in 2009-2011. We report an updated single-lined spectroscopic solution for the orbit of the primary star based on 20 years of monitoring at the CfA, combined with historical velocity observations dating back to 1897. There are 518 new CfA observations obtained between 1989 and 2009. Two solutions are presented. One uses the velocities outside the eclipse phases together with mid-times of previous eclipses, from photometry dating back to 1842, which provide the strongest constraint on the ephemeris. This yields a period of 9896.0 {+-} 1.6 days (27.0938 {+-} 0.0044 years) with a velocity semi-amplitude of 13.84 {+-} 0.23 km s{sup -1} and an eccentricity of 0.227 {+-} 0.011. The middle of the current ongoing eclipse predicted by this combined fit is JD 2,455,413.8 {+-} 4.8, corresponding to 2010 August 5. If we use only the radial velocities, we find that the predicted middle of the current eclipse is nine months earlier. This would imply that the gravitating companion is not the same as the eclipsing object. Alternatively, the purely spectroscopic solution may be biased by perturbations in the velocities due to the short-period oscillations of the supergiant.

  5. Interleukin-4 production by Fc epsilon R+ cells.

    PubMed

    Paul, W E

    1991-01-01

    Among non-B, non-T cells in the spleen and among unfractionated bone marrow cells, there is a population of cells that are capable of producing IL-4 in response to cross-linkage of FC epsilon RI or Fc gamma RII. Their IL-4-producing capacity is strikingly enhanced by treatment of the cells or of the animals donating such cells with interleukin-3 (IL-3). Fc epsilon R+ cells constitute 1-2% of splenic non-B, non-T cells and of bone marrow cells from normal donors but they contain all the capacity to produce IL-4 in response to cross-linkage of Fc epsilon RI or Fc gamma RII or to treatment with ionomycin. Fc epsilon R- cells fail to make such responses. Electron-microscopic analysis indicates that virtually all the granulated or vacuolated Fc epsilon R+ cells are of the basophil lineage. However, it has not yet been resolved whether these cells or immature mast cells, presumably in the Fc epsilon R+ granule/vacuole-negative cell population, are the principal producers of IL-4 in response to these stimuli. PMID:1837220

  6. Naturally acquired antibodies against Clostridium perfringens epsilon toxin in goats.

    PubMed

    Veschi, Josir Laine A; Bruzzone, Octavio A; Losada-Eaton, Daniela M; Dutra, Iveraldo S; Fernandez-Miyakawa, Mariano E

    2008-09-15

    Clostridium perfringens type D-producing epsilon toxin is a common cause of death in sheep and goats worldwide. Although anti-epsilon toxin serum antibodies have been detected in healthy non-vaccinated sheep, the information regarding naturally acquired antibodies in ruminants is scanty. The objective of the present report was to characterize the development of naturally acquired antibodies against C. perfringens epsilon toxin in goats. The levels of anti-epsilon toxin antibodies in blood serum of goat kids from two different herds were examined continuously for 14 months. Goats were not vaccinated against any clostridial disease and received heterologous colostrums from cows that were not vaccinated against any clostridial disease. During the survey one of these flocks suffered an unexpectedly severe C. perfringens type D enterotoxemia outbreak. The results showed that natural acquired antibodies against C. perfringens epsilon toxin can appear as early as 6 weeks in young goats and increase with the age without evidence of clinical disease. The enterotoxemia outbreak was coincident with a significant increase in the level of anti-epsilon toxin antibodies. PMID:18538416

  7. INFRARED STUDIES OF EPSILON AURIGAE IN ECLIPSE

    SciTech Connect

    Stencel, Robert E.; Kloppenborg, Brian K.; Wall, Randall E.; Hopkins, Jeffrey L.; Howell, Steve B.; Hoard, D. W.; Rayner, John; Bus, Schelte; Tokunaga, Alan; Sitko, Michael L.; Bradford, Suellen; Russell, Ray W.; Lynch, David K.; Hammel, Heidi; Whitney, Barbara; Orton, Glenn; Yanamandra-Fisher, Padma; Hora, Joseph L.; Hinz, Philip; Hoffmann, William; and others

    2011-11-15

    We report here on a series of medium resolution spectro-photometric observations of the enigmatic long period eclipsing binary epsilon Aurigae, during its eclipse interval of 2009-2011, using near-infrared spectra obtained with SpeX on the Infrared Telescope Facility (IRTF), mid-infrared spectra obtained with BASS on AOES and IRTF, MIRSI on IRTF, and MIRAC4 on the MMT, along with mid-infrared photometry using MIRSI on IRTF and MIRAC4 on the MMT, plus 1995-2000 timeframe published photometry and data obtained with Denver's TNTCAM2 at WIRO. The goals of these observations included: (1) comparing eclipse depths with prior eclipse data, (2) confirming the re-appearance of CO absorption bands at and after mid-eclipse, associated with sublimation in the disk, (3) seeking evidence for any mid-infrared solid state spectral features from particles in the disk, and (4) providing evidence that the externally irradiated disk has azimuthal temperature differences. IR eclipse depths appear similar to those observed during the most recent (1983) eclipse, although evidence for post-mid-eclipse disk temperature increase is present, due to F star heated portions of the disk coming into view. Molecular CO absorption returned 57 days after nominal mid-eclipse, but was not detected at mid-eclipse plus 34 days, narrowing the association with differentially heated sub-regions in the disk. Transient He I 10830A absorption was detected at mid-eclipse, persisting for at least 90 days thereafter, providing a diagnostic for the hot central region. The lack of solid-state features in Spitzer Infrared Spectrograph, BASS, and MIRAC spectra to date suggests the dominance of large particles (micron-sized) in the disk. Based on these observations, mid-infrared studies out of eclipse can directly monitor and map the disk thermal changes, and better constrain disk opacity and thermal conductivity.

  8. Enhanced PKC beta II translocation and PKC beta II-RACK1 interactions in PKC epsilon-induced heart failure: a role for RACK1.

    PubMed

    Pass, J M; Gao, J; Jones, W K; Wead, W B; Wu, X; Zhang, J; Baines, C P; Bolli, R; Zheng, Y T; Joshua, I G; Ping, P

    2001-12-01

    Recent investigations have established a role for the beta II-isoform of protein kinase C (PKC beta II) in the induction of cardiac hypertrophy and failure. Although receptors for activated C kinase (RACKs) have been shown to direct PKC signal transduction, the mechanism through which RACK1, a selective PKC beta II RACK, participates in PKC beta II-mediated cardiac hypertrophy and failure remains undefined. We have previously reported that PKC epsilon activation modulates the expression of RACKs, and that altered epsilon-isoform of PKC (PKC epsilon)-RACK interactions may facilitate the genesis of cardiac phenotypes in mice. Here, we present evidence that high levels of PKC epsilon activity are commensurate with impaired left ventricular function (dP/dt = 6,074 +/- 248 mmHg/s in control vs. 3,784 +/- 269 mmHg/s in transgenic) and significant myocardial hypertrophy. More importantly, we demonstrate that high levels of PKC epsilon activation induce a significant colocalization of PKC beta II with RACK1 (154 +/- 7% of control) and a marked redistribution of PKC beta II to the particulate fraction (17 +/- 2% of total PKC beta II in control mice vs. 49 +/- 5% of total PKC beta II in hypertrophied mice), without compensatory changes of the other eight PKC isoforms present in the mouse heart. This enhanced PKC beta II activation is coupled with increased RACK1 expression and PKC beta II-RACK1 interactions, demonstrating PKC epsilon-induced PKC beta II signaling via a RACK1-dependent mechanism. Taken together with our previous findings regarding enhanced RACK1 expression and PKC epsilon-RACK1 interactions in the setting of cardiac hypertrophy and failure, these results suggest that RACK1 serves as a nexus for at least two isoforms of PKC, the epsilon-isoform and the beta II-isoform, thus coordinating PKC-mediated hypertrophic signaling. PMID:11709417

  9. Differential Protein Expression in Honeybee (Apis mellifera L.) Larvae: Underlying Caste Differentiation

    PubMed Central

    Li, Jianke; Wu, Jing; Begna Rundassa, Desalegn; Song, Feifei; Zheng, Aijuan; Fang, Yu

    2010-01-01

    Honeybee (Apis mellifera) exhibits divisions in both morphology and reproduction. The queen is larger in size and fully developed sexually, while the worker bees are smaller in size and nearly infertile. To better understand the specific time and underlying molecular mechanisms of caste differentiation, the proteomic profiles of larvae intended to grow into queen and worker castes were compared at 72 and 120 hours using two dimensional electrophoresis (2-DE), network, enrichment and quantitative PCR analysis. There were significant differences in protein expression between the two larvae castes at 72 and 120 hours, suggesting the queen and the worker larvae have already decided their fate before 72 hours. Specifically, at 72 hours, queen intended larvae over-expressed transketolase, aldehyde reductase, and enolase proteins which are involved in carbohydrate metabolism and energy production, imaginal disc growth factor 4 which is a developmental related protein, long-chain-fatty-acid CoA ligase and proteasome subunit alpha type 5 which metabolize fatty and amino acids, while worker intended larvae over-expressed ATP synthase beta subunit, aldehyde dehydrogenase, thioredoxin peroxidase 1 and peroxiredoxin 2540, lethal (2) 37 and 14-3-3 protein epsilon, fatty acid binding protein, and translational controlled tumor protein. This differential protein expression between the two caste intended larvae was more pronounced at 120 hours, with particular significant differences in proteins associated with carbohydrate metabolism and energy production. Functional enrichment analysis suggests that carbohydrate metabolism and energy production and anti-oxidation proteins play major roles in the formation of caste divergence. The constructed network and validated gene expression identified target proteins for further functional study. This new finding is in contrast to the existing notion that 72 hour old larvae has bipotential and can develop into either queen or worker based on

  10. Prediction of two-dimensional momentumless wake by k- epsilon - gamma model

    NASA Astrophysics Data System (ADS)

    Ahn, Jong Woo; Sung, Hyung Jin

    1995-04-01

    The k-epsilon-gamma model performance is extended to resolve the jet/wake anomaly, which is encountered in calculating free shear flows by the standard k-epsilon model. In the k-epsilon-gamma model, the relation between the rate of dissipation epsilon and the level of intermittency gamma is well incorporated. The model is run to predict the two-dimensional momentumless wake where a jet and a wake coexist. The predictions of the k-epsilon-gamma model are compared with those of the k-epsilon model as well as the experiment. It is shown that the model performance is generally satisfactory.

  11. Epsilon-near-zero mode for active optoelectronic devices.

    PubMed

    Vassant, S; Archambault, A; Marquier, F; Pardo, F; Gennser, U; Cavanna, A; Pelouard, J L; Greffet, J J

    2012-12-01

    The electromagnetic modes of a GaAs quantum well between two AlGaAs barriers are studied. At the longitudinal optical phonon frequency, the system supports a phonon polariton mode confined in the thickness of the quantum well that we call epsilon-near-zero mode. This epsilon-near-zero mode can be resonantly excited through a grating resulting in a very large absorption localized in the single quantum well. We show that the reflectivity can be modulated by applying a voltage. This paves the way to a new class of active optoelectronic devices working in the midinfrared and far infrared at ambient temperature. PMID:23368264

  12. The Challenge of Observing the Recent Eclipse of Epsilon Aurigae

    NASA Astrophysics Data System (ADS)

    Melillo, Frank J.

    2013-07-01

    This author participated in the 'International Epsilon Aurigae Campaign' in 2009. A total of 100 V-band observations were made in Holtsville, New York for the 2009-2011 eclipse of Epsilon Aurigae. A lightcurve has been plotted using data from these observations, which cover the phase before, during and after the eclipse. The lightcurve shows precise timing during the first, second, third and fourth contacts and possibly mid-eclipse brightening. The magnitude and the duration of the eclipse in photometric V band are discussed. This poster represents the work by Frank J Melillo and the observations were close enough to generate the true shape of the lightcurve.

  13. An improved k-epsilon model for near wall turbulence

    NASA Technical Reports Server (NTRS)

    Shih, T. H.; Hsu, Andrew T.

    1991-01-01

    An improved k-epsilon model for low Reynolds number turbulence near a wall is presented. In the first part of this work, the near-wall asymptotic behavior of the eddy viscosity and the pressure transport term in the turbulent kinetic energy equation are analyzed. Based on these analyses, a modified eddy viscosity model with the correct near-wall behavior is suggested, and a model for the pressure transport term in the k-equation is proposed. In addition, a modeled dissipation rate equation is reformulated, and a boundary condition for the dissipation rate is suggested. In the second part of the work, one of the deficiencies of the existing k-epsilon models, namely, the wall distance dependency of the equations and the damping functions, is examined. An improved model that does not depend on any wall distance is introduced. Fully developed turbulent channel flows and turbulent boundary layers over a flat plate are studied as validations for the proposed new models. Numerical results obtained from the present and other previous k-epsilon models are compared with data from direct numerical simulation. The results show that the present k-epsilon model, with added robustness, performs as well as or better than other existing models in predicting the behavior of near-wall turbulence.

  14. Detection of Clostridium perfringens epsilon toxin by ELISA.

    PubMed

    Naylor, R D; Martin, P K; Sharpe, R T

    1987-03-01

    An enzyme-linked immunosorbent assay (ELISA) has been developed as an alternative to neutralisation tests in mice to detect Clostridium perfringens type D epsilon toxin in the intestinal contents of animals which have died from suspected enterotoxaemia. The test was sensitive and quantitative and gave excellent agreement with the mouse protection test. PMID:2884704

  15. Observations of Epsilon Lyrae by the Video Drift Method

    NASA Astrophysics Data System (ADS)

    Wasson, Rick; Nelson, Nancy; Nelson, Eric; Buehlman, William; Wilson, Earl; Zapata, Deanna

    2015-01-01

    The major components of the famous "double-double" star Epsilon Lyrae, STF2382AB and STF2383CD, were measured by the Video Team at the Apple Valley Double Star Workshop in 2013, using the Video Drift Method. The results are in reasonable agreement with other recent measures and predictions of the latest orbital solutions.

  16. Fate of the Epsilon Phase in the Oklo Natural Reactors

    SciTech Connect

    S. Utsunomiya; R.C. Ewing

    2005-03-31

    In spent nuclear fuel (SNF), the micron- to submicron-sized epsilon phase (Mo-Ru-Pd-Tc-Rh) is an important host of {sup 99}Tc which has a long half life (2.13 x 10{sup 5} years) and can be an important contributor to dose in safety assessments of nuclear waste repositories. In addition, Tc is predominantly present as TcO{sub 4}{sup -} under oxidizing conditions at wide range of pH, weakly adsorbed onto mineral surfaces, and unlikely to be incorporated into alteration uranyl minerals. In the Oklo natural reactor (2.0 Ga), essentially all of the {sup 99}Tc has decayed to {sup 99}Ru. Thus, this study focuses on Ru and the other metals of the epsilon phase in order to investigate the occurrence and the fate of the epsilon phase during the corrosion of this natural SNF. Samples from reactor zone (RZ)-10 (836, 819, 687); from RZ-13 (864, 910); were investigated using TEM (transmission electron microscopy). Within the UO{sub 2} matrix, a Bi-Pd particle (40-60 nm), fioodite, PdBi{sub 2}, was observed with trace amounts of As, Fe, and Te surrounded by an amorphous Pb-rich area. (Pd,Rh){sub 2}As, palladodymite or rhodarsenide, was observed (400-500 nm in size). Ruthenarsinite, (Ru,Ni)As, was identified in most samples: with a representative composition of As, 59.9: Co, 2.5: Ni, 5.2; Ru, 18.6; Rh, 8.4; Pd, 3.1; Sb, 2.4 in atomic percent. The particles diameters are a few hundred nanometers and, in most cases, surrounded by a Pb-rich phase (400-500 nm). Typically, the ruthenarsenite does not occur as single particle but an aggregate of {approx}200 nm-sized particles. Some Ru-particles revealed a complex phase separation within the grain such as a Ru-particle (600-700 nm) with Pb at the core of the particle and enrichment of Ni, Co, and As at the rim. Some ruthenarsenite crystals were embedded in chlorite immediately adjacent to uraninite. A few particles were still coated by Pb. These results suggest a history for the epsilon phases: (1) The original epsilon phase was

  17. Association of apolipoprotein E allele {epsilon}4 with late-onset sporadic Alzheimer`s disease

    SciTech Connect

    Lucotte, G.; David, F.; Berriche, S.

    1994-09-15

    Apolipoprotein E, type {epsilon}4 allele (ApoE {epsilon}4), is associated with late-onset sporadic Alzheimer`s disease (AD) in French patients. The association is highly significant (0.45 AD versus 0.12 controls for {epsilon}4 allele frequencies). These data support the involvement of ApoE {epsilon}4 allele as a very important risk factor for the clinical expression of AD. 22 refs., 1 fig., 3 tabs.

  18. A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

    PubMed Central

    Pigaglio, Emmanuelle; Durand, Nathalie; Meyer, Christian

    1999-01-01

    It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II. PMID:9880364

  19. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

    DOEpatents

    Houtz, Robert L.

    1999-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

  20. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

    DOEpatents

    Houtz, R.L.

    1999-02-02

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS){sup {epsilon}}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 8 figs.

  1. Crystal structure of [alpha]-COP in complex with [epsilon]-COP provides insight into the architecture of the COPI vesicular coat

    SciTech Connect

    Hsia, Kuo-Chiang; Hoelz, André

    2010-07-23

    The heptameric coatomer complex forms the protein shell of membrane-bound vesicles that are involved in transport from the Golgi to the endoplasmatic reticulum and in intraGolgi trafficking. The heptamer can be dissected into a heterotetrameric F-subcomplex, which displays similarities to the adapter complex of the 'inner' coat in clathrin-coated vesicles, and a heterotrimeric B-subcomplex, which is believed to form an 'outer' coat with a morphology distinct from that of clathrin-coated vesicles. We have determined the crystal structure of the complex between the C-terminal domain (CTD) of {alpha}-COP and full-length {epsilon}-COP, two components of the B-subcomplex, at a 2.9 {angstrom} resolution. The {alpha}-COP{sup CTD} {center_dot} {epsilon}-COP heterodimer forms a rod-shaped structure, in which {epsilon}-COP adopts a tetratricopeptide repeat (TPR) fold that deviates substantially from the canonical superhelical conformation. The {alpha}-COP CTD adopts a U-shaped architecture that complements the TPR fold of {epsilon}-COP. The {epsilon}-COP TPRs form a circular bracelet that wraps around a protruding {beta}-hairpin of the {alpha}-COP CTD, thus interlocking the two proteins. The {alpha}-COPCTD {center_dot} {epsilon}-COP complex forms heterodimers in solution, and we demonstrate biochemically that the heterodimer directly interacts with the Dsl1 tethering complex. These data suggest that the heterodimer is exposed on COPI vesicles, while the remaining part of the B-subcomplex oligomerizes underneath into a cage.

  2. How to measure and predict the molar absorption coefficient of a protein.

    PubMed Central

    Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T.

    1995-01-01

    The molar absorption coefficient, epsilon, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring epsilon for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-326] and is based on data from Edelhoch [1967, Biochemistry 6:1948-1954]). The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average epsilon values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the epsilon values in water, propanol, 6 M guanidine hydrochloride (GdnHCl), and 8 M urea have been measured. For Trp, the average epsilon values for the proteins are less than the epsilon values measured in any of the solvents. For Tyr, the average epsilon values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured epsilon values for 80 proteins, the epsilon at 280 nm of a folded protein in water, epsilon (280), can best be predicted with this equation: epsilon (280) (M-1 cm-1) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125) These epsilon (280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict epsilon. PMID:8563639

  3. Quarks with Twisted Boundary Conditions in the Epsilon Regime

    SciTech Connect

    Thomas Mehen; Brian C. Tiburzi

    2005-05-01

    We study the effects of twisted boundary conditions on the quark fields in the epsilon regime of chiral perturbation theory. We consider the SU(2){sub L} x SU(2){sub R} chiral theory with non-degenerate quarks and the SU(3){sub L} x SU(3){sub R} chiral theory with massless up and down quarks and massive strange quarks. The partition function and condensate are derived for each theory. Because flavor-neutral Goldstone bosons are unaffected by twisted boundary conditions chiral symmetry is still restored in finite volumes. The dependence of the condensate on the twisting parameters can be used to extract the pion decay constant from simulations in the epsilon regime. The relative contribution to the partition function from sectors of different topological charge is numerically insensitive to twisted boundary conditions.

  4. A k-epsilon modeling of near wall turbulence

    NASA Technical Reports Server (NTRS)

    Yang, Z.; Shih, T. H.

    1991-01-01

    A k-epsilon model is proposed for turbulent bounded flows. In this model, the turbulent velocity scale and turbulent time scale are used to define the eddy viscosity. The time scale is shown to be bounded from below by the Kolmogorov time scale. The dissipation equation is reformulated using the time scale, removing the need to introduce the pseudo-dissipation. A damping function is chosen such that the shear stress satisfies the near wall asymptotic behavior. The model constants used are the same as the model constants in the commonly used high turbulent Reynolds number k-epsilon model. Fully developed turbulent channel flows and turbulent boundary layer flows over a flat plate at various Reynolds numbers are used to validate the model. The model predictions were found to be in good agreement with the direct numerical simulation data.

  5. EISCAT observations during MAC/SINE and MAC/Epsilon

    NASA Technical Reports Server (NTRS)

    Roettger, J.; Hoppe, U.-P.; Hall, C.

    1989-01-01

    The EISCAT incoherent scatter radar facility in Tromsoe, Norway was operated during the MAC/SINE campaign for 78 hours in the period 10 June to 17 July 1987, and during the MAC/Epsilon campaign for 90 hours in the period 15 October to 5 November 1987. The VHF (224 MHz) radar operations during MAC/SINE yielded most interesting observations of strong coherent echoes from the mesopause region. Characteristic data of these polar mesospheric summer echoes are presented. The UHF (933 MHz) radar operations during MAC/Epsilon were done with 18 deg off zenith beam and allows the deduction of meridonal and horizontal wind components as well as radial velocity spectra in addition to the usual electron density profiles in the D and lower E regions. Some results from the VHF and UHF radars indicating the presence of gravity waves are examined.

  6. Structure of the Martian atmosphere from Epsilon Gem occultation observations

    NASA Technical Reports Server (NTRS)

    Hubbard, W. B.

    1982-01-01

    Information about Martian atmospheric scale heights derived from observations of the occultation of Epsilon Gem by Mars on April 8, 1976 has been collected. The observations give data in the altitude range of about 50 to 80 km. A rough, unweighted average of results so far available yields a temperatue of approximately 165 K. Excursions of about + or - 4 K about this mean may be present as a function of both altitude and areographic coordinates.

  7. Structure of the Martian atmosphere from Epsilon Gem occultation observations

    NASA Technical Reports Server (NTRS)

    Hubbard, W. B.

    1978-01-01

    Information has been collected on Martian atmospheric scale heights derived from observations of the occultation of Epsilon Gem by Mars on Apr. 8, 1976; the observations give data in the altitude range of approximately 50-80 km. A rough, unweighted average of results so far available yields a temperature of approximately 165 K. Excursions of plus or minus 40 K about this mean may be present as a function of both altitude and areographic coordinates.

  8. MAGNETIC ACTIVITY CYCLES IN THE EXOPLANET HOST STAR {epsilon} ERIDANI

    SciTech Connect

    Metcalfe, T. S.; Mathur, S.; Buccino, A. P.; Mauas, P. J. D.; Petrucci, R.; Brown, B. P.; Soderblom, D. R.; Henry, T. J.; Hall, J. C.; Basu, S.

    2013-02-01

    The active K2 dwarf {epsilon} Eri has been extensively characterized both as a young solar analog and more recently as an exoplanet host star. As one of the nearest and brightest stars in the sky, it provides an unparalleled opportunity to constrain stellar dynamo theory beyond the Sun. We confirm and document the 3-year magnetic activity cycle in {epsilon} Eri originally reported by Hatzes and coworkers, and we examine the archival data from previous observations spanning 45 years. The data show coexisting 3-year and 13-year periods leading into a broad activity minimum that resembles a Maunder minimum-like state, followed by the resurgence of a coherent 3-year cycle. The nearly continuous activity record suggests the simultaneous operation of two stellar dynamos with cycle periods of 2.95 {+-} 0.03 years and 12.7 {+-} 0.3 years, which, by analogy with the solar case, suggests a revised identification of the dynamo mechanisms that are responsible for the so-called 'active' and 'inactive' sequences as proposed by Boehm-Vitense. Finally, based on the observed properties of {epsilon} Eri, we argue that the rotational history of the Sun is what makes it an outlier in the context of magnetic cycles observed in other stars (as also suggested by its Li depletion), and that a Jovian-mass companion cannot be the universal explanation for the solar peculiarities.

  9. Advanced k-epsilon modeling of heat transfer

    NASA Technical Reports Server (NTRS)

    Kwon, Okey; Ames, Forrest E.

    1995-01-01

    This report describes two approaches to low Reynolds-number k-epsilon turbulence modeling which formulate the eddy viscosity on the wall-normal component of turbulence and a length scale. The wall-normal component of turbulence is computed via integration of the energy spectrum based on the local dissipation rate and is bounded by the isotropic condition. The models account for the anisotropy of the dissipation and the reduced mixing length due to the high strain rates present in the near-wall region. The turbulent kinetic energy and its dissipation rate were computed from the k and epsilon transport equations of Durbin. The models were tested for a wide range of turbulent flows and proved to be superior to other k-epsilon models, especially for nonequilibrium anisotropic flows. For the prediction of airfoil heat transfer, the models included a set of empirical correlations for predicting laminar-turbulent transition and laminar heat transfer augmentation due to the presence of freestream turbulence. The predictions of surface heat transfer were generally satisfactory.

  10. The ClpXP protease is responsible for the degradation of the Epsilon antidote to the Zeta toxin of the streptococcal pSM19035 plasmid.

    PubMed

    Brzozowska, Iwona; Zielenkiewicz, Urszula

    2014-03-14

    Most bacterial genomes contain different types of toxin-antitoxin (TA) systems. The ω-ε-ζ proteinaceous type II TA cassette from the streptococcal pSM19035 plasmid is a member of the ε/ζ family, which is commonly found in multiresistance plasmids and chromosomes of various human pathogens. Regulation of type II TA systems relies on the proteolysis of antitoxin proteins. Under normal conditions, the Epsilon antidote neutralizes the Zeta toxin through the formation of a tight complex. In this study, we show, using both in vivo and in vitro analyses, that the ClpXP protease is responsible for Epsilon antitoxin degradation. Using in vivo studies, we examined the stability of the plasmids with active or inactive ω-ε-ζ TA cassettes in B. subtilis mutants that were defective for different proteases. Using in vitro assays, the degradation of purified His6-Epsilon by the His6-LonBs, ClpPBs, and ClpXBs proteases from B. subtilis was analyzed. Additionally, we showed that purified Zeta toxin protects the Epsilon protein from rapid ClpXP-catalyzed degradation. PMID:24492616

  11. New prediction for the direct CP-violating parameter {var_epsilon}{prime}/{var_epsilon} and the {Delta}I=1/2 rule

    SciTech Connect

    Wu, Yue-Liang

    2001-07-01

    The low-energy dynamics of QCD is investigated with special attention paid to the matching between QCD and chiral perturbation theory (ChPT), and also to some useful algebraic chiral operator relations which survive even when we include chiral loop corrections. It then allows us to evaluate the hadronic matrix elements below the energy scale {Lambda}{sub {chi}}{approx_equal}1GeV. Based on the new analyses, we present a consistent prediction for both the direct CP-violating parameter {var_epsilon}{prime}/{var_epsilon} and the {Delta}I=1/2 rule in kaon decays. In the leading 1/N{sub c} approximation, the isospin amplitudes A{sub 0} and A{sub 2} are found to agree well with the data, and the direct CP-violating parameter {var_epsilon}{prime}/{var_epsilon} is predicted to be large, which also confirms our earlier conclusion. Its numerical value is {var_epsilon}{prime}/{var_epsilon}=23.6{sub {minus}7.8}{sup +12.4}{times}10{sup {minus}4}(Im{lambda}{sub t}/1.2{times}10{sup {minus}4}) which is no longer sensitive to the strange quark mass due to the matching conditions. Taking into account a simultaneous consistent analysis on the isospin amplitudes A{sub 0} and A{sub 2}, the ratio {var_epsilon}{prime}/{var_epsilon} is in favor of the values {var_epsilon}{prime}/{var_epsilon}=(20{+-}9){times}10{sup {minus}4}.

  12. {epsilon} expansion of the conductivity at the superconductor{endash}Mott-insulator transition

    SciTech Connect

    Fazio, R.; Zappala, D.

    1996-04-01

    We study the critical behavior of the conductivity {sigma}({omega}) at the zero-temperature superconductor{endash}Mott-insulator transition in {ital d} spacetime dimensions for a model of bosons with short-range interaction and no disorder. We obtain {sigma}({omega}{sub {ital n}})=(4{ital e}{sup 2}/{h_bar}){sigma}{sub {epsilon}}{omega}{sub {ital n}}{sup 1{minus}{epsilon}}, as predicted by the scaling theory, and the prefactor {sigma}{sub {epsilon}} is calculated in the {epsilon} expansion, to order {epsilon}{sup 2} ({epsilon}=4{minus}{ital d}). In two spatial dimensions ({ital d}=3), we find a value of the universal conductance {sigma}{sup {sq_bullet}}=0.315(4{ital e}{sup 2}/{ital h}), in good agreement with the known Monte Carlo results. {copyright} {ital 1996 The American Physical Society.}

  13. Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxgenase large subunit .epsilon. n-methyltransferase and method of inactivating ribulose-1,5-bishosphatase .epsilon. n-methyltransferase activity

    DOEpatents

    Houtz, Robert L.

    2001-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltansferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

  14. Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

    DOEpatents

    Houtz, Robert L.

    1999-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

  15. Epsilon Canis Majoris and the ionization of the local cloud

    NASA Technical Reports Server (NTRS)

    Vallerga, J. V.; Welsh, B. Y.

    1995-01-01

    The Lyman continuum radiation from the brightest extreme ultraviolet (EUV) source, the B2 II star epsilon Canis Majoris (Adara), is so intense that it dominates the local stellar EUV radiation field at wavelengths longer than 450 A and therefore sets a lower limit to the ionization of hydrogen in the Local Cloud. Using the EUV (70-730 A) spectrum of epsilon CMa taken with the Extreme Ultraviolet Explorer Satellite (EUVE) and simple models that extrapolate this spectrum to the Lyman edge at 912 A, we have determined the local interstellar hydrogen photionizatin parameter Gamma solely from epsilon CMa to be 1.1 x 10(exp -15)/s. This fiugre is a factor of 7 greater than previous estimates of Gamma calculated for all nearby stars combined (Bruhweiler & Cheng 1988). Using measured values of the density and temperature of neutral interstellar hydrogen gas in the Local Cloud, we derive a particle density of ionized hydrogen n(H(+)) and electrons n(sub e) of 0.015-0.019/cu cm assuming ionization equilibrium and a helium ionization fraction of less than 20%. These values correspond to a hydrogen ionizatin fraction, chi(sub H) from 19% to 15%, respectively. The range of these derived quantities is due to the uncertainties in the local values of the neutral hydrogen and helium interstellar densities derived from both (1) solar backscatter measurements of Ly alpha lines of hydrogen and helium (1216 and 584 A), and (2) the average neutral densities along the line of sight to nearby stars. The local proton density produced by epsilon CMa is enough to allow the ionization mechanism of Ripken & Fahr (1983) to work at the heliopause and explain the discrepancy between the neutral hydrogen density derived from solar backscatter measurements and line-of-sight averages to nearby stars. A large value of electron density in the Local Cloud of n(sub e) is approximately 0.3-0.7/cu cm (T = 7000 K) has recently been reported by Lallement et al. (1994) using observations of Mg II and Mg I

  16. The 1982-1984 Eclipse of Epsilon Aurigae

    NASA Technical Reports Server (NTRS)

    Stencel, R. E. (Editor)

    1985-01-01

    A workshop proceedings concerned with the new data collected during the 1982-1984 eclipse period of the 27-year system Epsilon Aurigae is presented. This binary star has been a classic problem in astrophysics because the opaque eclipsing object is nonstellar, and probably disk shaped. Invited papers concerning the history of the system, optical, infrared and ultraviolet photometry, optical polarimetry and ultraviolet spectroscopy are included. An invited paper concerning comprehensive theoretical interpretation in the context of stellar evolution also is included. The information collected herein is unparalleled in scope and will remain a standard reference until the next eclipse cycle in the year 2009 A.D., in all probability.

  17. Observations on the formation of [var epsilon] martensite in an Fe-23. 2%Mn alloy

    SciTech Connect

    Akguen, I.; Durlu, T.N. . Dept. of Physics)

    1994-11-15

    In Fe-Mn binary alloys the formation behavior of [var epsilon] martensite is quite sensitive to the Mn percentage and although both [var epsilon] and [alpha][prime] type martensites are formed in low Mn alloys, mostly [gamma] [yields] [var epsilon] transformation occur as the Mn concentration is increased. The present study was undertaken to examine the formation of thermally induced and also strain-induced [var epsilon] martensites, and their intersections in a Fe-23.2%Mn alloy by using transmission electron microscopy techniques.

  18. Structure of Epsilon15 Phage Reveals Organization of Genome and DNA Packaging/Injection Apparatus

    PubMed Central

    Jiang, Wen; Chang, Juan; Jakana, Joanita; Weigele, Peter; King, Jonathan; Chiu, Wah

    2006-01-01

    Using single particle electron cryomicroscopy that does not impose icosahedral averaging, we determined the structure of the entire infectious Salmonella phage Epsilon151, including both icosahedral and non-icosahedral components. At least three layers of condensed viral DNA were observed to pack in coaxial coils with local 25 Å hexagonal inter-strand spacing. At one of the five-fold vertices, a portal complex with twelve subunits replaces a capsid pentamer. A tail hub with six projecting trimeric tailspikes sits on the external face of the portal. Below the portal is a cylindrical protein core. An extended shaft of density fills the central channel of the protein core and the portal complex and appears to consist of about 90 nucleotides at the terminus of the packaged DNA poised for injection. Using an icosahedral symmetry imposed reconstruction, the fold of the capsid shell protein is seen to resemble the capsid protein fold of other tailed double-stranded DNA phages2–5 and human herpesvirus6. These common structural features suggest a common evolutionary origin among these viruses. PMID:16452981

  19. Upgrade of the time-of-flight diffractometer EPSILON

    NASA Astrophysics Data System (ADS)

    Ulyanov, V.; Walther, K.; Kuznetsov, I.; Frischbutter, A.; Medvedev, E.; Scheffzük, Ch.; Schebetov, A.

    At beam line 7A of the pulsed reactor IBR-2 (JINR Dubna) the strain diffractometer EPSILON has been working for several years. Due to a flight path of 102m a good resolution (Δd/d<3×10-3) was achieved. Nevertheless, further improvements of the instrument were required because of intended studies on geological samples. The modernisation of EPSILON is a renewal of the whole recording system, including collimators and detectors. The two previously used Soller collimators are replaced by nine radial collimators. Each collimator consists of 48 gadolinium oxide coated mylar foils with an angular distance of about 20' of arc to each other and therefore covers a range of nearly 16° for 2Θ. In the other direction a range of about 18° is covered. Three collimators in each case are grouped into a unit covering 66° in the plane perpendicular to the incident beam. Single 3He counter tubes with a diameter of 10mm and an active length of 120mm are used. Each collimator/detector block is mounted on a ring carrier with its surface perpendicular to the incident neutron beam. All blocks have the possibility of adjustment in three directions. The transparency of the collimators is about 90-95%.

  20. An Amateur's Spatial Model of the epsilon Aurigae System

    NASA Astrophysics Data System (ADS)

    Gemma, Marina

    This project has its origins in a summer program I attended in astrophysics (COSMOS) last year at the University of California, Irvine. The Intel Corporation offered a small number of 500 grants to students that submitted proposals for an independent research project that had to span the length of the next academic year. I received an initial exposure to astronomical data analysis in the COSMOS summer program, and was interested in expanding my experience with astronomy because I plan to major in astronomy or astrophysics in college. I had heard about the upcoming epsilon Aurigae eclipse, and decided that the eclipse was the perfect option for a research project. I ended up submitting a proposal, and was awarded the 500 grant. The original question I was attempting to answer was: Is it possible for an amateur astronomer to build a spatial model of the object that periodically eclipses the star epsilon Aurigae? One of the requirements of the grant was that the project be presented in the spring of 2010. This paper describes my efforts thus far.

  1. High Precision Polarimetry of the Epsilon Aurigae Eclipse

    NASA Astrophysics Data System (ADS)

    Wiktorowicz, Sloane

    2013-07-01

    Polarimetry of the epsilon Aurigae eclipse has the potential to discern the stellar latitude occulted by the companion's dusty disk, which may directly test interferometric results. In addition, the limb polarization of the primary star may be measured, which is an effect predicted by S. Chandrasekhar and verified by spatially resolved observations of the Sun. I will present B band, polarimetric observations of epsilon Aurigae taken over six nights in September and October 2009 using the POLISH high precision polarimeter at the Lick 3-m telescope. Polarimetric precision achieved during each night is of order 1 part in 10^5. Extensive post-eclipse observations have been taken with the significantly upgraded POLISH2 polarimeter at Lick Observatory. This instrument simultaneously measures all four Stokes parameters (I, Q, U, and V) and achieves precision within 2.0 times the photon shot noise limit over an entire observing run. This work is supported by a NExScI Sagan Fellowship, UC Lab Fees Research Grant, and UCO/Lick Observatory.

  2. Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide

    PubMed Central

    Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

    2015-01-01

    Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology. PMID:26452234

  3. Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

    PubMed

    Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

    2015-01-01

    Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology. PMID:26452234

  4. The strong isospin-breaking correction for the gluonic penguin contribution to {epsilon}{prime}/{epsilon} at next-to-leading order in the chiral expansion

    SciTech Connect

    Wolfe, Carl E.; Maltman, Kim

    2001-01-01

    The strong isospin-breaking correction {Omega}{sub st}, which appears in estimates of the standard model value for the direct CP-violating ratio {epsilon}{prime}/{epsilon}, is evaluated to next-to-leading order (NLO) in the chiral expansion using chiral perturbation theory. The relevant linear combinations of the unknown NLO CP-odd weak low-energy constants (LEC's) which, in combination with one-loop and strong LEC contributions, are required for a complete determination at this order, are estimated using two different models. It is found that, to NLO, {Omega}{sub st}=0.08{+-}0.05, significantly reduced from the ''standard'' value, 0.25{+-}0.08, employed in recent analyses. The potentially significant numerical impact of this decrease on standard model predictions for {epsilon}{prime}/{epsilon}, associated with the decreased cancellation between gluonic penguin and electroweak penguin contributions, is also discussed.

  5. Ultraviolet observations of cool stars. IV - Intensities of Lyman-alpha and Mg II in epsilon Pegasi and epsilon Eridani, and line width-luminosity correlations

    NASA Technical Reports Server (NTRS)

    Mcclintock, W.; Linsky, J. L.; Henry, R. C.; Moos, H. W.

    1975-01-01

    A spectrometer on the Copernicus satellite has been used to confirm the existence of a line width-luminosity relation for the Ly-alpha and Mg II 2800-A chromospheric emission lines in K-type stars by observation of a K2 dwarf (epsilon Eri) and a K2 supergiant (epsilon Peg). Combined with previously reported observations of lines in three K giants (alpha Boo, alpha Tau, and beta Gem), the data are consistent with an identical dependence of line width on absolute visual magnitude for the Ca II K, Ly-alpha, and Mg II 2795-A lines. Surface fluxes of Ly-alpha, Mg II 2800-A, and O V 1218-A (upper limit) for epsilon Eri, and of Mg II 2800-A for epsilon Peg are also compared with values reported previously for the three giant stars.

  6. Preferential emission into epsilon-near-zero metamaterial [Invited

    SciTech Connect

    Galfsky, Tal; Sun, Zheng; Jacob, Zubin; Menon, Vinod M.

    2015-11-23

    We report the use of epsilon near zero (ENZ) metamaterial to control spontaneous emission from Zinc-Oxide (ZnO) excitons. The ENZ material consists of alternating layers of silver and alumina with subwavelength thicknesses, resulting in an effective medium where one of the components of the dielectric constant approach zero between 370nm-440nm wavelength range. Bulk ZnO with photoluminescence maximum in the ENZ regime was deposited via atomic layer deposition to obtain a smooth film with near field coupling to the ENZ metamaterial. Preferential emission from the ZnO layer into the metamaterial with suppression of forward emission by 90% in comparison to ZnO on silicon is observed. We attribute this observation to the presence of dispersionless plasmonic modes in the ENZ regime as shown by the results of theoretical modeling presented here. Integration of ENZ metamaterials with light emitters is an attractive platform for realizing a low threshold subwavelength laser.

  7. Shock initiation of an {epsilon}-CL-20-estane formulation

    SciTech Connect

    Tarver, C.M.; Simpson, R.L.; Urtiew, P.A.

    1995-07-19

    The shock sensitivity of a pressed solid explosive formulation, LX-19, containing 95.2% by weight epsilon phase 2,4,6,8,10,12-hexanitrohexaazaisowurtzitane (HNIW) and 4.8% Estane binder, was determined using the wedge test and embedded manganin pressure gauge techniques. This formulation was shown to be slightly more sensitive than LX-14, which contains 95.5% HMX and 4.5% Estane binder. The measured pressure histories for LX-19 were very similar to those obtained using several HMX-inert binder formulations. An Ignition and Growth reactive model for LX-19 was developed which differed from those for HMX-inert binder formulations only by a 25% higher hot spot growth rate.

  8. Shock initiation of an {epsilon}-CL-20-estane formulation

    SciTech Connect

    Tarver, C.M.; Simpson, R.L.; Urtiew, P.A.

    1996-05-01

    The shock sensitivity of a pressed solid explosive formulation, LX-19, containing 95.2{percent} by weight epsilon phase 2,4,6,8,10,12-hexanitrohexaazaisowurtzitane (HNIW) and 4.8{percent} Estane binder, was determined using the wedge test and embedded manganin pressure gauge techniques. This formulation was shown to be slightly more sensitive than LX-14, which contains 95.5{percent} HMX and 4.5{percent} Estane binder. The measured pressure histories for LX-19 were very similar to those obtained using several HMX-inert binder formulations. An Ignition and Growth reactive flow model for LX-19 was developed which differed from those for HMX-inert binder formulations only by a 25{percent} higher hot spot growth rate. {copyright} {ital 1996 American Institute of Physics.}

  9. Data Evaluation for 56Co epsilon + beta+ Decay

    SciTech Connect

    Baglin, Coral M.; MacMahon, T. Desmond

    2005-02-28

    Recommended values for nuclear and atomic data pertaining to the {var_epsilon} + {beta}{sup +} decay of {sup 56}Co are provided here, followed by comments on evaluation procedures and a summary of all available experimental data. {sup 56}Co is a radionuclide which is potentially very useful for Ge detector efficiency calibration because it is readily produced via the {sup 56}Fe(p,n) reaction, its half-life of 77.24 days is conveniently long, and it provides a number of relatively strong {gamma} rays with energies up to {approx}3500 keV. The transition intensities recommended here for the strongest lines will be included in the forthcoming International Atomic Energy Agency Coordinated Research Programme document ''Update of X- and Gamma-ray Decay Data Standards for Detector Calibration and Other Applications'', and the analysis for all transitions along with relevant atomic data have been provided to the Decay Data Evaluation Project.

  10. K-epsilon models of the lower overshoot layer

    NASA Astrophysics Data System (ADS)

    Petrovay, K.

    Apart from the solar core, the overshoot layer below the convective zone is the layer where some discrepancies between the standard and seismic solar models continue to exist. Non-local mixing length models are in clear conflict with the seismic evidence. A major difficulty for the development of more sophisticated models is the large degree of arbitrariness in formulating expressions for input parameters like the length scale. We propose that the application of the k-epsilon modelling approach, widely used in physics and engineering, could help in alleviating these difficulties. We present some simple overshoot models calculated with this approach, and we point out that the resulting expression of the length scale naturally reduces to the usual mixing length expression (proportional to the pressure scale height) well inside the convectively unstable region. Implications for the solar Li problem and for dynamo theory will also be discussed.

  11. The 5' flanking region of human epsilon-globin gene.

    PubMed Central

    Baralle, F E; Shoulders, C C; Goodbourn, S; Jeffreys, A; Proudfoot, N J

    1980-01-01

    The structural analysis of the 2.0 kb region upstream from the epsilon-globin gene has been carried out. A genomic DNA map around the gene was worked out in some detail to ensure that the cloned DNA was representative of the actual chromosomal arrangement. Furthermore, a new technique was developed to precisely map a reiterated DNA sequence present 1.5 kb to the 5' side of the gene. The complete nucleotide sequence of the 2.0 kb 5' flanking region was then determined and overlapped with the gene. The sequence included the reiterated DNA sequence which is homologous to the so-called AluI family of repeats. Unusual stretches of sequence 50 nucleotides long, where A + T represent about 90% of the bases, are present at both the 5' and 3' sides of the repeat. Images PMID:6253916

  12. Scattered light in the IUE spectra of Epsilon Aurigae

    NASA Technical Reports Server (NTRS)

    Altner, B.; Chapman, R. D.; Kondo, Y.; Stencel, R. E.

    1986-01-01

    Recent infrared photometry indicates that the alleged disk of particulate matter surrounding the mysterious secondary object in the Epsilon Aur system is cold, around 500 K. IUE spectra, on the other hand, contain significant flux in excess of that expected from an F0 Ia star in the far UV, which if interpreted as a hot secondary star leads to a possible contradiction with the IR data. Other models of the UV excess have been proposed, including the idea that the bulk of the short-wavelength flux is light scattered into the SWP camera from longer wavelengths. With the recent availability of a detailed generalized IUE descattering algorithm it is possible to thoroughly investigate the scattered-light contribution to the short-wavelength continuum. It is found that the IUE spectra are indeed partially contaminated by scattered light, but that even after correction for this instrumental effect a significant time-dependent UV excess is still present.

  13. The mid-ultraviolet spectrum of Epsilon Aurigae

    NASA Technical Reports Server (NTRS)

    Castelli, F.; Hoekstra, R.; Kondo, Y.

    1982-01-01

    An LTE synthetic spectrum with Te = 7800 K and log g = 1 has been computed for the 2455-to-3230 A range of Epsilon Aurigae (F0 1a). This has been compared with the spectra observed with 0.2 A resolution with the IUE experiment and with 0.1 A resolution with the BUSS experiment. As a consequence of this comparison, identifications and abundance values have been obtained. Ions definitely present include Mg I, Mg II, Al I, Al II, Si I, Ca II, Ti II, V II, Cr II, Mn II, Fe I, Fe II, Co II, Ni I, Ni II, Zr II. Ions which are possibly present include Ti III, Mn I, Fe III, Nb II, Mo I. The abundances are nearly 10 times the solar values for the iron group, with the exception of Fe, which has solar abundance. The abundances of the other elements are solar.

  14. Terahertz epsilon-near-zero graded-index lens.

    PubMed

    Torres, Víctor; Pacheco-Peña, Víctor; Rodríguez-Ulibarri, Pablo; Navarro-Cía, Miguel; Beruete, Miguel; Sorolla, Mario; Engheta, Nader

    2013-04-01

    An epsilon-near-zero graded-index converging lens with planar faces is proposed and analyzed. Each perfectly-electric conducting (PEC) waveguide comprising the lens operates slightly above its cut-off frequency and has the same length but different cross-sectional dimensions. This allows controlling individually the propagation constant and the normalized characteristic impedance of each waveguide for the desired phase front at the lens output while Fresnel reflection losses are minimized. A complete theoretical analysis based on the waveguide theory and Fermat's principle is provided. This is complemented with numerical simulation results of two-dimensional and three-dimensional lenses, made of PEC and aluminum, respectively, and working in the terahertz regime, which show good agreement with the analytical work. PMID:23572004

  15. Campaign Photometry During The 2010 Eclipse Of Epsilon Aurigae

    NASA Astrophysics Data System (ADS)

    Hopkins, Jeff; Stencel, R. E.

    2011-01-01

    Epsilon Aurigae is a long period (27.1 years) eclipsing binary star system with an eclipse that lasts nearly 2 years, but with severe ambiguities about component masses and shape. The current eclipse began on schedule in August of 2009. During the previous, 1982-1984 eclipse, an International Campaign was formed to coordinate a detailed study of the system. While that Campaign was deemed successful, the evolutionary status of the star system remained unclear. Epsilon Aurigae has been observed nearly continuously since the 1982 eclipse. The current Campaign was officially started in 2006. In addition to a Yahoo forum we have a dedicated web site and more than 18 online newsletters reporting photometry, spectroscopy, interferometry and polarimetry data. High quality UBVRIJH band photometric data since before the start of the current eclipse has been submitted. We explore the color differences among the light curves in terms of eclipse phases and archival data. At least one new model of the star system has been proposed since the current Campaign began: a low mass but very high luminosity F star plus a B star surrounded by a debris disk. The current eclipse and in particular the interferometry and spectroscopic data have caused new thoughts on defining eclipsing variable star contact points and phases of an eclipse. Second contact may not be the same point as start of totality and third contact may not be the same point as the start of egress and end of totality. In addition, the much awaited mid-eclipse brightening may or may not have appeared. This paper identifies the current Campaign contributors and the photometric data. This work was supported in part by the bequest of William Herschel Womble in support of astronomy at the University of Denver, by NSF grant 1016678 to the University of Denver.

  16. CONSTRAINTS FROM ASYMMETRIC HEATING: INVESTIGATING THE EPSILON AURIGAE DISK

    SciTech Connect

    Pearson, Richard L. III; Stencel, Robert E. E-mail: robert.stencel@du.edu

    2015-01-01

    Epsilon Aurigae is a long-period eclipsing binary that likely contains an F0Ia star and a circumstellar disk enshrouding a hidden companion, assumed to be a main-sequence B star. High uncertainty in its parallax has kept the evolutionary status of the system in question and, hence, the true nature of each component. This unknown, as well as the absence of solid state spectral features in the infrared, requires an investigation of a wide parameter space by means of both analytic and Monte Carlo radiative transfer (MCRT) methods. The first MCRT models of epsilon Aurigae that include all three system components are presented here. We seek additional system parameter constraints by melding analytic approximations with MCRT outputs (e.g., dust temperatures) on a first-order level. The MCRT models investigate the effects of various parameters on the disk-edge temperatures; these include two distances, three particle size distributions, three compositions, and two disk masses, resulting in 36 independent models. Specifically, the MCRT temperatures permit analytic calculations of effective heating and cooling curves along the disk edge. These are used to calculate representative observed fluxes and corresponding temperatures. This novel application of thermal properties provides the basis for utilization of other binary systems containing disks. We find degeneracies in the model fits for the various parameter sets. However, the results show a preference for a carbon disk with particle size distributions ≥10 μm. Additionally, a linear correlation between the MCRT noon and basal temperatures serves as a tool for effectively eliminating portions of the parameter space.

  17. Constraints from Asymmetric Heating: Investigating the Epsilon Aurigae Disk

    NASA Astrophysics Data System (ADS)

    Pearson, Richard L., III; Stencel, Robert E.

    2015-01-01

    Epsilon Aurigae is a long-period eclipsing binary that likely contains an F0Ia star and a circumstellar disk enshrouding a hidden companion, assumed to be a main-sequence B star. High uncertainty in its parallax has kept the evolutionary status of the system in question and, hence, the true nature of each component. This unknown, as well as the absence of solid state spectral features in the infrared, requires an investigation of a wide parameter space by means of both analytic and Monte Carlo radiative transfer (MCRT) methods. The first MCRT models of epsilon Aurigae that include all three system components are presented here. We seek additional system parameter constraints by melding analytic approximations with MCRT outputs (e.g., dust temperatures) on a first-order level. The MCRT models investigate the effects of various parameters on the disk-edge temperatures; these include two distances, three particle size distributions, three compositions, and two disk masses, resulting in 36 independent models. Specifically, the MCRT temperatures permit analytic calculations of effective heating and cooling curves along the disk edge. These are used to calculate representative observed fluxes and corresponding temperatures. This novel application of thermal properties provides the basis for utilization of other binary systems containing disks. We find degeneracies in the model fits for the various parameter sets. However, the results show a preference for a carbon disk with particle size distributions >=10 μm. Additionally, a linear correlation between the MCRT noon and basal temperatures serves as a tool for effectively eliminating portions of the parameter space.

  18. Transglutaminase Polymerization of Peanut Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    INTRODUCTION: Transglutaminase (TGase) [protein-glutamine:amine gamma-glutamyl-transferase, EC 2.3.2.13]promotes protein cross-linking reactions through an acyl transferase mechanism involving protein-bound glutaminyl residues and primary amines (1), including the epsilon-amino group of lysine resid...

  19. A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell.

    PubMed

    So, Anthony K-C; Espie, George S; Williams, Eric B; Shively, Jessup M; Heinhorst, Sabine; Cannon, Gordon C

    2004-02-01

    A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO(2) fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO(2)-concentrating mechanism and are essential components for autotrophic growth. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (epsilon-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle. PMID:14729686

  20. Protein-protein interactions as drug targets.

    PubMed

    Skwarczynska, Malgorzata; Ottmann, Christian

    2015-10-01

    Modulation of protein-protein interactions (PPIs) is becoming increasingly important in drug discovery and chemical biology. While a few years ago this 'target class' was deemed to be largely undruggable an impressing number of publications and success stories now show that targeting PPIs with small, drug-like molecules indeed is a feasible approach. Here, we summarize the current state of small-molecule inhibition and stabilization of PPIs and review the active molecules from a structural and medicinal chemistry angle, especially focusing on the key examples of iNOS, LFA-1 and 14-3-3. PMID:26510391

  1. Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test.

    PubMed

    Gonçalves, Luciana A; Lobato, Zélia I P; Silva, Rodrigo O S; Salvarani, Felipe M; Pires, Prhiscylla S; Assis, Ronnie A; Lobato, Francisco C F

    2009-11-01

    Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin. PMID:19779698

  2. Clostridium perfringens type-D enterotoxaemia in cattle: the diagnostic significance of intestinal epsilon toxin.

    PubMed

    Jones, A L; Dagleish, M P; Caldow, G L

    2015-10-17

    The aims of this study were to describe 42 cases of Clostridium perfringens type-D enterotoxaemia in cattle seen between 2003 and 2014 and to determine the diagnostic value of detecting epsilon toxin in bovine intestinal content. All cases in the series had histological brain changes considered pathognomonic for C. perfringens type-D enterotoxaemia in sheep and goats and the epsilon toxin of C. perfringens was concurrently detected in the intestinal contents of 15 (36 per cent) cases. The data from the case series indicate that intestinal epsilon toxin has a sensitivity of 56 per cent compared with histology of the brain for diagnosis of bovine C. perfringens type-D enterotoxaemia. The diagnostic specificity of detecting epsilon toxin in bovine intestinal content was investigated by screening intestinal contents of 60 bovine carcases submitted for postmortem examination. Epsilon toxin was detected in 11 (18 per cent) carcases but no pathognomonic histological brain change was found in any. The specificity of intestinal epsilon toxin was estimated to be 80.4 per cent. These studies demonstrate that for a definitive diagnosis of C. perfringens type-D enterotoxaemia in cattle histological examination of the brain is essential as the presence of epsilon toxin in the intestinal contents alone is neither sensitive nor specific enough. PMID:26428898

  3. epsilon-Poly-L: -lysine producer, Streptomyces albulus, has feedback-inhibition resistant aspartokinase.

    PubMed

    Hamano, Y; Nicchu, I; Shimizu, T; Onji, Y; Hiraki, J; Takagi, H

    2007-09-01

    Streptomyces albulus NBRC14147 produces epsilon-poly-L: -lysine (epsilon-PL), which is an amino acid homopolymer antibiotic. Despite the commercial importance of epsilon-PL, limited information is available regarding its biosynthesis; the L: -lysine molecule is directly utilized for epsilon-PL biosynthesis. In most bacteria, L: -lysine is biosynthesized by an aspartate pathway. Aspartokinase (Ask), which is the first enzyme in this pathway, is subject to complex regulation such as through feedback inhibition by the end-product amino acids such as L: -lysine and/or L: -threonine. S. albulus NBRC14147 can produce a large amount of epsilon-PL (1-3 g/l). We therefore suspected that Ask(s) of S. albulus could be resistant to feedback inhibition to provide sufficient L: -lysine for epsilon-PL biosynthesis. To address this hypothesis, in this study, we cloned the ask gene from S. albulus and investigated the feedback inhibition of its gene product. As predicted, we revealed the feedback resistance of the Ask; more than 20% relative activity of Ask was detected in the assay mixture even with extremely high concentrations of L: -lysine and L: -threonine (100 mM each). We further constructed a mutated ask gene for which the gene product Ask (M68V) is almost fully resistant to feedback inhibition. The homologous expression of Ask (M68V) further demonstrated the increase in epsilon-PL productivity. PMID:17611754

  4. Factorial combinations of protein interactions generate a multiplicity of florigen activation complexes in wheat and barley.

    PubMed

    Li, Chengxia; Lin, Huiqiong; Dubcovsky, Jorge

    2015-10-01

    The FLOWERING LOCUS T (FT) protein is a central component of a mobile flowering signal (florigen) that is transported from leaves to the shoot apical meristem (SAM). Two FT monomers and two DNA-binding bZIP transcription factors interact with a dimeric 14-3-3 protein bridge to form a hexameric protein complex. This complex, designated as the 'florigen activation complex' (FAC), plays a critical role in flowering. The wheat homologue of FT, designated FT1 (= VRN3), activates expression of VRN1 in the leaves and the SAM, promoting flowering under inductive long days. In this study, we show that FT1, other FT-like proteins, and different FD-like proteins, can interact with multiple wheat and barley 14-3-3 proteins. We also identify the critical amino acid residues in FT1 and FD-like proteins required for their interactions, and demonstrate that 14-3-3 proteins are necessary bridges to mediate the FT1-TaFDL2 interaction. Using in vivo bimolecular fluorescent complementation (BiFC) assays, we demonstrate that the interaction between FT1 and 14-3-3 occurs in the cytoplasm, and that this complex is then translocated to the nucleus, where it interacts with TaFDL2 to form a FAC. We also demonstrate that a FAC including FT1, TaFDL2 and Ta14-3-3C can bind to the VRN1 promoter in vitro. Finally, we show that relative transcript levels of FD-like and 14-3-3 genes vary among tissues and developmental stages. Since FD-like proteins determine the DNA specificity of the FACs, variation in FD-like gene expression can result in spatial and temporal modulation of the effects of mobile FT-like signals. PMID:26252567

  5. Transcription in vivo of an Alu family member upstream from the human epsilon-globin gene.

    PubMed Central

    Allan, M; Paul, J

    1984-01-01

    The more distal Alu repeat flanking the epsilon-globin gene is transcribed in K562 cells to generate transcripts 350-400 nucleotides in length. Initiation occurs at the start of the repeat, upstream of a putative PolIII control signal. These transcripts originate from the strand which does not code epsilon-globin and are oriented in the opposite direction from the gene. They are non-polyadenylated, nucleus-confined and are only detectable in association with expression of the epsilon-globin gene. Images PMID:6320117

  6. Simulations of free shear layers using a compressible kappa-epsilon model

    NASA Technical Reports Server (NTRS)

    Yu, S. T.; Chang, C. T.; Marek, C. J.

    1991-01-01

    A two-dimensional, compressible Navier-Stokes equation with a k-epsilon turbulence model is solved numerically to simulate the flow of a compressible free shear layer. The appropriate form of k and epsilon equations for compressible flow is discussed. Sarkar's modeling is adopted to simulate the compressibility effects in the k and epsilon equations. The numerical results show that the spreading rate of the shear layers decreases with increasing convective Mach number. In addition, favorable comparison was found between the calculated results and experimental data.

  7. Citizen Sky, Solving the Mystery of epsilon Aurigae

    NASA Astrophysics Data System (ADS)

    Turner, Rebecca; Price, A.; Kloppenborg, B.; Henden, A.

    2010-01-01

    Citizen Sky is a multi-year, NSF funded citizen science project involving the bright star eps Aur. The project was conceived by the IYA 2009 working group on Research Experiences for Students, Teachers, and Citizen-Scientists. Citizen Sky goes beyond simple observing to include a major data analysis component. The goal is to introduce the participant to the full scientific process from background research to paper writing for a peer-reviewed journal. It begins with a 10 Star Training Program of several types of binary and transient variable stars that are easy to observe from suburban locations with the naked eye. Participants then move on to monitoring the rare and mysterious 2009-2011 eclipse (already underway) of epsilon Aurigae. This object undergoes eclipses only every 27.1 years and each eclipse lasts nearly two years. The star is bright enough to be seen with the naked eye from most urban areas. Training will be provided in observing techniques as well as basic data analysis of photometric and visual datasets (light curve and period analysis). The project also involves two public workshops, one on observing (already held in August of 2009) and one on data analysis and scientific paper writing (to be held in 2010.) This project has been made possible by the National Science Foundation.

  8. Copernicus observations of the Ap star Epsilon Ursae Majoris

    NASA Technical Reports Server (NTRS)

    Mallama, A. D.; Molnar, M. R.

    1977-01-01

    Spectral scans of the Ap star Epsilon UMa made with the Copernicus satellite show strong line blanketing from profuse Cr II and Fe II lines. In the spectral region covering 1900 to 3000 A, about 500 lines are present which suppress the apparent continuum by at least 15-30%. An accurate line-identification list is compiled showing Eu II present in addition to Mn II and Ni II. The identification of Eu II, however, rests on very stringent identification limits for Fe II. If these are relaxed, the existence of Eu II is dubious. There are no broad features in this spectral region which would suggest strong photoionization discontinuities by metals, but one feature near 2137 A might contain the photoionization edge due to Cr I 5S lying 0.94 eV above the ground level. However, a significant correlation between the line-blanketing strength and the amplitude of the OAO-2 ultraviolet light curves was found such that both monotonically increase in the same proportion toward shorter wavelengths. This gives additional strength to the suggestion that variations in the metal line-blanketing cause the observed photometric variations.

  9. The Fourier analysis technique and epsilon-pseudo-eigenvalues

    SciTech Connect

    Donato, J.M.

    1993-07-01

    The spectral radii of iteration matrices and the spectra and condition numbers of preconditioned systems are important in forecasting the convergence rates for iterative methods. Unfortunately, the spectra of iteration matrices or preconditioned systems is rarely easily available. The Fourier analysis technique has been shown to be a useful tool in studying the effectiveness of iterative methods by determining approximate expressions for the eigenvalues or condition numbers of matrix systems. For non-symmetric matrices the eigenvalues may be highly sensitive to perturbations. The spectral radii of nonsymmetric iteration matrices may not give a numerically realistic indication of the convergence of the iterative method. Trefethen and others have presented a theory on the use of {epsilon}-pseudo-eigenvalues in the study of matrix equations. For Toeplitz matrices, we show that the theory of c-pseudo-eigenvalues includes the Fourier analysis technique as a limiting case. For non-Toeplitz matrices, the relationship is not clear. We shall examine this relationship for non-Toeplitz matrices that arise when studying preconditioned systems for methods applied to a two-dimensional discretized elliptic differential equation.

  10. Infrared Studies of Epsilon Aurigae in Eclipse 2010

    NASA Astrophysics Data System (ADS)

    Stencel, Robert E.; Kloppenborg, B.; Wall, R.; Howell, S.; Hoard, D.; Rayner, J.; Bus, S.; Tokunaga, A.; Sitko, M.; Russell, R.; Lynch, D.; Brafford, S.; Hammel, H.; Whitney, B.; Orton, G.; Yanamandra-Fisher, P.; Hora, J.; Hoffman, W.; Skemer, A.

    2011-01-01

    We report a series of observations of the enigmatic long period eclipsing binary epsilon Aurigae during its eclipse interval 2009-2011, using near-infrared spectra & photometry obtained with SpeX/IRTF, Spitzer/IRAC, mid-infrared data with BASS on IRTF & AEOS, MIRSI on IRTF and MIRAC4 on MMT, along with MIRSI on IRTF and MIRAC4 on MMT & Denver's TNTCAM2 at WIRO, and an Optec SSP-4 J&H photometer at Mt.Evans Observatory. The objective of these observations include: (1) confirm the appearance of CO absorption bands at and after mid-eclipse, due to the dark disk, and (2) seek evidence for any mid-infrared solid state spectral features from particles in the disk, seen during different portions of total eclipse. The results to date show that the infrared eclipse is less deep than the optical one, and the implied disk temperature has begun to increase from 550K toward 1100K as eclipse progresses past midpoint and heated portions of the disk come into view. Material properties of the disk are consistent with large particles. This work was supported in part by the bequest of William Herschel Womble in support of astronomy at the University of Denver, by NSF grant 1016678 and JPL RSA 1414715 to the University of Denver, by NASA ADP grant NNX09AC73G to the University of Cincinnati, by The Aerospace Corporation's Independent Research and Development Program.

  11. Epsilon toxin: a fascinating pore-forming toxin.

    PubMed

    Popoff, Michel R

    2011-12-01

    Epsilon toxin (ETX) is produced by strains of Clostridium perfringens classified as type B or type D. ETX belongs to the heptameric β-pore-forming toxins including aerolysin and Clostridium septicum alpha toxin, which are characterized by the formation of a pore through the plasma membrane of eukaryotic cells consisting in a β-barrel of 14 amphipatic β strands. By contrast to aerolysin and C. septicum alpha toxin, ETX is a much more potent toxin and is responsible for enterotoxemia in animals, mainly sheep. ETX induces perivascular edema in various tissues and accumulates in particular in the kidneys and brain, where it causes edema and necrotic lesions. ETX is able to pass through the blood-brain barrier and stimulate the release of glutamate, which accounts for the symptoms of nervous excitation observed in animal enterotoxemia. At the cellular level, ETX causes rapid swelling followed by cell death involving necrosis. The precise mode of action of ETX remains to be determined. ETX is a powerful toxin, however, it also represents a unique tool with which to vehicle drugs into the central nervous system or target glutamatergic neurons. PMID:21535407

  12. Preferential emission into epsilon-near-zero metamaterial [Invited

    DOE PAGESBeta

    Galfsky, Tal; Sun, Zheng; Jacob, Zubin; Menon, Vinod M.

    2015-11-23

    We report the use of epsilon near zero (ENZ) metamaterial to control spontaneous emission from Zinc-Oxide (ZnO) excitons. The ENZ material consists of alternating layers of silver and alumina with subwavelength thicknesses, resulting in an effective medium where one of the components of the dielectric constant approach zero between 370nm-440nm wavelength range. Bulk ZnO with photoluminescence maximum in the ENZ regime was deposited via atomic layer deposition to obtain a smooth film with near field coupling to the ENZ metamaterial. Preferential emission from the ZnO layer into the metamaterial with suppression of forward emission by 90% in comparison to ZnOmore » on silicon is observed. We attribute this observation to the presence of dispersionless plasmonic modes in the ENZ regime as shown by the results of theoretical modeling presented here. Integration of ENZ metamaterials with light emitters is an attractive platform for realizing a low threshold subwavelength laser.« less

  13. The Epsilon Eridani Debris Disk Resolved by Millimeter Interferometry

    NASA Astrophysics Data System (ADS)

    Wilner, David J.; MacGregor, Meredith A.; Andrews, Sean M.; Jean-Francois, Lestrade; Tahli Maddison, Sarah

    2016-01-01

    At a distance of only 3.22 pc, epsilon Eridani hosts the closest debris disk to the Sun. We present the first millimeter interferometric observations of this system, using the Submillimeter Array (SMA) at 1.3 mm and the Australia Telescope Compact Array (ATCA) at 7 mm, reaching 4 arcsec (13 AU) resolution. These observations reveal two distinct emission components: (1) the well-known outer dust belt, which is resolved in the radial direction, and (2) a compact source coincident with the position of the star. Model-fitting the visibilities constrains the basic properties of these components. The outer belt is located at 64 +/- 3 AU with fractional width 0.3, wider than the classical Kuiper Belt. This belt shows no significant azimuthal structure, or stellocentric offset, that might result from the presence of unseen giant planets on wide orbits in the system. The flux density of the unresolved central component exceeds predictions for thestellar photosphere; this excess may arise from a stellar chromosphere.

  14. Polarimetry of Epsilon Aurigae from Mid Eclipse to Third Contact

    NASA Astrophysics Data System (ADS)

    Cole, Gary M.; Stencel, Robert E.

    2011-05-01

    In a previous paper, the author discussed the construction of an automated dual beam imaging polarimeter and of observations made in the November 2009 to February 2010 period. Here, we discuss observations and instrumental improvements that span the period from late August 2010 through third and into fourth contacts in Spring 2011. Approximately 930 linear polarization measurements of the target star in BVR bands were obtained during 99 nights of observation. Additional measurements were made of both known polarization standards and zero polarization stars to verify instrument calibration. The polarization of Epsilon Aurigae was observed to vary by nearly 0.4% peak to valley during this period. These variations occurred in several major cycles of varying duration. Measurement error is estimated to be on the order of +/-0.05%. The observed variations resemble excess polarization seen during the 1984 eclipse egress, but may show some differences in detail. During this project, a new optical rotator was developed in conjunction with Optec, Inc., and used for the last two months of observations. This project was initiated at the suggestion of Dr. Robert Stencel at the May 2009 SAS meeting to extend measurements done during the 1984 eclipse by Dr. Jack Kemp and followed up by his student, Dr. Gary Henson thereafter.

  15. Electrically Tunable Epsilon-Near-Zero (ENZ) Metafilm Absorbers

    PubMed Central

    Park, Junghyun; Kang, Ju-Hyung; Liu, Xiaoge; Brongersma, Mark L.

    2015-01-01

    Enhancing and spectrally controlling light absorption is of great practical and fundamental importance. In optoelectronic devices consisting of layered semiconductors and metals, absorption has traditionally been manipulated with the help of Fabry-Pérot resonances. Even further control over the spectral light absorption properties of thin films has been achieved by patterning them into dense arrays of subwavelength resonant structures to form metafilms. As the next logical step, we demonstrate electrical control over light absorption in metafilms constructed from dense arrays of actively tunable plasmonic cavities. This control is achieved by embedding indium tin oxide (ITO) into these cavities. ITO affords significant tuning of its optical properties by means of electrically-induced carrier depletion and accumulation. We demonstrate that particularly large changes in the reflectance from such metafilms (up to 15% P) can be achieved by operating the ITO in the epsilon-near-zero (ENZ) frequency regime where its electrical permittivity changes sign from negative to positive values. PMID:26549615

  16. Electrically Tunable Epsilon-Near-Zero (ENZ) Metafilm Absorbers.

    PubMed

    Park, Junghyun; Kang, Ju-Hyung; Liu, Xiaoge; Brongersma, Mark L

    2015-01-01

    Enhancing and spectrally controlling light absorption is of great practical and fundamental importance. In optoelectronic devices consisting of layered semiconductors and metals, absorption has traditionally been manipulated with the help of Fabry-Pérot resonances. Even further control over the spectral light absorption properties of thin films has been achieved by patterning them into dense arrays of subwavelength resonant structures to form metafilms. As the next logical step, we demonstrate electrical control over light absorption in metafilms constructed from dense arrays of actively tunable plasmonic cavities. This control is achieved by embedding indium tin oxide (ITO) into these cavities. ITO affords significant tuning of its optical properties by means of electrically-induced carrier depletion and accumulation. We demonstrate that particularly large changes in the reflectance from such metafilms (up to 15% P) can be achieved by operating the ITO in the epsilon-near-zero (ENZ) frequency regime where its electrical permittivity changes sign from negative to positive values. PMID:26549615

  17. Coherent perfect absorption in epsilon-near-zero metamaterials

    NASA Astrophysics Data System (ADS)

    Feng, Simin; Halterman, Klaus

    2012-10-01

    In conventional materials, strong absorption usually requires that the material have either high loss or a large thickness-to-wavelength ratio (d/λ≫1). We find the situation to be vastly different for bilayer structures composed of a metallic substrate and an anisotropic epsilon-near-zero (ENZ) metamaterial, where the permittivity in the direction perpendicular to its surface, ɛz, vanishes. Remarkably, perfect absorption can occur in situations where the metamaterial is arbitrarily thin (d/λ≪1) and arbitrarily low loss. Our numerical and analytical solutions reveal that under the conditions ɛz→0 and ℑ(ɛz)≫ℜ(ɛz), at perfect absorption there is a linear relationship between the thickness and the loss, which means the thickness of the absorber can be pushed to zero by reducing the material loss to zero. This counterintuitive behavior is explained in terms of coherent perfect absorption (or stimulated absorption) via critical coupling to a fast wave propagating along the ENZ layer.

  18. Preclinical memory profile in Alzheimer patients with and without allele APOE-epsilon4.

    PubMed

    Estévez-González, Armando; García-Sánchez, Carmen; Boltes, Anunciación; Otermín, Pilar; Baiget, Montserrat; Escartín, Antonio; del Rio, Elisabeth; Gironell, Alex; Kulisevsky, Jaime

    2004-01-01

    To investigate the association between APOE-epsilon4 allele and memory phenotype in the preclinical stage of Alzheimer's disease (AD). We compared an extensive preclinical memory profile at the baseline evaluation of 2 AD genotype groups: APOE-epsilon4 allele carriers and patients with APOE-epsilon3 homozygosity. Baseline memory performance was carried out at least 2 years (interval of 27.7 +/- 4 months) before AD diagnosis was established, and analysis included different modalities of working memory (visuoperceptive, visuospatial, digit span and processing speed), of declarative memory (recent, verbal learning, prospective and semantic) and of nondeclarative memory (procedural, incidental and priming). We found no significant differences: memory performance was similar in both genotype groups. The presence of the APOE-epsilon4 allele does not seem to be sufficient to cause a distinctive preclinical memory phenotype in AD patients. PMID:15159600

  19. Epsilon carbide - A low-temperature component of interplanetary dust particles

    NASA Technical Reports Server (NTRS)

    Christoffersen, R.; Buseck, P. R.

    1983-01-01

    Transmission electron microscope study of a chondritic interplanetary dust particle has revealed the presence of epsilon iron-nickel carbide, a low-temperature carbide previously encountered only in metallurgical studies. In these studies epsilon-carbide was synthesized by carburization of iron or nickel grains in a stream of carbon monoxide or carbon monoxide plus hydrogen. Similar carburization of an iron-nickel metal in situ may have produced epsilon-carbide during particle heating on atmospheric entry or in solar orbit. Alternatively, the epsilon-carbide may be a by-product of Fischer-Tropsch reactions in the solar nebula. Such reactions have been proposed as the mechanism of hydrocarbon formation in the early solar system.

  20. T{sub {epsilon}}{sup *} integral under plane stress crack growth

    SciTech Connect

    Omori, Yoshika; Ma, Leong; Kobayashi, A.S.; Okada, Hiroshi; Atluri, S.N.

    1997-12-01

    The T{sub {epsilon}}{sup *} integral values associated with stable crack growth in A606 HSLA steel single-edge-notched (SEN) specimens were determined experimentally and numerically. The displacement fields obtained through Moire interferometry and elastic-plastic finite element analysis were used to evaluate T{sub {epsilon}}{sup *} associated with the moving crack tip. T{sub {epsilon}}{sup *} decreased with the reduction in the size of the integration contour, {Gamma}{sub {epsilon}}, and continually increased with stable crack growth in this specimen. The measured and computed crack-tip opening angle (CTOA), on the other hand, was a constant 15{degree} during stable crack growth after dropping from the measured and computed initial high values of 35 and 57{degree}, respectively.

  1. Specification of an IF1 to PG translator for the Epsilon-2 dataflow machine

    SciTech Connect

    Boehm, W.; Hoch, J.E.

    1992-11-01

    This report describes a mechanism for compiling the functional language SISAL for Sandia`s Epsilon-2 hybrid dataflow machine. The strategy couples the front-end of the standard SISAL compiler (which generates a data dependence graph intermediate form called IF1) with an optimizing code-generator for Epsilon-2. The Epsilon-2 code-generator is the back-end of a compiler for the functional language Id. It translates a data dependence graph intermediate form called Program Graphs into Epsilon-2 machine code. This report describes a translation path from IF1 graphs to Program Graphs. This report also comments on the relative merits of the IF1 and Program Graph representations.

  2. Specification of an IF1 to PG translator for the Epsilon-2 dataflow machine

    SciTech Connect

    Boehm, W. . Dept. of Computer Science); Hoch, J.E. )

    1992-11-01

    This report describes a mechanism for compiling the functional language SISAL for Sandia's Epsilon-2 hybrid dataflow machine. The strategy couples the front-end of the standard SISAL compiler (which generates a data dependence graph intermediate form called IF1) with an optimizing code-generator for Epsilon-2. The Epsilon-2 code-generator is the back-end of a compiler for the functional language Id. It translates a data dependence graph intermediate form called Program Graphs into Epsilon-2 machine code. This report describes a translation path from IF1 graphs to Program Graphs. This report also comments on the relative merits of the IF1 and Program Graph representations.

  3. Low Reynolds number k-epsilon modelling with the aid of direct simulation data

    NASA Technical Reports Server (NTRS)

    Rodi, W.; Mansour, N. N.

    1993-01-01

    The constant C sub mu and the near-wall damping function f sub mu in the eddy-viscosity relation of the k-epsilon model are evaluated from direct numerical simulation (DNS) data for developed channel and boundary layer flow at two Reynolds numbers each. Various existing f sub mu model functions are compared with the DNS data, and a new function is fitted to the high-Reynolds-number channel flow data. The epsilon-budget is computed for the fully developed channel flow. The relative magnitude of the terms in the epsilon-equation is analyzed with the aid of scaling arguments, and the parameter governing this magnitude is established. Models for the sum of all source and sink terms in the epsilon-equation are tested against the DNS data, and an improved model is proposed.

  4. Low Reynolds number kappa-epsilon modeling with the aid of direct simulation data

    NASA Technical Reports Server (NTRS)

    Rodi, W.; Mansour, N. N.

    1990-01-01

    The constant C(sub mu) and the near-wall damping function f(sub mu) in the eddy-viscosity relation of the kappa-epsilon model are evaluated from direct numerical simulation (DNS) data for developed channel and boundary layer flow at two Reynolds numbers each. Various existing f(sub mu) model functions are compared with the DNS data, and a new function is fitted to the high-Reynolds-number channel flow data. The epsilon-budget is computed for the fully developed channel flow. The relative magnitude of the terms in the epsilon-equation is analyzed with the aid of scaling arguments, and the parameter governing this magnitude is established. Models for the sum of all source and sink terms in the epsilon-equation are tested against the DNS data, and an improved model is proposed.

  5. DNA replication: polymerase epsilon as a non-catalytic converter of the helicase.

    PubMed

    Zegerman, Philip

    2013-04-01

    In eukaryotes DNA polymerase epsilon (ε) synthesises the leading DNA strand during replication. A new study provides insight into how this polymerase also functions independently of its enzyme activity to assemble and activate the replicative helicase. PMID:23578873

  6. Calculations of diffuser flows with an anisotropic K-epsilon model

    NASA Astrophysics Data System (ADS)

    Zhu, J.; Shih, T.-H.

    1995-10-01

    A newly developed anisotropic K-epsilon model is applied to calculate three axisymmetric diffuser flows with or without separation. The new model uses a quadratic stress-strain relation and satisfies the realizability conditions, i.e., it ensures both the positivity of the turbulent normal stresses and the Schwarz' inequality between any fluctuating velocities. Calculations are carried out with a finite-element method. A second-order accurate, bounded convection scheme and sufficiently fine grids are used to ensure numerical credibility of the solutions. The standard K-epsilon model is also used in order to highlight the performance of the new model. Comparison with the experimental data shows that the anisotropic K-epsilon model performs consistently better than does the standard K-epsilon model in all of the three test cases.

  7. Calculations of Diffuser Flows with an Anisotropic K-Epsilon Model

    NASA Technical Reports Server (NTRS)

    Zhu, J.; Shih, T.-H.

    1995-01-01

    A newly developed anisotropic K-epsilon model is applied to calculate three axisymmetric diffuser flows with or without separation. The new model uses a quadratic stress-strain relation and satisfies the realizability conditions, i.e., it ensures both the positivity of the turbulent normal stresses and the Schwarz' inequality between any fluctuating velocities. Calculations are carried out with a finite-element method. A second-order accurate, bounded convection scheme and sufficiently fine grids are used to ensure numerical credibility of the solutions. The standard K-epsilon model is also used in order to highlight the performance of the new model. Comparison with the experimental data shows that the anisotropic K-epsilon model performs consistently better than does the standard K-epsilon model in all of the three test cases.

  8. Navier-Stokes cascade analysis with a stiff Kappa-Epsilon turbulence solver

    NASA Technical Reports Server (NTRS)

    Liu, Jong-Shang; Sockol, Peter M.; Prahl, Joseph M.

    1987-01-01

    The two dimensional, compressible, thin layer Navier-Stokes equations with the Baldwin-Lomax turbulence model and the kinetic energy-energy dissipation (k-epsilon) model are solved numerically to simulate the flow through a cascade. The governing equations are solved for the entire flow domain, without the boundary layer assumptions. The stiffness of the k-epsilon equations is discussed. A semi-implicit, Runge-Kutta, time-marching scheme is developed to solve the k-epsilon equations. The impact of the k-epsilon solver on the explicit Runge-Kutta Navier-Stokes solver is discussed. Numerical solutions are presented for two dimensional turbulent flow over a flat plate and a double circular arc cascade and compared with experimental data.

  9. Low cytoplasmic casein kinase 1 epsilon expression predicts poor prognosis in patients with hepatocellular carcinoma.

    PubMed

    Lin, Shu-Hui; Yeh, Chung-Min; Hsieh, Ming-Ju; Lin, Yueh-Min; Chen, Mei-Wen; Chen, Chih-Jung; Lin, Cheng-Yu; Hung, Hsiao-Fang; Yeh, Kun-Tu; Yang, Shun-Fa

    2016-03-01

    Casein kinase 1 epsilon (CK1ε) is a member of the casein kinase 1 (CK1) family, which comprises highly conserved and ubiquitous serine/threonine protein kinases. Recent studies have demonstrated that CK1ε plays a role in human cancers; however, the role of CK1ε in hepatocellular carcinoma (HCC) remains unclear. The study used immunohistochemistry to examine CK1ε expression in 230 HCC specimens by tissue microarray (TMA) and assessed the effect of CK1ε knockdown on migration of human hepatoma cells in vitro. The immunohistochemical analyses showed that low CK1ε expression was significantly correlated with tumor differentiation (p = 0.008), T classification (p = 0.016), tumor vascular invasion (p = 0.002), and cancer stage (p = 0.010). The univariate and multivariate analyses showed that patients with low CK1ε expression had a considerably lower OS rate than that of the patients with high CK1ε expression (p = 0.041, hazard ratio = 1.4; p = 0.039, hazard ratio = 1.4). Moreover, CK1ε small interfering RNA (siRNA) treatment exerted an invasion-promoting effect in human hepatoma cells. In conclusion, our data indicated that low CK1ε expression is correlated with a low survival rate and CK1ε may play a role as a tumor suppressor in hepatocarcinogenesis. PMID:26482619

  10. Attenuation of epsilon(sub eff) of coplanar waveguide transmission lines on silicon substrates

    NASA Technical Reports Server (NTRS)

    Taub, Susan R.; Young, Paul G.

    1993-01-01

    Attenuation and epsilon(sub eff) of Coplanar Waveguide (CPW) transmission lines were measured on Silicon substrates with resistivities ranging from 400 to greater than 30,000 ohm-cm, that have a 1000 angstrom coating of SiO2. Both attenuation and epsilon(sub eff) are given over the frequency range 5 to 40 GHz for various strip and slot widths. These measured values are also compared to the theoretical values.

  11. Phase stability of {epsilon} and {gamma} HNIW (CL-20) at high-pressure and temperature

    SciTech Connect

    Gump, Jared C.; Stoltz, Chad A.; Peiris, Suhithi M.

    2007-12-12

    Hexanitrohexaazaisowurtzitane (CL-20) is one of the few ingredients developed since World War II to be considered for transition to military use. Five polymorphs have been identified for CL-20 by FTIR measurements ({alpha}, {beta}, {gamma}, {epsilon}, {zeta}). As CL-20 is transitioned into munitions it will become necessary to predict its response under conditions of detonation, for performance evaluation. Such predictive modeling requires a phase diagram and basic thermodynamic properties of the various phases at high pressure and temperature. Therefore, the epsilon and gamma phases of CL-20 at static high-pressure and temperature were investigated using synchrotron angle-dispersive x-ray diffraction experiments. The samples were compressed and heated using diamond anvil cells (DAC). Pressures and temperatures achieved were around 5 GPa and 240 deg. C, respectively. The epsilon phase was stable to 6.3 GPa at ambient temperature. When heated at ambient pressure the epsilon phase was sustained to a temperature of 120 deg. C then underwent a transition to the gamma phase above 125 deg. C and then thermal decomposition occurred above 150 deg. C. Upon compression, the gamma phase underwent a phase transition at both ambient temperature and 140 deg. C. Pressure--volume data for the epsilon and gamma phase at ambient temperature and the epsilon phase at 75 deg. C were fit to the Birch-Murnaghan formalism to obtain isothermal equations of state.

  12. Carnitine biosynthesis in Neurospora crassa: enzymatic conversion of lysine to epsilon-N-trimethyllysine.

    PubMed Central

    Rebouche, C J; Broquist, H P

    1976-01-01

    The enzymatic conversion of L-lysine, epsilon-N-trimethyl-L-lysine the first series of reactions in the biosynthesis of carnitine in Neurospora crassa, proceeds via sequential methylation of free L-lysine, epsilon-N-methyl-L-lysine, and epsilon -N-dimethyl-L-lysine. The latter two compounds have been shown to be intermediates in the biosynthesis of carnitine by radioisotope dilution and incorporation experiments in growing cultures of N. crassa 33933 (lys-) and 38706 (met-). Methionine but not choline, has been recognized as an effective methyl donor in vivo. Inclusion of choline in the growth medium of strain 33933 does, however, enhance incorporation of the methyl groups of L-[methyl-3H]methionine into carnitine in an apparent "sparing" effect on methionine synthesis. Studies in cell-free extracts of the lysine auxotroph strain 33933 of N. crassa have established that lysine and epsilon-N-methyl and epsilon-N-dimethyllysine are enzymatically methylated, with S-adenosyl-L-methionine as the methyl group donor. The enzyme system appears to have no essential cofactors. Lysine does not induce synthesis of the enzyme system in the wild-type strain 262, whereas both carnitine and epsilon-N-trimethyllysine repress its synthesis in strain 33933. PMID:133101

  13. Lessons Learned During the Recent Epsilon Aurigae Eclipse Observing Campaign

    NASA Astrophysics Data System (ADS)

    Stencel, Robert E.

    2011-05-01

    The 18 month long eclipse of the 3rd magnitude star, epsilon Aurigae, is forecast to end during May 2011, based on six eclipse events, in 2010, 1982, 1955, 1930, 1902 and 1874. In partnership with AAVSO, Hopkins Phoenix Observatory and others, we have organized observing campaigns during the past several years in order to maximize data acquired during this rare event and to promote reporting and analysis of observations of all kinds. Hundreds of registered participants have signed up for alert notices and newsletters, and many dozens of observers have contributed photometry, spectra and ideas to the ongoing effort - see websites: www.CitizenSky.org and www.hposoft.com/Campaign09.html . In this presentation, I will provide an update on the participation leading to extensive photometric results. Similarly, bright star spectroscopy has greatly benefited from small telescope plus spectrometer capabilities, now widely available, that complement traditional but less-frequent large telescope high dispersion work. Polarimetry provided key insights during the last eclipse, and we promoted the need for new data using this method. Finally, interferometry has come of age since the last eclipse, leading to the direct detection of the transiting dark disk causing the eclipse. Along with these traditional measurements, I will outline campaign-related efforts to promote Citizen Science opportunities among the public. Support for these efforts derives in part from AAVSO/NSF-Informal Science Education, NSF AAG grant 10-16678 and a bequest to the University of Denver Astronomy Program by alumnus William Herschel Womble, for which I am grateful.

  14. Pharmacokinetics of Epsilon-Aminocaproic Acid in Neonates

    PubMed Central

    Eaton, Michael P.; Alfieris, George M; Sweeney, Dawn M; Angona, Ronald E; Cholette, Jill M; Venuto, Charles; Anderson, Brian

    2016-01-01

    Background Antifibrinolytic medications such as epsilon-aminocaproic acid (EACA) are used in pediatric heart surgery to decrease surgical bleeding and transfusion. Dosing schemes for neonates are often based on adult regimens, or are simply empiric, in part due to the lack of neonatal pharmacokinetic information. We sought to determine the pharmacokinetics of EACA in neonates undergoing cardiac surgery and to devise a dosing regimen for this population. Methods Ten neonates undergoing cardiac surgery with cardiopulmonary bypass were given EACA according to standard practice, and blood was drawn at 10 time points to determine drug concentrations. Time-concentration profiles were analyzed using nonlinear mixed effects models. Parameter estimates (standardized to a 70 kg person) were used to develop a dosing regimen intended to maintain a target concentration shown to inhibit fibrinolysis in neonatal plasma (50 mg/L). Results Pharmacokinetics were described using a two compartment model plus an additional compartment for the cardiopulmonary bypass pump. First order elimination was described with a clearance of 5.07 L/h*(WT/70) 0.75. Simulation showed a dosing regimen with a loading dose of 40 mg/kg, and an infusion of 30 mg/kg/h, with a pump prime concentration of 100 mg/L maintained plasma concentrations above 50 mg/L in 90% of neonates during cardiopulmonary bypass surgery. Conclusions EACA clearance, expressed using allometry, is reduced in neonates compared to older children and adults. Loading dose and infusion dose are approximately half those required in children and adults. PMID:25723765

  15. Discovery of the February Epsilon Virginids (FEV, IAU#506)

    NASA Astrophysics Data System (ADS)

    Steakley, Kathryn; Jenniskens, P. M.

    2013-01-01

    Halley type comets are relatively few, but at Earth they are sampled over a large part of the inner solar system because dust accumulates in comparatively stable orbits. We have detected a new meteor shower with a Halley-type orbit, the February epsilon Virginids (FEV), from video observations with the Cameras for All-Sky Meteor Surveillance (CAMS) and by examining orbits listed in the SonataCo Japanese database. Twenty-two meteors were detected during the period from February 1st through February 9th of 2008 to 2012 that are part of this shower. The FEVs originate from the geocentric radiant of R.A. = 201.66° and Dec = +10.39° with a mean geocentric velocity of 63.01 km/s. The mean orbital elements of these meteoroids are q = (0.488 ± 0.021) AU, 1/a = ( 0.085 ± 0.095) 1/AU, e = 0.958 ± 0.046, i = 138.05° ± 1.28°, ω = 271.15° ± 3.70°, Ω = 315.26 ± 0.86°, and Π = 228.12°. We investigated whether this meteoroid stream could have originated from comets C/1978 T3 (Bradfield), C/1808 F1 (Pons), or C/1939 H1 (Jurlof-Achmarof-Hassel). If the parent body can be identified, we can determine when the comet was first captured into a low perihelion distance orbit. Future examination of the shower will allow us to examine the physical properties of the parent comet.

  16. Monoclonal antibody (H107) inhibiting IgE binding to Fc epsilon R(+) human lymphocytes.

    PubMed

    Noro, N; Yoshioka, A; Adachi, M; Yasuda, K; Masuda, T; Yodoi, J

    1986-08-15

    A hybridoma-producing monoclonal antibody blocking the binding of human IgE to lymphocytes Fc receptor (Fc epsilon R) was established by the fusion of murine myeloma cells. P3X63.653.Ag8, with BALB/c spleen cells immunized with Fc epsilon R(+) human B lymphoblastoid cell line cells, RPMI1788. A clone of the hybridoma (H107) produced a monoclonal IgG2b antibody that inhibited the rosette formation of Fc epsilon R(+) human B lymphoblastoid cell line cells (RPMI1788, RPMI8866, CESS, Dakiki, and IM9) with fixed ox red blood cells (ORBC) conjugated with human IgE (IgE-ORBC). In contrast, the rosette formation with IgG-conjugated ORBC (IgG-ORBC) on Fc gamma R(+), Fc epsilon R(-) Daudi cells were not affected by the H107 antibodies. A close association of Fc epsilon R and the antigenic determinant recognized by H107 antibody was suggested by the following results. First, the bindings of 125I-labeled IgE (125I-IgE) or 125I-labeled H107 IgG2b antibody (125I-H107) to RPMI8866 cells were inhibited by cold human IgE and H107 IgG2b but not by other classes of human Ig (IgA and IgG), MPC11 IgG2b, or unrelated monoclonal antibodies. Second, H107 antibody reacted with Fc epsilon R(+) B cell lines but not with Fc epsilon R(-) B cell lines as determined by an indirect immunofluorescence. Third, Fc epsilon R(+) cells were depleted by the incubation in the dish coated with H107 antibody or IgE but not in the dish coated with unrelated antibodies. Finally, there was a correlation between the increase of Fc epsilon R(+) cells and that of H107(+) cells in the peripheral blood lymphocytes of the patients with atopic dermatitis. The surface antigens on Fc epsilon R(+) RPMI8866 cells recognized by H107 antibodies had the molecular size of 45,000 as determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PMID:2942602

  17. Resistance of ovine, caprine and bovine endothelial cells to Clostridium perfringens type D epsilon toxin in vitro.

    PubMed

    Uzal, F A; Rolfe, B E; Smith, N J; Thomas, A C; Kelly, W R

    1999-08-01

    Ovine, caprine and bovine endothelial cells were grown in vitro and challenged with Clostridium perfringens type D epsilon toxin to compare their susceptibility to this toxin. Madin Darby canine kidney (MDCK) cells, which are known to be susceptible to epsilon toxin, were used as a positive control. No morphological alterations were observed in any of the endothelial cell cultures tested, even after challenging with doses as high as 1200 MLD50/ml of epsilon toxin. MDCK cells showed contour rounding and nuclear condensation as early as 30 min after exposure to 100 MLD50/ml of epsilon toxin and after 60 min of exposure to 12.5 MLD50/ml of the same toxin. All the MDCK cells were dead after 3 h of exposure to all concentrations of epsilon toxin. The results indicate that ovine, caprine and bovine endothelial cells are not morphologically responsive to the action of epsilon toxin in vitro. PMID:10493114

  18. Allele doses of apolipoprotein E type {epsilon}4 in sporadic late-onset Alzheimer`s disease

    SciTech Connect

    Lucotte, G.; Aouizerate, A.; Gerard, N.

    1995-12-18

    Apoliprotein E, type {epsilon}4 allele (ApoE-{epsilon}4) is associated with late-onset sporadic Alzheimer`s disease (AD). We have found that the cumulative probability of remaining unaffected over time decreases for each dose of ApoE-{epsilon}4 in sporadic, late-onset French AD. The effect of genotypes on age at onset of AD was analyzed using the product limit method, to compare unaffected groups during aging. 26 refs., 2 figs., 1 tab.

  19. The early effects of Clostridium perfringens type D epsilon toxin in ligated intestinal loops of goats and sheep.

    PubMed

    Fernandez Miyakawa, M E; Uzal, F A

    2003-04-01

    Clostridium perfringens type D produces enterotoxaemia in goats, sheep and other animals. The disease is caused by C. perfringens epsilon toxin and, while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by the ovine than by the caprine intestine. In an attempt to clarify this, we examined the early effects of epsilon toxin on caprine and ovine intestine. Intestinal loop assays were performed to analyse the physiological and morphological changes induced by epsilon toxin in the intestine of these species. Fluid accumulation was observed in caprine and ovine ileum and colon treated with epsilon toxin. Ileal loops from goats treated with epsilon toxin retained sodium and water earlier than ovine ileal loops treated with the same toxin. Histological analysis showed morphological alterations in the colon of both species as early as 2 h after the commencement of epsilon toxin treatment: these changes were more marked in goats than in sheep. No morphological changes were observed in the ileum of either species after 4 h incubation with epsilon toxin. These results suggest that epsilon toxin modifies ion and water transport in the small and the large intestine of goats and sheep through different mechanisms. PMID:12777097

  20. Stabilization of the HCP [var epsilon] phase in an Fe-21%Mn alloy subjected to cathodic hydrogen charging

    SciTech Connect

    Aikawa, T.; Nishino, Y.; Asano, S. )

    1993-07-01

    Cathodic hydrogen charging induces some phase transformation in the surface layer of FCC iron alloys. Such a hydrogen-induced phase transformation has extensively been studied for austenitic stainless steels, where the FCC [gamma] phase transforms partially into an HCP [var epsilon][sub H] phase. The formation of the [var epsilon][sub H] phase has also been found in FCC Fe-Mn alloys and Fe-Ni-Mn alloys. The [var epsilon][sub H] phase is unstable in the absence of hydrogen and decomposes to an [var epsilon] martensite in the metastable FCC alloys. In order to make clear the nature of the [var epsilon][sub H] phase, it is necessary to reveal the stability of the [var epsilon] phase in FCC iron alloys during cathodic hydrogen charging. In the present study, the authors employed an Fe-21%Mn alloy which consisted of a mixture of the [gamma] and [var epsilon] phases in an as-quenched state. For this two-phase alloy, the [var epsilon] [r arrow] [gamma] transformation has so far been examined by a high-pressure equilibration method in a hydrogen-gas atmosphere. The purpose of this study is to compare the stability of the [gamma] phases in the two-phase alloy subjected to cathodic hydrogen charging in an aqueous solution. The influence of hydrogen on the [var epsilon] phase stability will be discussed, including the transformation in a hydrogen-gas atmosphere.

  1. Development of a hybrid k-epsilon turbulence model for swirling recirculating flows under moderate to strong swirl intensities

    NASA Astrophysics Data System (ADS)

    Chang, Keh-Chin; Chen, Ching-Shun

    1993-03-01

    A hybrid k-epsilon turbulence model, based on the concept that the modification of anisotropic effects should not be made in the flow regions inherent to small streamline curvatures, has been developed and examined with the swirling recirculating flows, with the swirl levels ranging from 0.6 to 1.23 in an abrupt pipe expansion. A fairly satisfactory agreement of model predictions with the experimental data shows that this hybrid k-epsilon model can perform better simulation of swirling recirculating flows as compared to the standard k-epsilon model and the modified k-epsilon model proposed by Abujelala and Lilley (1984).

  2. Clostridium perfringens epsilon toxin increases the small intestinal permeability in mice and rats.

    PubMed

    Goldstein, Jorge; Morris, Winston E; Loidl, César Fabián; Tironi-Farinati, Carla; Tironi-Farinatti, Carla; McClane, Bruce A; Uzal, Francisco A; Fernandez Miyakawa, Mariano E

    2009-01-01

    Epsilon toxin is a potent neurotoxin produced by Clostridium perfringens types B and D, an anaerobic bacterium that causes enterotoxaemia in ruminants. In the affected animal, it causes oedema of the lungs and brain by damaging the endothelial cells, inducing physiological and morphological changes. Although it is believed to compromise the intestinal barrier, thus entering the gut vasculature, little is known about the mechanism underlying this process. This study characterizes the effects of epsilon toxin on fluid transport and bioelectrical parameters in the small intestine of mice and rats. The enteropooling and the intestinal loop tests, together with the single-pass perfusion assay and in vitro and ex vivo analysis in Ussing's chamber, were all used in combination with histological and ultrastructural analysis of mice and rat small intestine, challenged with or without C. perfringens epsilon toxin. Luminal epsilon toxin induced a time and concentration dependent intestinal fluid accumulation and fall of the transepithelial resistance. Although no evident histological changes were observed, opening of the mucosa tight junction in combination with apoptotic changes in the lamina propria were seen with transmission electron microscopy. These results indicate that C. perfringens epsilon toxin alters the intestinal permeability, predominantly by opening the mucosa tight junction, increasing its permeability to macromolecules, and inducing further degenerative changes in the lamina propria of the bowel. PMID:19763257

  3. Clostridium perfringens Epsilon Toxin Increases the Small Intestinal Permeability in Mice and Rats

    PubMed Central

    Goldstein, Jorge; Morris, Winston E.; Loidl, César Fabián; Tironi-Farinatti, Carla; McClane, Bruce A.; Uzal, Francisco A.; Fernandez Miyakawa, Mariano E.

    2009-01-01

    Epsilon toxin is a potent neurotoxin produced by Clostridium perfringens types B and D, an anaerobic bacterium that causes enterotoxaemia in ruminants. In the affected animal, it causes oedema of the lungs and brain by damaging the endothelial cells, inducing physiological and morphological changes. Although it is believed to compromise the intestinal barrier, thus entering the gut vasculature, little is known about the mechanism underlying this process. This study characterizes the effects of epsilon toxin on fluid transport and bioelectrical parameters in the small intestine of mice and rats. The enteropooling and the intestinal loop tests, together with the single-pass perfusion assay and in vitro and ex vivo analysis in Ussing's chamber, were all used in combination with histological and ultrastructural analysis of mice and rat small intestine, challenged with or without C. perfringens epsilon toxin. Luminal epsilon toxin induced a time and concentration dependent intestinal fluid accumulation and fall of the transepithelial resistance. Although no evident histological changes were observed, opening of the mucosa tight junction in combination with apoptotic changes in the lamina propria were seen with transmission electron microscopy. These results indicate that C. perfringens epsilon toxin alters the intestinal permeability, predominantly by opening the mucosa tight junction, increasing its permeability to macromolecules, and inducing further degenerative changes in the lamina propria of the bowel. PMID:19763257

  4. Investigations of the adjustment of nitrogen and carbon in the {epsilon}-phase

    SciTech Connect

    Pietzsch, S.; Bohmer, S.; Spies, H.J.

    1995-12-31

    The phase field of {epsilon}-(carbo)nitride together with its equilibrium solubility of nitrogen and carbon can be calculated by thermodynamic models as a function of the temperature and the nitriding potential K{sub N} = {var_phi}(NH{sub 3})/{var_phi}(H{sub 2}) and carburizing potential K{sub c} = {var_phi}(CO){var_phi}(H{sub 2})/{var_phi}(H{sub 2}O). The results are presented in the form of modified Lehrer diagrams. The calculations show that a non-coupled adjustment of nitrogen and carbon is possible in the {epsilon}-phase by a suitable choice of the nitriding and carburizing potential. However, the experimental adjustment of the interstitials in the {epsilon}-phase is not satisfactory. Therefore, iron foils (thickness: 10 {micro}m) were nitrided in retort-type furnaces (150 liters) with precisely controlled atmospheres. The gas mixtures were controlled by gas chromatographic analysis and oxygen probes. The complete transformation of the iron foils into the {epsilon}-(carbo)nitride was controlled by X-ray analysis. The nitrogen and carbon concentrations in the {epsilon}-phase were determined by chemical analysis. The experimental results are in good agreement with the calculations. The investigations of nitrided specimens allow conclusions for the production of compound layers with various nitrogen and carbon concentrations by the purposeful adjustment of the nitriding and carburizing potential. This is of interest for the corrosion and wear resistance of nitrided components.

  5. Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination

    PubMed Central

    Linden, Jennifer R.; Ma, Yinghua; Zhao, Baohua; Harris, Jason Michael; Rumah, Kareem Rashid; Schaeren-Wiemers, Nicole

    2015-01-01

    ABSTRACT Clostridium perfringens epsilon toxin (ε-toxin) is responsible for a devastating multifocal central nervous system (CNS) white matter disease in ruminant animals. The mechanism by which ε-toxin causes white matter damage is poorly understood. In this study, we sought to determine the molecular and cellular mechanisms by which ε-toxin causes pathological changes to white matter. In primary CNS cultures, ε-toxin binds to and kills oligodendrocytes but not astrocytes, microglia, or neurons. In cerebellar organotypic culture, ε-toxin induces demyelination, which occurs in a time- and dose-dependent manner, while preserving neurons, astrocytes, and microglia. ε-Toxin specificity for oligodendrocytes was confirmed using enriched glial culture. Sensitivity to ε-toxin is developmentally regulated, as only mature oligodendrocytes are susceptible to ε-toxin; oligodendrocyte progenitor cells are not. ε-Toxin sensitivity is also dependent on oligodendrocyte expression of the proteolipid myelin and lymphocyte protein (MAL), as MAL-deficient oligodendrocytes are insensitive to ε-toxin. In addition, ε-toxin binding to white matter follows the spatial and temporal pattern of MAL expression. A neutralizing antibody against ε-toxin inhibits oligodendrocyte death and demyelination. This study provides several novel insights into the action of ε-toxin in the CNS. (i) ε-Toxin causes selective oligodendrocyte death while preserving all other neural elements. (ii) ε-Toxin-mediated oligodendrocyte death is a cell autonomous effect. (iii) The effects of ε-toxin on the oligodendrocyte lineage are restricted to mature oligodendrocytes. (iv) Expression of the developmentally regulated proteolipid MAL is required for the cytotoxic effects. (v) The cytotoxic effects of ε-toxin can be abrogated by an ε-toxin neutralizing antibody. PMID:26081637

  6. Packaging of DNA by bacteriophage epsilon15: structure, forces, and thermodynamics.

    PubMed

    Petrov, Anton S; Lim-Hing, Krista; Harvey, Stephen C

    2007-07-01

    The structure of bacteriophage epsilon15 has recently been determined by 3D reconstruction of single particle cryo-electron microscopy images. Although this study revealed that the viral genome inside the bacteriophage is on average coaxially spooled, individual DNA conformations inside the capsid could not be determined. In the current study, we present the results of 40 independent simulations of DNA packaging into epsilon15 using the previously described low-resolution model for bacteriophages. In addition to coaxially spooled conformations, we also observe a number of folded-toroidal patterns, but the density averaged over all conformations closely resembles the experimental density. Thermodynamic analysis of the simulations predicts that a force of approximately 125 pN would be required to package DNA into epsilon15. We also show that the origin of this force is predominantly due to electrostatic and entropic contributions. However, the DNA conformation is determined primarily by the need to minimize the DNA bending energy. PMID:17637341

  7. The fundamental parameters of the chromospherically active K2 dwarf Epsilon Eridani

    NASA Technical Reports Server (NTRS)

    Drake, Jeremy J.; Smith, Geoffrey

    1993-01-01

    A silicon array detector was used to record regions exhibiting calcium and iron lines in the spectrum of the chromospherically active K2 dwarf Epsilon Eri at a resolution of 120,000 and with an SNR of not less than 200. The effective temperature, surface gravity, logarithmic iron and calcium abundances, and microturbulence are determined. Three high-excitation lines of Fe I were found to yield anomalously low iron abundances; it is postulated that the origin of the anomaly lies in the nonthermal excitation of the upper photosphere caused by chromospheric emission. It is shown that Epsilon Eri is in an evolutionary stage consistent with an M/solar mass of 0.85 theoretical zero-age main-sequence model. It is suggested that Epsilon Eri is almost certainly a young star of slightly less than one solar mass.

  8. Computation of turbulent flows using an extended k-epsilon turbulence closure model

    NASA Technical Reports Server (NTRS)

    Chen, Y.-S.; Kim, S.-W.

    1987-01-01

    An extended kappa-epsilon turbulence model is proposed and tested with successful results. An improved transport equation for the rate of dissipation of the turbulent kinetic energy, epsilon, is proposed. The proposed model gives more effective response to the energy production rate than does the standard kappa-epsilon turbulence model. An extra time scale of the production range is included in the dissipation rate equation. This enables the present model to perform equally well for several turbulent flows with different characteristics, e.g., plane and axisymmetric jets, turbulent boundary layer flow, turbulent flow over a backward-facing step, and a confined turbulent swirling flow. A second-order accurate finite difference boundary layer code and a nearly second-order accurate finite difference elliptic flow solver are used for the present numerical computations.

  9. Lattice study of meson correlators in the {epsilon}-regime of two-flavor QCD

    SciTech Connect

    Fukaya, H.; Aoki, S.; Hashimoto, S.; Kaneko, T.; Yamada, N.; Matsufuru, H.; Noaki, J.; Ogawa, K.; Onogi, T.

    2008-04-01

    We calculate mesonic two-point functions in the {epsilon}-regime of two-flavor QCD on the lattice with exact chiral symmetry. We use gauge configurations of size 16{sup 3}x32 at a{approx}0.11 fm generated with dynamical overlap fermions. The sea quark mass is fixed at around 3 MeV and the valence quark mass is varied in the range 1-4 MeV, both of which are in the {epsilon}-regime. We find a good consistency with the expectations from the next-to-leading order calculation in the {epsilon}-expansion of (partially quenched) chiral perturbation theory. From a fit we obtain the pion decay constant F=87.3(5.6) MeV and the chiral condensate {sigma}{sup MS}=[239.8(4.0) MeV]{sup 3} up to next-to-next-to-leading order contributions.

  10. Node-avoiding Levy flight - A numerical test of the epsilon expansion. [random walk

    NASA Technical Reports Server (NTRS)

    Halley, J. W.; Nakanishi, H.

    1985-01-01

    A study is conducted of an extension of Levy flight to include self-repulsion in the path of the walk. The extension is called node-avoiding Levy flight and its equivalence to the n approaches 0 limit of a statistical mechanical model for a magnetic system with long-range interactions between the spins is shown. By use of this equivalence it is possible to make a detailed comparison beween the results of the epsilon expansion for the magnetic model, a Monte Carlo simulation of the Levy flight model, and the results of a Flory-type argument. This is the first comparison of the epsilon expansion for epsilon much less than 1 with a numerical simulation for any model. Some speculations are made on applications of the model of node-avoiding Levy flight.

  11. Evidence on primate phylogeny from epsilon-globin gene sequences and flanking regions.

    PubMed

    Porter, C A; Sampaio, I; Schneider, H; Schneider, M P; Czelusniak, J; Goodman, M

    1995-01-01

    Phylogenetic relationships among various primate groups were examined based on sequences of epsilon-globin genes. epsilon-globin genes were sequenced from five species of strepsirhine primates. These sequences were aligned and compared with other known primate epsilon-globin sequences, including data from two additional strepsirhine species, one species of tarsier, 19 species of New World monkeys (representing all extant genera), and five species of catarrhines. In addition, a 2-kb segment upstream of the epsilon-globin gene was sequenced in two of the five strepsirhines examined. This upstream sequence was aligned with five other species of primates for which data are available in this segment. Domestic rabbit and goat were used as outgroups. This analysis supports the monophyly of order Primates but does not support the traditional prosimian grouping of tarsiers, lorisoids, and lemuroids; rather it supports the sister grouping of tarsiers and anthropoids into Haplorhini and the sister grouping of lorisoids and lemuroids into Strepsirhini. The mouse lemur (Microcebus murinus) and dwarf lemur (Cheirogaleus medius) appear to be most closely related to each other, forming a clade with the lemuroids, and are probably not closely related to the lorisoids, as suggested by some morphological studies. Analysis of the epsilon-globin data supports the hypothesis that the aye-aye (Daubentonia madagascariensis) shares a sister-group relationship with other Malagasy strepsirhines (all being classified as lemuroids). Relationships among ceboids agree with findings from a previous epsilon-globin study in which fewer outgroup taxa were employed. Rates of molecular evolution were higher in lorisoids than in lemuroids. PMID:7714911

  12. Anomalous nonlinear absorption in epsilon-near-zero materials: optical limiting and all-optical control.

    PubMed

    Vincenti, M A; de Ceglia, D; Scalora, Michael

    2016-08-01

    We investigate nonlinear absorption in films of epsilon-near-zero materials. The combination of large local electric fields at the fundamental frequency and material losses at the harmonic frequencies induce unusual intensity-dependent phenomena. We predict that the second-order nonlinearity of a low-damping, epsilon-near-zero slab produces an optical limiting effect that mimics a two-photon absorption process. Anomalous absorption profiles that depend on low permittivity values at the pump frequency are also predicted for third-order nonlinearities. These findings suggest new opportunities for all-optical light control and novel ways to design reconfigurable and tunable nonlinear devices. PMID:27472631

  13. Correlation between In Vitro Cytotoxicity and In Vivo Lethal Activity in Mice of Epsilon Toxin Mutants from Clostridium perfringens

    PubMed Central

    Dorca-Arévalo, Jonatan; Pauillac, Serge; Díaz-Hidalgo, Laura; Martín-Satué, Mireia; Popoff, Michel R.; Blasi, Juan

    2014-01-01

    Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB. PMID:25013927

  14. Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens.

    PubMed

    Dorca-Arévalo, Jonatan; Pauillac, Serge; Díaz-Hidalgo, Laura; Martín-Satué, Mireia; Popoff, Michel R; Blasi, Juan

    2014-01-01

    Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB. PMID:25013927

  15. Modification of the Two-equation Turbulence Model in NPARC to a Chien Low Reynolds Number K-epsilon Formulation

    NASA Technical Reports Server (NTRS)

    Georgiadis, Nicholas J.; Chitsomboon, Tawit; Zhu, Jiang

    1994-01-01

    This report documents the changes that were made to the two-equation k-epsilon turbulence model in the NPARC (National-PARC) code. The previous model based on the low Reynolds number model of Speziale, was replaced with the low Reynolds number k-epsilon model of Chien. The most significant difference was in the turbulent Prandtl numbers appearing in the diffusion terms of the k and epsilon transport equations. A new inflow boundary condition and stability enhancements were also implemented into the turbulence model within NPARC. The report provides the rationale for making the change to the Chien model, code modifications required, and comparisons of the performances of the new model with the previous k-epsilon model and algebraic models used most often in PARC/NPARC. The comparisons show that the Chien k-epsilon model installed here improves the capability of NPARC to calculate turbulent flows.

  16. Numerical solution of turbulent flow past a backward facing step using a nonlinear K-epsilon model

    NASA Technical Reports Server (NTRS)

    Speziale, C. G.; Ngo, Tuan

    1987-01-01

    The problem of turbulent flow past a backward facing step is important in many technological applications and has been used as a standard test case to evaluate the performance of turbulence models in the prediction of separated flows. It is well known that the commonly used kappa-epsilon (and K-l) models of turbulence yield inaccurate predictions for the reattachment points in this problem. By an analysis of the mean vorticity transport equation, it will be argued that the intrinsically inaccurate prediction of normal Reynolds stress differences by the Kappa-epsilon and K-l models is a major contributor to this problem. Computations using a new nonlinear kappa-epsilon model (which alleviates this deficiency) are made with the TEACH program. Comparisons are made between the improved results predicted by this nonlinear kappa-epsilon model and those obtained from the linear kappa-epsilon model as well as from second-order closure models.

  17. N epsilon-acetyl-beta-lysine: an osmolyte synthesized by methanogenic archaebacteria.

    PubMed Central

    Sowers, K R; Robertson, D E; Noll, D; Gunsalus, R P; Roberts, M F

    1990-01-01

    Methanosarcina thermophila, a nonmarine methanogenic archaebacterium, can grow in a range of saline concentrations. At less than 0.4 M NaCl, Ms. thermophila accumulated glutamate in response to increasing osmotic stress. At greater than 0.4 M NaCl, this organism synthesized a modified beta-amino acid that was identified as N epsilon-acetyl-beta-lysine by NMR spectroscopy and ion-exchange HPLC. This beta-amino acid derivative accumulated to high intracellular concentrations (up to 0.6 M) in Ms. thermophila and in another methanogen examined--Methanogenium cariaci, a marine species. The compound has features that are characteristic of a compatible solute: it is neutrally charged at physiological pH and it is highly soluble. When the cells were grown in the presence of exogenous glycine betaine, a physiological compatible solute, N epsilon-acetyl-beta-lysine synthesis was repressed and glycine betaine was accumulated. N epsilon-acetyl-beta-lysine was synthesized by species from three phylogenetic families when grown in high solute concentrations, suggesting that it may be ubiquitous among the methanogens. The ability to control the biosynthesis of N epsilon-acetyl-beta-lysine in response to extracellular solute concentration indicates that the methanogenic archaebacteria have a unique beta-amino acid biosynthetic pathway that is osmotically regulated. PMID:2123548

  18. IKK{epsilon} modulates RSV-induced NF-{kappa}B-dependent gene transcription

    SciTech Connect

    Bao Xiaoyong; Indukuri, Hemalatha; Liu Tianshuang; Liao Suiling; Tian, Bing; Brasier, Allan R.; Garofalo, Roberto P.; Casola, Antonella

    2010-12-20

    Respiratory syncytial virus (RSV), a negative-strand RNA virus, is the most common cause of epidemic respiratory disease in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-{kappa}B (NF-{kappa}B). In this study we have investigated the role of the non canonical I{kappa}B kinase (IKK){epsilon} in modulating RSV-induced NF-{kappa}B activation. Our results show that inhibition of IKK{epsilon} activation results in significant impairment of viral-induced NF-{kappa}B-dependent gene expression, through a reduction in NF-{kappa}B transcriptional activity, without changes in nuclear translocation or DNA-binding activity. Absence of IKK{epsilon} results in a significant decrease of RSV-induced NF-{kappa}B phosphorylation on serine 536, a post-translational modification important for RSV-induced NF-{kappa}B-dependent gene expression, known to regulate NF-{kappa}B transcriptional activity without affecting nuclear translocation. This study identifies a novel mechanism by which IKK{epsilon} regulates viral-induced cellular signaling.

  19. N. sup. var epsilon. -acetyl-. beta. -lysine: An osmolyte synthesized by mothanogenic archaebacteria

    SciTech Connect

    Sowers, K.R.; Gunsalus, R.P. ); Robertson, D.E.; Noll, D.; Roberts, M.F. )

    1990-12-01

    Methanosarcina thermophila, a nonmarine methanogenic archaebacterium, can grow in a range of saline concentrations. At less than 0.4 M NaCl, Ms. thermophila accumulated glutamate in response to increasing osmotic stress. At greater than 0.4 M NaCl, this organism synthesized a modified {beta}-amino acid that was identified as N{sup {var epsilon}}-acetyl-{beta}-lysine by NMR spectroscopy and ion-exchange HPLC. This {beta}-amino acid derivative accumulated to high intracellular concentrations (up to 0.6 M) in Ms. thermophila and in another methanogen examined - Methanogenium cariaci, a marine species. The compound has features that are characteristic of a compatible solute: it is neutrally charged at physiological pH and it is highly soluble. When the cells were grown in the presence of exogenous glycine betaine, a physiological pH and it is highly soluble. When the cells were grown in the presence of exogenous glycine betaine, a physiological compatible solute, N{sup {var epsilon}}-acetyl-{beta}-lysine synthesis was repressed and glycine betaine was accumulated. N{sup {var epsilon}}-Acetyl-{beta}-lysine was synthesized by species from three phylogenetic families when grown in high solute concentrations, suggesting that it may be ubiquitous among the methanogens. The ability to control the biosynthesis of N{sup {var epsilon}}-acetyl-{beta}-lysine in response to extracellular solute concentration indicates that the methanogenic archaebacteria have a unique {beta}-amino acid biosynthetic pathway that is osmotically regulated.

  20. Using contour plots in elecgroproduction to examine regions in {epsilon}, Q{sup 2}, W space

    SciTech Connect

    Funsten, H.

    1994-04-01

    In determining incident CEBAF beam energies for CLAS electroproduction experiments that separate the longitudinal and transverse cross section components, contour plots of {epsilon} defined over a 2 dimensional Q{sup 2}, W space can be useful. This note describes an approximate method of constructing such plots.

  1. Epsilon Metal Waste Form for Immobilization of Noble Metals from Used Nuclear Fuel

    SciTech Connect

    Crum, Jarrod V.; Strachan, Denis M.; Rohatgi, Aashish; Zumhoff, Mac R.

    2013-10-01

    Epsilon metal (ε-metal), an alloy of Mo, Pd, Rh, Ru, and Tc, is being developed as a waste form to treat and immobilize the undissolved solids and dissolved noble metals from aqueous reprocessing of commercial used nuclear fuel. Epsilon metal is an attractive waste form for several reasons: increased durability relative to borosilicate glass, it can be fabricated without additives (100% waste loading), and in addition it also benefits borosilicate glass waste loading by eliminating noble metals from the glass and thus the processing problems related there insolubility in glass. This work focused on the processing aspects of the epsilon metal waste form development. Epsilon metal is comprised of refractory metals resulting in high reaction temperatures to form the alloy, expected to be 1500 - 2000°C making it a non-trivial phase to fabricate by traditional methods. Three commercially available advanced technologies were identified: spark-plasma sintering, microwave sintering, and hot isostatic pressing, and investigated as potential methods to fabricate this waste form. Results of these investigations are reported and compared in terms of bulk density, phase assemblage (X-ray diffraction and elemental analysis), and microstructure (scanning electron microscopy).

  2. The Students' Take on the Epsilon-Delta Definition of a Limit

    ERIC Educational Resources Information Center

    Fernandez, Eileen

    2004-01-01

    This paper describes a sequence of lessons from two Calculus I classes for teaching the epsilon-delta definition of a limit. In these lessons, the author elicited students' misconceptions and perceptions of this definition through a reading/writing lesson and then used these student ideas to design a lesson aimed at addressing these misconceptions…

  3. Perfect electromagnetic absorption using graphene and epsilon-near-zero metamaterials

    NASA Astrophysics Data System (ADS)

    Lobet, Michaël; Majerus, Bruno; Henrard, Luc; Lambin, Philippe

    2016-06-01

    The ability of graphene/polymer heterostructures to absorb GHz electromagnetic radiation was recently evidenced both theoretically and experimentally [Batrakov et al., Sci. Rep. 4, 7191 (2014), 10.1038/srep07191 and Lobet et al., Nanotechnology 26, 285702 (2015), 10.1088/0957-4484/26/28/285702]. Maximum absorption was shown to depend solely on refractive indices of incident and emergence media once impedance matching conditions are fulfilled. In this paper, analytical models and numerical simulations are performed for both semi-infinite and finite slab substrate. We evidenced that only three graphene layers separated by a dielectric spacer and an epsilon-near-zero metamaterial as emergence medium allow a perfect absorption for normal incidence. The use of lossless epsilon-near-zero metamaterial prevents radiations to go through the device, because of infinite impedance, and forces them to be totally absorbed in the dissipative medium (graphene). The device is proved to be robust regarding angular incidence up to 45 deg for a semi-infinite epsilon-near-zero metamaterial. The proposed strategy is universal and can be applied to any kind of two-dimensional dissipative materials lying on epsilon-near-zero metamaterial. The proposed absorber does not rely on surface patterning or texturing and hence is more appealing for device applications.

  4. Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin.

    PubMed

    McClain, Mark S; Cover, Timothy L

    2007-04-01

    The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin. PMID:17261609

  5. An Optimality Theoretic Account of Hungarian ESL Learners' Acquisition of /[epsilon]/ and /[ash]/

    ERIC Educational Resources Information Center

    Bunta, Ferenc; Major, Roy C.

    2004-01-01

    This paper provides an Optimality Theoretic account of how Hungarian learners of English acquire /[epsilon]/ and /[ash]/. It is hypothesized that as the learners' pronunciation becomes more nativelike, L1 transfer substitutions will diminish; non-transfer substitutions will be especially prevalent in the intermediate stages, and that all learners…

  6. Experience with k-epsilon turbulence models for heat transfer computations in rotating

    NASA Technical Reports Server (NTRS)

    Tekriwal, Prabbat

    1995-01-01

    This viewgraph presentation discusses geometry and flow configuration, effect of y+ on heat transfer computations, standard and extended k-epsilon turbulence model results with wall function, low-Re model results (the Lam-Bremhorst model without wall function), a criterion for flow reversal in a radially rotating square duct, and a summary.

  7. Experience with k-epsilon turbulence models for heat transfer computations in rotating

    NASA Astrophysics Data System (ADS)

    Tekriwal, Prabbat

    1995-03-01

    This viewgraph presentation discusses geometry and flow configuration, effect of y+ on heat transfer computations, standard and extended k-epsilon turbulence model results with wall function, low-Re model results (the Lam-Bremhorst model without wall function), a criterion for flow reversal in a radially rotating square duct, and a summary.

  8. Two-flavor lattice QCD in the {epsilon} regime and chiral random matrix theory

    SciTech Connect

    Fukaya, H.; Aoki, S.; Chiu, T. W.; Ogawa, K.; Hashimoto, S.; Kaneko, T.; Yamada, N.; Matsufuru, H.; Noaki, J.; Onogi, T.

    2007-09-01

    The low-lying eigenvalue spectrum of the QCD Dirac operator in the {epsilon} regime is expected to match with that of chiral random matrix theory (ChRMT). We study this correspondence for the case including sea quarks by performing two-flavor QCD simulations on the lattice. Using the overlap fermion formulation, which preserves exact chiral symmetry at finite lattice spacings, we push the sea quark mass down to {approx}3 MeV on a 16{sup 3}x32 lattice at a lattice spacing a{approx_equal}0.11 fm. We compare the low-lying eigenvalue distributions and find a good agreement with the analytical predictions of ChRMT. By matching the lowest-lying eigenvalue we extract the chiral condensate, {sigma}{sup MS}(2 GeV)=(251{+-}7{+-}11 MeV){sup 3}, where errors represent statistical and higher order effects in the {epsilon} expansion. We also calculate the eigenvalue distributions on the lattices with heavier sea quarks at two lattice spacings. Although the {epsilon} expansion is not applied for those sea quarks, we find a reasonable agreement of the Dirac operator spectrum with ChRMT. The value of {sigma}, after extrapolating to the chiral limit, is consistent with the estimate in the {epsilon} regime.

  9. [Bacteria that degrade low-molecular linear epsilon-caprolactam olygomers].

    PubMed

    Esikova, T Z; Akatova, E V; Taran, S A

    2014-01-01

    Five bacterial strains with the unique ability to utilize low-molecular linear caprolactam olygomers (nylon olygomers) were isolated from soil samples contaminated with industrial wastes of epsilon-caprolactam. Based on the properties studied and also on the analysis of 16S rRNA gene nucleotide sequences, the strains BS2,BS3, BS9, BS38, and BS57 were classified to the general Arthrobacter, Brevibacterium, Microbacteriun, Gulosibacter, and Achromobacter, respectively. All of the strains also utilized 6-aminohexanoic and adipic acids, which are intermidiates of the epsilon-caprolactam catabolism. This indirectly points to the fact that degradation of olygomers in these bacteria occurs via the monomer degradation pathway. The BS9 and BS57 strains utilized only olygomers of the epsilon-caprolactam, while BS2, BS3, and BS38 also degraded epsilon-caprolactam and its homologs, enantolactam and caprylolactam, which differentiates the latter from the previously known degraders of olygomers and suggests the presence in these strains of enzymes with lactam hydrolase activity, in addition to 6-aminohexanoate-dimer hydrolase. PMID:25707105

  10. The Use of Visual Approach in Teaching and Learning the Epsilon-Delta Definition of Continuity

    ERIC Educational Resources Information Center

    Pešic, Duška; Pešic, Aleksandar

    2015-01-01

    In this paper we introduce a new collaborative technique in teaching and learning the epsilon-delta definition of a continuous function at the point from its domain, which connects mathematical logic, combinatorics and calculus. This collaborative approach provides an opportunity for mathematical high school students to engage in mathematical…

  11. THE FATE OF THE EPSILON PHASE IN UO2 OF THE OKLO NATURAL FISSON REACTORS

    SciTech Connect

    S. Utsunomiya; R.C. Ewing

    2005-09-08

    In spent nuclear fuel (SNF), the micron- to nano-sized epsilon phase (Mo-Ru-Pd-Tc-Rh) is an important host of {sup 99}Tc which has a long half life (2.13 x 10{sup 5} years) and can be an important contributor to dose in safety assessments of nuclear waste repositories. In order to examine the occurrence and the fate of the epsilon phase during the corrosion of SNF over long time periods, samples of uraninite from the Oklo natural reactors ({approx}2.0 Ga) have been investigated using transmission electron microscopy (TEM). Because essentially all of the {sup 99}Tc has decayed to {sup 99}Ru, this study focuses on 4d-elements of the epsilon phase. Samples were obtained from the research collection at University of Michigan representing reactor zone (RZ) 10 (836, 819,687) and from RZ 13 (864,910). Several phases with 4d-metals have been identified within UO{sub 2} matrix at the scale of 50-700 nm; fioodite, PdBi{sub 2}, with trace amounts of As, Fe, and Te, and palladodymite or rhodarsenide, (Pd,Rh){sub 2}As. The most abundant 4d-metal phase is ruthenarsinite, (Ru,Ni)As, which has a representative composition: As, 59.9; Coy 2.5; Ni, 5.2; Ru, 18.6; Rh, 8.4; Pd, 3.1; Sb, 2.4 in atomic%. Ruthenarsenite nanoparticles are typically surrounded by Pb-rich domains, galena in most cases; whereas, some particles reveal a complexly zoned composition within the grain, such as a Pb-rich domain at the core and enrichment of Ni, Co, and As at the rim. Some ruthenarsenites and Rh-Bi-particles are embedded in surrounding alteration products, e.g., chlorite, adjacent to uraninite (no further than {approx}5 {micro}m). A few of those particles are still coated by a Pb-rich layer. Based on these results, the history that epsilon phases have experienced can be described as follows: (1) The original epsilon phase was changed to, in most cases, ruthenarsenite, by As-rich fluids with other trace metals. Dissolution and a simultaneous precipitation may be responsible for the phase change. (2

  12. Apolipoprotein E epsilon4 allele differentiates the clinical response to donepezil in Alzheimer's disease.

    PubMed

    Bizzarro, A; Marra, C; Acciarri, A; Valenza, A; Tiziano, F D; Brahe, C; Masullo, C

    2005-01-01

    The existence of an association between apolipoprotein E (APOE) and Alzheimer's disease (AD) has been reported in several studies. The possession of an ApoE epsilon4 allele is now considered a genetic risk factor for sporadic AD. There has been a growing agreement about the role exerted by the ApoE epsilon4 allele on the neuropsychological profile and the rate of cognitive decline in AD patients. However, a more controversial issue remains about a possible influence of the APOE genotype on acetylcholinesterase inhibitor therapy response in AD patients. In order to address this issue, 81 patients diagnosed as having probable AD were evaluated by a complete neuropsychological test battery at the time of diagnosis (baseline) and after 12-16 months (retest). Patients were divided into two subgroups: (1) treated with donepezil at a dose of 5 mg once a day (n = 41) and (2) untreated (n = 40). Donepezil therapy was started after baseline evaluation. The APOE genotype was determined according to standardized procedures. We evaluated the possible effect of the APOE genotype on the neuropsychological tasks in relation to donepezil therapy. The statistical analysis of the results showed a global worsening of cognitive performances for all AD patients at the retest. Differences in the clinical outcome were analysed in the four subgroups of AD patients for each neuropsychological task. ApoE epsilon4 carriers/treated patients had improved or unchanged scores at retest evaluation for the following tasks: visual and verbal memory, visual attention and inductive reasoning and Mini Mental State Examination. These results indicate an effect of donepezil on specific cognitive domains (attention and memory) in the ApoE epsilon4 carriers with AD. This might suggest an early identification of AD patients carrying at least one epsilon4 allele as responders to donepezil therapy. PMID:16103669

  13. Denitrification with epsilon-caprolactam by acclimated mixed culture and by pure culture of bacteria isolated from polyacrylonitrile fibre manufactured wastewater treatment system.

    PubMed

    Lee, C M; Wang, C C

    2004-01-01

    The aim of this study is to isolate denitrifying bacteria utilizing epsilon-caprolactam as the substrate, from a polyacrylonitrile fibre manufactured wastewater treatment system. The aim is also to compare the performance of PAN (polyacrylonitrile) mixed bacteria cultures acclimated to epsilon-caprolactam and isolated pure strain for treating different initial epsilon-caprolactam concentrations from synthetic wastewater under anoxic conditions. The result showed that the PAN mixed bacteria cultures acclimated to epsilon-caprolactam could utilize 1538.5 mg/l of epsilon-caprolactam as a substrate for denitrification. Sufficient time and about 2200 mg/l of nitrate were necessary for the complete epsilon-caprolactam removal. Paracoccus thiophilus was isolated from the polyacrylonitrile fibre manufactured wastewater treatment system and it could utilize 1722.5 mg/l of epsilon-caprolactam as a substrate for denitrification. About 3500 mg/l of nitrate was necessary for the complete removal of epsilon-caprolactam. When the initial epsilon-caprolactam concentration was below 784.3 mg/l, the removal efficiency of epsilon-caprolactam by Paracoccus thiophilus was better than that for the PAN mixed bacteria cultures. The growth of Paracoccus thiophilus was better. However, when the initial epsilon-caprolactam concentration was as high as 1445.8 mg/l, both the epsilon-caprolactam removal efficiency by Paracoccus thiophilus and Paracoccus thiophilus specific growth rate were similar to the PAN mixed bacteria cultures. PMID:15137443

  14. Does one need the O({epsilon})- and O({epsilon}{sup 2})-terms of one-loop amplitudes in a next-to-next-to-leading order calculation ?

    SciTech Connect

    Weinzierl, Stefan

    2011-10-01

    This article discusses the occurrence of one-loop amplitudes within a next-to-next-to-leading-order calculation. In a next-to-next-to-leading-order calculation, the one-loop amplitude enters squared and one would therefore naively expect that the O({epsilon})- and O({epsilon}{sup 2})-terms of the one-loop amplitudes are required. I show that the calculation of these terms can be avoided if a method is known, which computes the O({epsilon}{sup 0})-terms of the finite remainder function of the two-loop amplitude.

  15. Evaluation of different fluids for detection of Clostridium perfringens type D epsilon toxin in sheep with experimental enterotoxemia.

    PubMed

    Layana, Jorge E; Fernandez Miyakawa, Mariano E; Uzal, Francisco A

    2006-08-01

    Enterotoxemia caused by Clostridium perfringens type D is a highly lethal disease of sheep, goats and other ruminants. The diagnosis of this condition is usually confirmed by detection of epsilon toxin, a major exotoxin produced by C. perfringens types B and D, in the intestinal content of affected animals. It has been suggested that other body fluids can also be used for detection of epsilon toxin. This study was performed to evaluate the usefulness of intestinal content versus other body fluids in detecting epsilon toxin in cases of sheep enterotoxemia. Samples of duodenal, ileal and colon contents, pericardial and abdominal fluids, aqueous humor and urine from 15 sheep with experimentally induced enterotoxemia, were analysed for epsilon toxin using a capture ELISA. Epsilon toxin was detected in 92% of the samples of ileal content, 64% of the samples of duodenal content, 57% of the samples of colon content and in 7% of the samples of pericardial fluid and aqueous humor. No epsilon toxin was found in samples of abdominal fluid or urine from the animals with enterotoxemia or in any samples from six clinically healthy sheep used as negative controls. The results of this study indicate that with the diagnostic capture ELISA used, intestinal content (preferably ileum) should be used for C. perfringens type D epsilon toxin detection in suspected cases of sheep enterotoxemia. PMID:16857397

  16. Reverse phase passive haemagglutination and single radial immunodiffusion to detect epsilon antigen of Clostridium perfringens type D.

    PubMed

    Beh, K J; Buttery, S H

    1978-11-01

    Two in vitro immunological assays were developed for detection of the epsilon (epsilon) antigen of Cl. perfringens type D. It was found that the reverse phase passive haemagglutination assay (RPHA) was able to detect concentrations of epsilon-antigen as low as 6 x 10-7 mg/ml whereas the single radial immunodiffusion techniques (SRID) was capable of detecting concentrations of epsilon-antigen above 0.01 mg/ml. When applied to gut contents from freshly dead infected sheep the RPHA test was found to be more sensitive than mouse toxicity assay in detecting the presence of epsilon-antigen. However, very low titres were detected in gut contents from normal sheep which meant that in a diagnostic situation interpretation of RPHA titres would be difficult. No epsilon-antigen was detected by SRID in gut contents from normal sheep or in gut contents from freshly dead infected sheep. The SRID assay could detect epsilon-antigen in gut contents from infected sheep allowed to decompose for 20 h post-mortem. PMID:223537

  17. Heat transfer predictions with extended kappa-epsilon turbulence model in radical cooling ducts rotating in orthogonal mode

    NASA Astrophysics Data System (ADS)

    Tekriwal, P.

    1994-05-01

    Standard and extended kappa-epsilon turbulence closure models have been employed for three-dimensional heat transfer calculations for radially outward flow in rectangular and square cooling passages rotating in orthogonal mode. The objective of this modeling effort is to validate the numerical model in an attempt to fill the gap between model predictions and the experimental data for heat transfer in rotating systems. While the trend of heat transfer predictions by the standard kappa-epsilon turbulence model is satisfactory, the differences between the data and the predictions are approximately 30 percent or so in the case of high rotation number flow. The extended kappa-epsilon turbulence model takes an approach where an extra 'source' term based on a second time scale of the turbulent kinetic energy production rate is added to the equation for the dissipation rate of turbulent kinetic energy. This yields a more effective calculation of turbulent kinetic energy as compared to the standard kappa-epsilon turbulence model in the case of high rotation number and high density ratio flow. As a result, comparison with the experimental data available in the literature shows that an improvement of up to a significant 15 percent (with respect to data) in the heat transfer coefficient predictions is achieved over the standard kappa-epsilon model in the case of high rotation number flow. Comparisons between the results of the standard kappa-epsilon model and the extended formulation are made at different rotation numbers, different Reynolds numbers, and varying temperature ratio. The results of the extended kappa-epsilon turbulence model are either as good or better than those of the standard kappa-epsilon model in all these cases of parametric study. Thus, the extended kappa-epsilon turbulence model proves to be more general and reduces the discrepancy between the model predictions and the experimental data for heat transfer in rotating systems.

  18. Diacylglycerol kinase epsilon in bovine and rat photoreceptor cells. Light-dependent distribution in photoreceptor cells.

    PubMed

    Natalini, Paola M; Zulian, Sandra E; Ilincheta de Boschero, Mónica G; Giusto, Norma M

    2013-07-01

    The present study shows the selective light-dependent distribution of 1,2-diacylglycerol kinase epsilon (DAGKɛ) in photoreceptor cells from bovine and albino rat retina. Immunofluorescence microscopy in isolated rod outer segments from bleached bovine retinas (BBROS) revealed a higher DAGKɛ signal than that found in rod outer segments from dark-adapted bovine retinas (BDROS). The light-dependent outer segment localization of DAGKɛ was also observed by immunohistochemistry in retinas from albino rats. DAGK activity, measured in terms of phosphatidic acid formation from a) [(3)H]DAG and ATP in the presence of EGTA and R59022, a type I DAGK inhibitor, or b) [γ-(32)P]ATP and 1-stearoyl, 2-arachidonoylglycerol (SAG), was found to be significantly higher in BBROS than in BDROS. Higher light-dependent DAGK activity (condition b) was also found when ROS were isolated from dark-adapted rat retinas exposed to light. Western blot analysis of isolated ROS proteins from bovine and rat retinas confirmed that illumination increases DAGKɛ content in the outer segments of these two species. Light-dependent DAGKɛ localization in the outer segment was not observed when U73122, a phospholipase C inhibitor, was present prior to the exposure of rat eyecups (in situ model) to light. Furthermore, no increased PA synthesis from [(3)H]DAG and ATP was observed in the presence of neomycin prior to the exposure of bovine eyecups to light. Interestingly, when BBROS were pre-phosphorylated with ATP in the presence of 1,2-dioctanoyl sn-glycerol (di-C8) or phorbol dibutyrate (PDBu) as PKC activation conditions, higher DAGK activity was observed than in dephosphorylated controls. Taken together, our findings suggest that the selective distribution of DAGKɛ in photoreceptor cells is a light-dependent mechanism that promotes increased SAG removal and synthesis of 1-stearoyl, 2-arachidonoyl phosphatidic acid in the sensorial portion of this cell, thus demonstrating a novel mechanism of light

  19. Alterations in Fc[epsilon]RI induced by protoporphyrin plus long-wavelength ultraviolet light in mouse bone marrow-derived mast cells

    SciTech Connect

    Yen, A.; Barrett, K.E.; Gigli, I. ); Liu, F.T. )

    1993-07-15

    As previously reported, protoporphyrin plus long-wavelength UV light (PP/UVA) inhibits IgE-mediated degranulation of mouse bone marrow-derived mast cells, as assessed by measurement of the release of [beta]-hexosaminidase. This inhibitory effect was seen with cells sensitized with IgE either before or after PP/UVA treatment (57.8 and 55.35 inhibition, respectively). PP/UVA did not dissociate IgE already bound to cells as assessed either by measure of release of bound [sup 125]I-IgE or by flow cytometric analysis. Results from immunoadsorption followed by SDS-PAGE analysis suggested that PP/UVA treatment may cause stable conjugation of IgE to its receptor. In unsensitized cells, PP/UVA did not cause conjugation of the unoccupied Fc[epsilon]RI to other proteins in the plasma membrane. Nevertheless, Scatchard analysis revealed that PP/UVA decreased the number of Fc[epsilon]Ri per cell by 37% (0.95 [times] 10[sup 5] vs 1.51 [times] 10[sup 5] cell), whereas affinity of the receptor for IgE was comparable between PP/UVA-treated and untreated cells (3.40 nM vs 3.27 nM). Flow cytometric analysis also confirmed the decrease in Fc[epsilon]RI number in PP/UVA-treated unsensitized mouse bone marrow-derived mast cells. Although 84% of PP/UVA-treated and 82% of untreated cells expressed positive fluorescence when stained with FITC-conjugated IgE, fluorescence intensity was reduced by 40% after PP/UVA treatment. The authors conclude that PP/UVA alters the conformational structure and/or number of Fc[epsilon]RI expressed on the mast cell surface. This effect could potentially explain the ability of PP/UVA to inhibit mast cell secretory function and may be related to an ability of PP/UVA to alter the properties of the plasma membrane. 29 refs., 8 figs.

  20. Development of an injection molded poly(epsilon-caprolactone) intravaginal insert for the delivery of progesterone to cattle.

    PubMed

    Rathbone, Michael J; Bunt, Craig R; Ogle, Colin R; Burggraaf, Shane; Macmillan, Keith L; Pickering, Kim

    2002-12-13

    This paper reports experiments conducted to research, develop and clinically evaluate an injection molded intravaginal insert manufactured from the biodegradable polyester poly(epsilon-caprolactone). The study demonstrated that it is possible to engineer poly(epsilon-caprolactone) into a shape that is well retained, and can be used as a platform for the controlled delivery of progesterone via the vagina of cows. Field evaluation showed that the poly(epsilon-caprolactone) intravaginal inserts containing 10% (w/w) progesterone were at least as effective clinically as the commercially available CIDR intravaginal insert. PMID:12480312

  1. Association of the apolipoprotein E {epsilon}4 allele with clinical subtypes of autopsy-confirmed Alzheimer`s Disease

    SciTech Connect

    Zubenko, G.S.; Stiffler, S.; Kopp, U.

    1994-09-15

    Consistent with previous reports, we observed a significant association of the APOE {epsilon}4 allele with Alzheimer`s Disease (AD) in a series of 91 autopsy-confirmed cases. The {epsilon}4 allele frequency was higher in cases with a family history of AD-like dementia (0.54 {+-} 0.07), although the {epsilon}4 allele frequency in the AD cases with a negative family history (0.38 {+-} 0.05) remained significantly greater than that for the non-AD control group (0.13 {+-} 0.03). A similar increase in {epsilon}4 allele frequency (0.54 {+-} 0.07) was observed in the AD cases with amyloid angiopathy, compared to those who did not have amyloid angiopathy (0.35 {+-} 0.04). Contrary to previous reports, no effect of the dosage of the {epsilon}4 allele was found on the age of onset of dementia among the AD cases and, contrary to reports suggesting an association of {epsilon}4 and atherosclerosis, the {epsilon}4 allele frequency was similar in cases with or without concurrent brain infarcts. Modest but consistent correlations were observed between the dosage of {epsilon}4 alleles and the cortical density of senile plaques, but not neurofibrillary tangles. The last finding suggests that the pathogenic events mediated by the {epsilon}4 allele may be more directly involved in the formation of senile plaques, the identifying lesions in AD, than neurofibrillary tangles. A robust association of both the presence of an {epsilon}4 allele and a family history of AD-like dementia with concurrent amyloid angiopathy occurred within our sample of AD cases. This association arose from an interaction of the {epsilon}4 allele with a separate familial factor for which a family history of dementia served as a surrogate. These results suggest that amyloid angiopathy may be a common or central feature of a form of familial AD that is associated with the transmission of the APOE {epsilon}4 allele. 22 refs., 2 figs., 5 tabs.

  2. Limitations and empirical extensions of the k-epsilon model as applied to turbulent confined swirling flows

    NASA Technical Reports Server (NTRS)

    Lilley, D. G.; Abujelala, M. T.

    1984-01-01

    Shortcomings and recommended corrections to the standard two-equation k-epsilon turbulence model suggested by previous investigators are presented. They are assessed regarding their applicability to turbulent swirling recirculating flow. Recent experimental data on swirling confined flows, obtained with a five-hole pitot probe and a six-orientation hot-wire probe, are used to obtain optimum values of the turbulence parameters C-mu, C2, and sigma-epsilon for swirling flows. General predictions of moderately and strongly swirling flows with these values are more accurate than predictions with the standard or previous simple extensions of the k-epsilon turbulence model.

  3. p16INK4A tumor suppressor gene expression and CD3epsilon deficiency but not pre-TCR deficiency inhibit TAL1-linked T-lineage leukemogenesis.

    PubMed

    Fasseu, Magali; Aplan, Peter D; Chopin, Martine; Boissel, Nicolas; Bories, Jean-Christophe; Soulier, Jean; von Boehmer, Harald; Sigaux, François; Regnault, Armelle

    2007-10-01

    Inactivation of the CDKN2 genes that encode the p16(INK4A) and p14(ARF) proteins occurs in the majority of human T-cell acute lymphoblastic leukemias (T-ALLs). Ectopic expression of TAL1 and LMO1 genes is linked to the development of T-ALL in humans. In TAL1xLMO1 mice, leukemia develops in 100% of mice at 5 months. To identify the molecular events crucial to leukemic transformation, we produced several mouse models. We report here that expression of P16(INK4A) in developing TAL1xLMO1 thymocytes blocks leukemogenesis in the majority of the mice, and the leukemias that eventually develop show P16(INK4A) loss of expression. Events related to the T-cell receptor beta selection process are thought to be important for leukemic transformation. We show here that the absence of the pTalpha chain only slightly delays the appearance of TAL1xLMO1-induced T-ALL, which indicates a minor role of the pTalpha chain. We also show that the CD3epsilon-mediated signal transduction pathway is essential for this transformation process, since the TAL1xLMO1xCD3epsilon-deficient mice do not develop T-ALL for up to 1 year. PMID:17507663

  4. Investigation of nanocomposites based on semi-interpenetrating network of [L-poly (epsilon-caprolactone)]/[net-poly (epsilon-caprolactone)] and hydroxyapatite nanocrystals.

    PubMed

    Hao, Jianyuan; Liu, Yu; Zhou, Shaobing; Li, Zhen; Deng, Xianmo

    2003-04-01

    In this paper the semi-interpenetrating network (semi-IPN) technique was used for the first time to prepare bone implant composites containing hydroxyapatite (HAP) nanocrystals. The prepared nanocomposites are expected to combine several property advantages including good mechanical strength, modified degradation rate and excellent osteoconductivity. The semi-IPN matrix based on the linear poly (epsilon-caprolactone) (L-PCL) and the network poly (epsilon-caprolactone) (net-PCL) structures are revealed to be phase separation structures. The morphology of net-PCL is featured by intracrosslinked microdomains (1-10 microm) that further interconnect with each other to form the network over the whole sample. The net-PCL component is totally amorphous at room temperature for the nanocomposites containing HAP up to 12.3 wt%. Further, the crystallinity of L-PCL is greatly decreased due to the presence of net-PCL as compared with that for pure L-PCL. The incorporation of L-PCL into the net-PCL network could significantly improve the mechanical properties of pure net-PCL. A great improvement in mechanical properties is observed for the nanocomposites if the HAP content is increased to 15.8 wt%. This transition is in agreement with that the net-PCL component changes from amorphous state to crystalline state at this composition. PMID:12559813

  5. Coherent production of {epsilon}{sup +} particles in crystal using proton beam from SSC

    SciTech Connect

    Okorokov, V.V.; Dubin, A.Yu.

    1995-05-01

    The unique possibilities of the SSC can be ideally used for a new generation of coherent generation experiments with relativistic protons which require 20 Tev energy of the incident beam. The availability of 20 Tev proton beam at SSC allows new experiments on coherent production of {var_epsilon}{sup +} particle by relativistic proton in crystal. Experiment carried out at low energies can now be extended with protons in very narrow energy region (resonance energy, which easy can be calculated) using the new accelerator facilities at SSC. We propose to study coherent production via the Coulomb field of the cristal atoms to excite the transition p + {gamma}{implies} {var_epsilon} {sup +} (1189).

  6. Implementation of a kappa-epsilon turbulence model to RPLUS3D code

    NASA Technical Reports Server (NTRS)

    Chitsomboon, Tawit

    1992-01-01

    The RPLUS3D code has been developed at the NASA Lewis Research Center to support the National Aerospace Plane (NASP) project. The code has the ability to solve three dimensional flowfields with finite rate combustion of hydrogen and air. The combustion process of the hydrogen-air system are simulated by an 18 reaction path, 8 species chemical kinetic mechanism. The code uses a Lower-Upper (LU) decomposition numerical algorithm as its basis, making it a very efficient and robust code. Except for the Jacobian matrix for the implicit chemistry source terms, there is no inversion of a matrix even though a fully implicit numerical algorithm is used. A k-epsilon turbulence model has recently been incorporated into the code. Initial validations have been conducted for a flow over a flat plate. Results of the validation studies are shown. Some difficulties in implementing the k-epsilon equations to the code are also discussed.

  7. Infrared photometry of the 1982-4 eclipse of Epsilon Aurigae

    NASA Technical Reports Server (NTRS)

    Backman, D. E.

    1985-01-01

    The infrared photometry of epsilon Aur performed prior to and during the ingress phase of the recent eclipse allowed the first solid determination of the temperature of the secondary object. The eclipse depth was significantly less at lambda 5 micrometers than in the near-infrared. This is explained by a model of the secondary as an opaque and very cool object with a temperature of approx. 500 K. During eclipse, the secondary blocks approximately 45% of the near infrared radiation from the primary star. At the same time, the radiation from the secondary remains completely unobscured, resulting in a shallower light curve at longer wavelengths. This phenomenon is well known in the study of eclipsing binary stars; if the two stars have different colors, then the net color of the system changes during eclipse. In the case of epsilon Aur, the eclipsing object has a color deep in the infrared, so the effect is only noticeable there.

  8. Martian occultation of epsilon Gem as observed from the C. E. Kenneth Mees Observatory

    NASA Technical Reports Server (NTRS)

    French, R. G.; Goguen, J. D.; Duthie, J. G.

    1978-01-01

    Ground-based observations of the occultation of epsilon Gem by Mars on April 8, 1976 have been reduced to yield the scale height and temperature profiles of the Martian atmosphere for number densities between 10 to the 13th and 10 to the 15th per cu cm. The deduced variations in temperature are remarkably similar to the in situ measurements from the Viking landers.

  9. Sequence of the dog immunoglobulin alpha and epsilon constant region genes

    SciTech Connect

    Patel, M.; Selinger, D.; Mark, G.E.; Hollis, G.F.; Hickey, G.J.

    1995-03-01

    The immunoglobulin alpha (IGHAC) and epsilon (IGHEC) germline constant region genes were isolated from a dog liver genomic DNA library. Sequence analysis indicates that the dog IGHEC gene is encoded by four exons spread out over 1.7 kilobases (kb). The IGHAC sequence encompasses 1.5 kb and includes all three constant region coding exons. The complete exon/intron sequence of these genes is described. 28 refs., 2 figs., 2 tabs.

  10. Optical response of dipole antennas on an epsilon-near-zero substrate

    NASA Astrophysics Data System (ADS)

    Schulz, Sebastian A.; Tahir, Asad A.; Alam, M. Zahirul; Upham, Jeremy; De Leon, Israel; Boyd, Robert W.

    2016-06-01

    Materials with vanishing permittivity (epsilon-near-zero or ENZ materials) show unconventional optical behavior. Here we show that plasmonic dipole antennas on an ultrathin ENZ substrate have properties significantly different from antennas on a traditional substrate. Specifically, the presence of a 23-nm-thick ENZ material strongly modifies the linear response of plasmonic antennas and, as a result, the resonant wavelength is independent of the linear dimensions of the dipole antenna.

  11. Clostridium perfringens epsilon-toxin increases permeability of single perfused microvessels of rat mesentery.

    PubMed

    Adamson, R H; Ly, J C; Fernandez-Miyakawa, M; Ochi, S; Sakurai, J; Uzal, F; Curry, F E

    2005-08-01

    Epsilon-toxin, the primary virulence factor of Clostridium perfringens type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (L(p)) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 microg ml(-1) the L(p) increased irreversibly to more than 15 times the control value by 10 min. At 0.3 microg ml(-1) no increase in L(p) was observed for up to 90 min. The toxin-induced increase in L(p) was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium. PMID:16041001

  12. Application of the k-epsilon-v(exp 2) model to multi-component airfoils

    NASA Technical Reports Server (NTRS)

    Iaccarino, G.; Durbin, P. A.

    1996-01-01

    Flow computations around two-element and three-element configurations are presented and compared to detailed experimental measurements. The k-epsilon-v(exp 2)(bar) model has been applied and the ability of the model to capture streamline curvature effects, wake-boundary layer confluence, and laminar/turbulent transition is discussed. The numerical results are compared to experimental datasets that include mean quantities (velocity and pressure coefficient) and turbulent quantities (Reynolds normal and shear stresses).

  13. Implementation and Validation of the Chien k-epsilon Turbulence Model in the Wind Navier-Stokes Code

    NASA Technical Reports Server (NTRS)

    Yoder, Dennis A.; Georgiadis, Nicholas J.

    1999-01-01

    The two equation k-epsilon turbulence model of Chien has been implemented in the WIND Navier-Stokes flow solver. Details of the numerical solution algorithm, initialization procedure, and stability enhancements are described. Results obtained with this version of the model are compared with those from the Chien k-epsilon model in the NPARC Navier-Stokes code and from the WIND SST model for three validation cases: the incompressible flow over a smooth flat plate, the incompressible flow over a backward facing step, and the shock-induced flow separation inside a transonic diffuser. The k-epsilon model results indicate that the WIND model functions very similarly to that in NPARC, though the WIND code appears to he slightly more accurate in the treatment of the near-wall region. Comparisons of the k-epsilon model results with those from the SST model were less definitive, as each model exhibited strengths and weaknesses for each particular case.

  14. An Improved K-Epsilon Model for Near-Wall Turbulence and Comparison with Direct Numerical Simulation

    NASA Technical Reports Server (NTRS)

    Shih, T. H.

    1990-01-01

    An improved k-epsilon model for low Reynolds number turbulence near a wall is presented. The near-wall asymptotic behavior of the eddy viscosity and the pressure transport term in the turbulent kinetic energy equation is analyzed. Based on this analysis, a modified eddy viscosity model, having correct near-wall behavior, is suggested, and a model for the pressure transport term in the k-equation is proposed. In addition, a modeled dissipation rate equation is reformulated. Fully developed channel flows were used for model testing. The calculations using various k-epsilon models are compared with direct numerical simulations. The results show that the present k-epsilon model performs well in predicting the behavior of near-wall turbulence. Significant improvement over previous k-epsilon models is obtained.

  15. The elastic constants and related properties of the epsilon polymorph of the energetic material CL-20 determined by Brillouin scattering.

    PubMed

    Haycraft, James J

    2009-12-01

    The acoustic phonons of the epsilon polymorph of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazatetracyclo [5.5.0.0(5,9).0(3,11)] dodecane (epsilon-CL-20) have been studied using Brillouin scattering spectroscopy. Analysis of the acoustic phonon velocities allowed determination of the complete stiffness tensor for this energetic material. The results are compared to a theoretical determination of the epsilon-CL-20 elastic constants, bulk moduli, and shear moduli. The observed ordering of elastic constants, C(22)>C(33)>C(11), is noted to be different from other nitramine energetic materials. Finally, the elasticity of epsilon-CL-20 is compared to recently published reports on cyclotrimethylene trinitramine's (RDX) elasticity and the beta polymorph of cyclotetramethylene tetranitramine's (beta-HMX) elasticity. PMID:19968345

  16. Sulfur Metabolisms in Epsilon- and Gamma-Proteobacteria in Deep-Sea Hydrothermal Fields

    PubMed Central

    Yamamoto, Masahiro; Takai, Ken

    2011-01-01

    In deep-sea hydrothermal systems, super hot and reduced vent fluids from the subseafloor blend with cold and oxidized seawater. Very unique and dense ecosystems are formed within these environments. Many molecular ecological studies showed that chemoautotrophic epsilon- and gamma-Proteobacteria are predominant primary producers in both free-living and symbiotic microbial communities in global deep-sea hydrothermal fields. Inorganic sulfur compounds are important substrates for the energy conservative metabolic pathways in these microorganisms. Recent genomic and metagenomic analyses and biochemical studies have contributed to the understanding of potential sulfur metabolic pathways for these chemoautotrophs. Epsilon-Proteobacteria use sulfur compounds for both electron-donors and -acceptors. On the other hand, gamma-Proteobacteria utilize two different sulfur-oxidizing pathways. It is hypothesized that differences between the metabolic pathways used by these two predominant proteobacterial phyla are associated with different ecophysiological strategies; extending the energetically feasible habitats with versatile energy metabolisms in the epsilon-Proteobacteria and optimizing energy production rate and yield for relatively narrow habitable zones in the gamma-Proteobacteria. PMID:21960986

  17. Modeling of lipase catalyzed ring-opening polymerization of epsilon-caprolactone.

    PubMed

    Sivalingam, G; Madras, Giridhar

    2004-01-01

    Enzymatic ring-opening polymerization of epsilon-caprolactone by various lipases was investigated in toluene at various temperatures. The determination of molecular weight and structural identification was carried out with gel permeation chromatography and proton NMR, respectively. Among the various lipases employed, an immobilized lipase from Candida antartica B (Novozym 435) showed the highest catalytic activity. The polymerization of epsilon-caprolactone by Novozym 435 showed an optimal temperature of 65 degrees C and an optimum toluene content of 50/50 v/v of toluene and epsilon-caprolactone. As lipases can degrade polyesters, a maximum in the molecular weight with time was obtained due to the competition of ring opening polymerization and degradation by specific chain end scission. The optimum temperature, toluene content, and the variation of molecular weight with time are consistent with earlier observations. A comprehensive model based on continuous distribution kinetics was developed to model these phenomena. The model accounts for simultaneous polymerization, degradation and enzyme deactivation and provides a technique to determine the rate coefficients for these processes. The dependence of these rate coefficients with temperature and monomer concentration is also discussed. PMID:15003027

  18. Variation in the expression of macrophage Fc epsilon receptors in relation to experimental rat schistosome infection.

    PubMed

    Pestel, J; Joseph, M; Dessaint, J P; Capron, A

    1988-01-01

    The relationship between IgE and rat peritoneal macrophages during the course of Schistosoma mansoni infection was examined. Immune macrophages exhibited an IgE-dependent schistosomulicidal activity mainly between the 5th and the 7th weeks. At this period the percentages of anti-IgE rosettes whose variations appeared consecutive to the variations in the serum IgE level were higher. Following incubation in a serum-free medium immune macrophages could release membrane-associated IgE, to a certain extent, and consequently formed rosettes with IgE-coated erythrocytes, indicating the existence of Fc epsilon receptors. Thus, in terms of rosettes, their number varied in the course of the disease. In fact IgE-rich serum (i.e. at day 42) as IgE molecules could induce this macrophage Fc epsilon receptor. Moreover when transferred to syngeneic rats in association with parasite-specific IgE Fc epsilon R2+ macrophages led to some protection against challenge infection. All the data reported here emphasize the determining role of macrophages and IgE in immunity to schistosomiasis. PMID:2962948

  19. The structure and ordering of {epsilon}-MnO{sub 2}

    SciTech Connect

    Kim, Chang-Hoon; Akase, Zentaro; Zhang Lichun; Heuer, Arthur H. . E-mail: heuer@case.edu; Newman, Aron E.; Hughes, Paula J.

    2006-03-15

    The presence of {epsilon}-MnO{sub 2} as a major component of electrolytic manganese dioxide (EMD) has been demonstrated by a combined X-ray diffraction/transmission electron microscopy (TEM) study. {epsilon}-MnO{sub 2} usually has a partially ordered defect NiAs structure containing 50% cation vacancies; these vacancies can be fully ordered by a low temperature (200 deg. C) heat treatment to form a pseudohexagonal but monoclinic superlattice. Numerous fine-scale anti-phase domain boundaries are present in ordered {epsilon}-MnO{sub 2} and cause extensive peak broadening and a massive shift of a very intense, 0.37 nm superlattice peak. This suggests a radically different explanation of the ubiquitous, very broad {approx}0.42 nm peak ({approx}21-22 deg. 2{theta}, CuK{alpha} radiation) in EMDs, which heretofore has been attributed to Ramsdellite containing numerous planar defects. This work confirms the multi-phase model of equiaxed EMDs proposed by Heuer et al. [ITE Lett. 1(6) (2000) B50; Proc. Seventh Int. Symp. Adv. Phys. Fields 92 (2001)], rather than the defective single-phase model of Chabre and Pannetier [Prog. Solid State Chem. 23 (1995) 1] and Bowden et al. [ITE Lett. 4(1) (2003) B1].

  20. Rat costochondral cell characteristics on poly (L-lactide-co-epsilon-caprolactone) scaffolds.

    PubMed

    Honda, M; Morikawa, N; Hata, K; Yada, T; Morita, S; Ueda, M; Kimata, K

    2003-09-01

    This study was designed to examine the adhesion, proliferation, and morphology of chondrocytes on new scaffolds; and to examine these cells histologically for the ability of the chondrocytes to maintain chondrogenic properties after subcutaneous implantation into nude mice. Both 75:25 poly (L-lactide-co-epsilon-caprolactone) (75PLC) and 50:50 poly (L-lactide-co-epsilon-capro-lactone) scaffold (50PLC) were tested as a scaffold for rat costochondral resting zone chondrocytes in comparison with a type I collagen sponge scaffold (collagen scaffold). Both of the poly (L-lactide-co-epsilon-caprolactone) scaffolds (75PLC and 50PLC) were coated with type I collagen solution and the effects of the collagen coat (hybrid-PLC) were also examined. The hybrid-75PLC bound the same number of cells as the collagen scaffold, whereas the 75PLC and the 50PLC bound 60% and 50% fewer cells than the collagen scaffold, respectively. The cell growth on the scaffolds progressed with culture time in all scaffolds. Cell morphology was assessed by scanning electron microscopy for differences in the structure of cellular interaction. Chondrocytes on every scaffold maintained a spherical shape. The hybrid-PLCs were superior to the PLCs with respect to the number of cells attached. The PLCs had an advantageous degradation characteristic in that they retained their original shape better than the collagen scaffold. Additionally, in the PLCs seeded, the cells retained their integrity 4 weeks after implantation, although the volume of collagen scaffold decreased by 50%. PMID:12809780

  1. Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase

    PubMed Central

    Diretto, Gianfranco; Tavazza, Raffaela; Welsch, Ralf; Pizzichini, Daniele; Mourgues, Fabienne; Papacchioli, Velia; Beyer, Peter; Giuliano, Giovanni

    2006-01-01

    Background Potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity. Results We silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e), by introducing, via Agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. Real Time measurements confirmed the tuber-specific silencing of Lcy-e. Antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase (up to 14-fold). Total carotenoids increased up to 2.5-fold. These changes were not accompanied by a decrease in lutein, suggesting that LCY-e is not rate-limiting for lutein accumulation. Tuber-specific changes in expression of several genes in the pathway were observed. Conclusion The data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. Upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified. PMID:16800876

  2. Development and application of a zonal k-epsilon turbulence model for complex 3-D flowfields

    NASA Astrophysics Data System (ADS)

    Ladd, J. A.; Kral, L. D.

    1992-07-01

    A compressible, low Reynolds number two-equation turbulence model is applied to complex engineering problems. An upwind, implicit, factored algorithm with an optional TVD operator is used to solve both the mean-flow equations and the k-epsilon equations for three-dimensional turbulenct flow. A zonal approach is used for solution of both the mean flow variables and the turbulence variables. The zonal method allows complex geometries to be broken down into smaller blocks which are then computed sequentially. Several low Reynolds number k-epsilon models are implemented and validated for a subsonic and supersonic flat plate boundary layer. Calculations using the k-epsilon turbulence model are also presented for an axisymmetric jet plume, a supersonic combusting shear layer, a multislot ejector nozzle, and an F/A-18 forebody at high angle of attack. Comparison of the two-equation turbulence model results is made with results using algebraic turbulence models as well as experimental measurements. The two-equation turbulence model predicts better many of the flowfield characteristics for these complex geometries when compared with the algebraic solutions.

  3. A 10-bp deletion in the apolipoprotein epsilon gene causing apolipoprotein E deficiency and severe type III hyperlipoproteinemia.

    PubMed Central

    Feussner, G.; Dobmeyer, J.; Gröne, H. J.; Lohmer, S.; Wohlfeil, S.

    1996-01-01

    Type III hyperlipoproteinemia (HLP) is usually associated with homozygosity for apolipoprotein (apo) E2. We identified a 30-year-old male German of Hungarian ancestry with severe type III HLP and apo E deficiency. The disease was expressed in an extreme phenotype with multiple cutaneous xanthomas. Apo E was detectable only in trace amounts in plasma but not in the different lipoprotein fractions. Direct sequencing of PCR-amplified segments of the apo epsilon gene identified a 10-bp deletion in exon 4 (bp 4037-4046 coding for amino acids 209-212 of the mature protein). The mutation is predictive for a reading frameshift introducing a premature stop codon (TGA) at amino acid 229. By western blot analysis, we found small amounts of a truncated apo E in the patient's plasma. Family analysis revealed that the proband was homozygous--and 10 of 24 relatives were heterozygous--for the mutation. Heterozygotes had, as compared to unaffected family members, significantly higher triglycerides (TG), very low-density lipoprotein (VLDL) cholesterol and a significantly higher VLDL cholesterol-to-serum TG ratio, which is indicative of a delayed remnant catabolism. We propose that the absence of a functionally active apo E is the cause of the severe type III HLP in the patient and that the mutation, even in a single dose in heterozygotes, predisposes in variable severity to the phenotypic expression of the disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8571954

  4. A quantitative model of the generation of N(epsilon)-(carboxymethyl)lysine in the Maillard reaction between collagen and glucose.

    PubMed Central

    Ferreira, António E N; Ponces Freire, Ana M J; Voit, Eberhard O

    2003-01-01

    The Maillard reaction between reducing sugars and amino groups of biomolecules generates complex structures known as AGEs (advanced glycation endproducts). These have been linked to protein modifications found during aging, diabetes and various amyloidoses. To investigate the contribution of alternative routes to the formation of AGEs, we developed a mathematical model that describes the generation of CML [ N(epsilon)-(carboxymethyl)lysine] in the Maillard reaction between glucose and collagen. Parameter values were obtained by fitting published data from kinetic experiments of Amadori compound decomposition and glycoxidation of collagen by glucose. These raw parameter values were subsequently fine-tuned with adjustment factors that were deduced from dynamic experiments taking into account the glucose and phosphate buffer concentrations. The fine-tuned model was used to assess the relative contributions of the reaction between glyoxal and lysine, the Namiki pathway, and Amadori compound degradation to the generation of CML. The model suggests that the glyoxal route dominates, except at low phosphate and high glucose concentrations. The contribution of Amadori oxidation is generally the least significant at low glucose concentrations. Simulations of the inhibition of CML generation by aminoguanidine show that this compound effectively blocks the glyoxal route at low glucose concentrations (5 mM). Model results are compared with literature estimates of the contributions to CML generation by the three pathways. The significance of the dominance of the glyoxal route is discussed in the context of possible natural defensive mechanisms and pharmacological interventions with the goal of inhibiting the Maillard reaction in vivo. PMID:12911334

  5. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  6. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  7. Molecular cloning and expression of epsilon toxin from Clostridium perfringens type D and tests of animal immunization.

    PubMed

    Souza, A M; Reis, J K P; Assis, R A; Horta, C C; Siqueira, F F; Facchin, S; Alvarenga, E R; Castro, C S; Salvarani, F M; Silva, R O S; Pires, P S; Contigli, C; Lobato, F C F; Kalapothakis, E

    2010-01-01

    Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, goats and calves. Enterotoxemia can cause acute or superacute disease, with sudden death of the affected animal. It provokes huge economic losses when large numbers of livestock are affected. Therapeutic intervention is challenging, because the disease progresses very rapidly. However, it can be prevented by immunization with specific immunogenic vaccines. We cloned the etx gene, encoding epsilon toxin, into vector pET-11a; recombinant epsilon toxin (rec-epsilon) was expressed in inclusion bodies and was used for animal immunization. Serum protection was evaluated and cross-serum neutralization tests were used to characterize the recombinant toxin. To analyze the potency of the toxin (as an antigen), rabbits were immunized with 50, 100 or 200 microg recombinant toxin, using aluminum hydroxide gel as an adjuvant. Titers of 10, 30 and 40 IU/mL were obtained, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia (5 IU/mL) and by the USA Code of Federal Regulation (2 IU/mL). This rec-epsilon is a good candidate for vaccine production against enterotoxemia caused by epsilon toxin of C. perfringens type D. PMID:20198582

  8. Initial Evaluation of Processing Methods for an Epsilon Metal Waste Form

    SciTech Connect

    Crum, Jarrod V.; Strachan, Denis M.; Zumhoff, Mac R.

    2012-06-11

    During irradiation of nuclear fuel in a reactor, the five metals, Mo, Pd, Rh, Ru, and Tc, migrate to the fuel grain boundaries and form small metal particles of an alloy known as epsilon metal ({var_epsilon}-metal). When the fuel is dissolved in a reprocessing plant, these metal particles remain behind with a residue - the undissolved solids (UDS). Some of these same metals that comprise this alloy that have not formed the alloy are dissolved into the aqueous stream. These metals limit the waste loading for a borosilicate glass that is being developed for the reprocessing wastes. Epsilon metal is being developed as a waste form for the noble metals from a number of waste streams in the aqueous reprocessing of used nuclear fuel (UNF) - (1) the {var_epsilon}-metal from the UDS, (2) soluble Tc (ion-exchanged), and (3) soluble noble metals (TRUEX raffinate). Separate immobilization of these metals has benefits other than allowing an increase in the glass waste loading. These materials are quite resistant to dissolution (corrosion) as evidenced by the fact that they survive the chemically aggressive conditions in the fuel dissolver. Remnants of {var_epsilon}-metal particles have survived in the geologically natural reactors found in Gabon, Africa, indicating that they have sufficient durability to survive for {approx} 2.5 billion years in a reducing geologic environment. Additionally, the {var_epsilon}-metal can be made without additives and incorporate sufficient foreign material (oxides) that are also present in the UDS. Although {var_epsilon}-metal is found in fuel and Gabon as small particles ({approx}10 {micro}m in diameter) and has survived intact, an ideal waste form is one in which the surface area is minimized. Therefore, the main effort in developing {var_epsilon}-metal as a waste form is to develop a process to consolidate the particles into a monolith. Individually, these metals have high melting points (2617 C for Mo to 1552 C for Pd) and the alloy is

  9. Novel role for cyclin-dependent kinase 2 in neuregulin-induced acetylcholine receptor epsilon subunit expression in differentiated myotubes.

    PubMed

    Lu, Gang; Seta, Karen A; Millhorn, David E

    2005-06-10

    Cyclin-dependent kinases (CDKs) are a family of evolutionarily conserved serine/threonine kinases. CDK2 acts as a checkpoint for the G(1)/S transition in the cell cycle. Despite a down-regulation of CDK2 activity in postmitotic cells, many cell types, including muscle cells, maintain abundant levels of CDK2 protein. This led us to hypothesize that CDK2 may have a function in postmitotic cells. We show here for the first time that CDK2 can be activated by neuregulin (NRG) in differentiated C2C12 myotubes. In addition, this activity is required for expression of the acetylcholine receptor (AChR) epsilon subunit. The switch from the fetal AChRgamma subunit to the adult-type AChRepsilon is required for synapse maturation and the neuromuscular junction. Inhibition of CDK2 activity with either the specific CDK2 inhibitory peptide Tat-LFG or by RNA interference abolished neuregulin-induced AChRepsilon expression. Neuregulin-induced activation of CDK2 also depended on the ErbB receptor, MAPK, and PI3K, all of which have previously been shown to be required for AChRepsilon expression. Neuregulin regulated CDK2 activity through coordinating phosphorylation of CDK2 on Thr-160, accumulation of CDK2 in the nucleus, and down-regulation of the CDK2 inhibitory protein p27 in the nucleus. In addition, we also observed a novel mechanism of regulation of CDK2 activity by a low molecular weight variant of cyclin E in response to NRG. These findings establish CDK2 as an intermediate molecule that integrates NRG-activated signals from both the MAPK and PI3K pathways to AChRepsilon expression and reveal an undiscovered physiological role for CDK2 in postmitotic cells. PMID:15824106

  10. Binary star systems with asymmetrically heated disks: Thermal phase curves for the disk in epsilon Aurigae

    NASA Astrophysics Data System (ADS)

    Pearson, Richard L., III

    Epsilon Aurigae is a long-period eclipsing binary that contains a warm F-star (~7750 K) and a circumstellar disk enshrouding a hidden companion, likely to be a hot B-star (≥15,000 K). The eclipse itself lasts just over two years---thanks, in part, to the size of the disk---and occurs every 27.1-years. Its evolutionary status is still debated, along with the true nature of each stellar component, due to the high uncertainty in its parallax. The disk is similarly debated from the near absence of solid state infrared spectral features indicating its composition, particle size distribution, and density. An investigation of a wide parameter space by means of analytic, Monte Carlo radiative transfer (MCRT), and thermal inertia-dependent methods are presented here in order to minimize the current parameter space. The first MCRT models including all of the epsilon Aurigae components (F-star, B-star, and disk) are included here. Additional parameter constraints are found by melding MCRT outputs (e.g. dust temperatures) with a thermal inertia-based extrapolation. The so-called MCRT-TI models investigate the effects of various parameters on the disk-edge temperatures; these include two distances, three particle size distributions, three compositions, and two disk masses, resulting in thirty-six independent models. Adding in the MCRT temperatures as possible solutions doubles the number of models to seventy-two. Additionally, infrared observations at 7 epochs, spanning nearly 1/3 of the orbit of epsilon Aurigae, are evaluated in order to extract phase-dependent disk temperatures. The resulting temperatures create a thermal phase curve, or TPC, for the system. The TPC correlates the observed disk temperature with orbital phase or date of observation. Then, the best-case MCRT and MCRT-TI models are compared against two different mid-eclipse temperatures. If one considers the evolutionary constraints on the models---where a smaller distance denotes an older system with a disk

  11. Epsilon Lyrae

    NASA Astrophysics Data System (ADS)

    Murdin, P.

    2000-11-01

    Though not very bright, this is a favorite test object for powerful binoculars and small telescopes. Situated 1.6° north-east of Vega, it is known as `the Double Double'. The star can just be seen to be double with the unaided eye, under good conditions by observers with acute eyesight. With binoculars it is easily separated, the component stars being 3.5' apart at a position angle of 173°. It is...

  12. EUVE spectroscopy of epsilon Canis Majoris (B2 II) from 70 to 730 A

    NASA Technical Reports Server (NTRS)

    Cassinelli, J. P.; Cohen, D. H.; Macfarlane, J. J.; Drew, J. E.; Lynas-Gray, A. E.; Hoare, M. G.; Vallerga, J. V.; Welsh, B. Y.; Vedder, P. W.; Hubeny, I.

    1995-01-01

    We present spectra of the brightest stellar source of extreme ultraviolet (EUV) radiation longward of 400 A, the B2 II star, epsilon CMa. These data were taken with the three spectrometers aboard the NASA Extreme Ultraviolet Explorer satellite (EUVE) during the first cycle of pointed observations. We report on our initial studies of the continuum and line spectrum of the stellar photosphere in the 320 to 730 A region, and on the wind emission lines observed in the 170-375 A region. This is the first EUV spectrum of an early-type star, and thus makes epsilon CMa the most comprehensively observed B star from the X-ray to infrared regimes. The radiation in both the H Lyman continuum and He I continuum (shortward of 504 A) are found to be significantly greater than predicted by both Local Thermodynamic Equilibrium (LTE) and non-LTE model atmospheres. Since epsilon CMa also exhibits a mid-infrared excess, this points to the outer layers being warmer than the models indicate. The anomalously large Lyman continuum flux, combined with the very low column density measured in the direction toward this star implies that it is the dominant source of hydrogen ionization of the local interstellar medium in the immediate vicinity of the sun. All of the lines predicted to be strong from model atmospheres are present and several wind absorption features are also identified. We have detected emission lines from highly ionized iron that are consistent with the ROSAT Position Sensitive Proportional Counter (PSPC) observations if a multi-temperature emission model is used, and the assumption is made that there is significant absorption beyond that of the neutral phase of the ISM. The spectrum shows strong O III 374 A line emission produced by the Bowen flourescence mechanism, which has not previously been observed in the spectra of hot stars.

  13. DNA replication defect in Salmonella typhimurium mutants lacking the editing (epsilon) subunit of DNA polymerase III.

    PubMed Central

    Lifsics, M R; Lancy, E D; Maurer, R

    1992-01-01

    In Salmonella typhimurium, dnaQ null mutants (encoding the epsilon editing subunit of DNA polymerase III [Pol III]) exhibit a severe growth defect when the genetic background is otherwise wild type. Suppression of the growth defect requires both a mutation affecting the alpha (polymerase) subunit of DNA polymerase III and adequate levels of DNA polymerase I. In the present paper, we report on studies that clarify the nature of the physiological defect imposed by the loss of epsilon and the mechanism of its suppression. Unsuppressed dnaQ mutants exhibited chronic SOS induction, indicating exposure of single-stranded DNA in vivo, most likely as gaps in double-stranded DNA. Suppression of the growth defect was associated with suppression of SOS induction. Thus, Pol I and the mutant Pol III combined to reduce the formation of single-stranded DNA or accelerate its maturation to double-stranded DNA. Studies with mutants in major DNA repair pathways supported the view that the defect in DNA metabolism in dnaQ mutants was at the level of DNA replication rather than of repair. The requirement for Pol I was satisfied by alleles of the gene for Pol I encoding polymerase activity or by rat DNA polymerase beta (which exhibits polymerase activity only). Consequently, normal growth is restored to dnaQ mutants when sufficient polymerase activity is provided and this compensatory polymerase activity can function independently of Pol III. The high level of Pol I polymerase activity may be required to satisfy the increased demand for residual DNA synthesis at regions of single-stranded DNA generated by epsilon-minus pol III. The emphasis on adequate polymerase activity in dnaQ mutants is also observed in the purified alpha subunit containing the suppressor mutation, which exhibits a modestly elevated intrinsic polymerase activity relative to that of wild-type alpha. Images PMID:1400246

  14. Wall functions for the kappa-epsilon turbulence model in generalized nonorthogonal curvilinear coordinates

    NASA Technical Reports Server (NTRS)

    Sondak, D. L.; Pletcher, R. H.; Vandalsem, W. R.

    1992-01-01

    A k-epsilon turbulence model suitable for compressible flow, including the new wall function formulation, has been incorporated into an existing compressible Reynolds-averaged Navier-Stokes code, F3D. The low Reynolds number k-epsilon model of Chien (1982) was added for comparison with the present method. A number of features were added to the F3D code including improved far-field boundary conditions and viscous terms in the streamwise direction. A series of computations of increasing complexity was run to test the effectiveness of the new formulation. Flow over a flat plate was computed by using both orthogonal and nonorthogonal grids, and the friction coefficients and velocity profiles compared with a semi-empirical equation. Flow over a body of revolution at zero angle of attack was then computed to test the method's ability to handle flow over a curved surface. Friction coefficients and velocity profiles were compared to test data. All models gave good results on a relatively fine grid, but only the wall function formulation was effective with coarser grids. Finally, in order to demonstrate the method's ability to handle complex flow fields, separated flow over a prolate spheroid at angle of attack was computed, and results were compared to test data. The results were also compared to a k-epsilon model by Kim and Patel (1991), in which one equation model patched in at the wall was employed. Both models gave reasonable solutions, but improvement is required for accurate prediction of friction coefficients in the separated regions.

  15. Arbitrary Steady-State Solutions with the K-epsilon Model

    NASA Technical Reports Server (NTRS)

    Rumsey, Christopher L.; Pettersson Reif, B. A.; Gatski, Thomas B.

    2006-01-01

    Widely-used forms of the K-epsilon turbulence model are shown to yield arbitrary steady-state converged solutions that are highly dependent on numerical considerations such as initial conditions and solution procedure. These solutions contain pseudo-laminar regions of varying size. By applying a nullcline analysis to the equation set, it is possible to clearly demonstrate the reasons for the anomalous behavior. In summary, the degenerate solution acts as a stable fixed point under certain conditions, causing the numerical method to converge there. The analysis also suggests a methodology for preventing the anomalous behavior in steady-state computations.

  16. Super-resolution with a positive epsilon multi-quantum-well super-lens

    SciTech Connect

    Bak, A. O.; Giannini, V.; Maier, S. A.; Phillips, C. C.

    2013-12-23

    We design an anisotropic and dichroic quantum metamaterial that is able to achieve super-resolution without the need for a negative permittivity. When exploring the parameters of the structure, we take into account the limits of semiconductor fabrication technology based on quantum well stacks. By heavily doping the structure with free electrons, we infer an anisotropic effective medium with a prolate ellipsoid dispersion curve which allows for near-diffractionless propagation of light (similar to an epsilon-near-zero hyperbolic lens). This, coupled with low absorption, allows us to resolve images at the sub-wavelength scale at distances 6 times greater than equivalent natural materials.

  17. Goos-Hänchen shift of partially coherent light fields in epsilon-near-zero metamaterials

    NASA Astrophysics Data System (ADS)

    Ziauddin; Chuang, You-Lin; Qamar, Sajid; Lee, Ray-Kuang

    2016-05-01

    The Goos-Hänchen (GH) shifts in the reflected light are investigated both for p and s polarized partial coherent light beams incident on epsilon-near-zero (ENZ) metamaterials. In contrary to the coherent counterparts, the magnitude of GH shift becomes non-zero for p polarized partial coherent light beam; while GH shift can be relatively large with a small degree of spatial coherence for s polarized partial coherent beam. Dependence on the beam width and the permittivity of ENZ metamaterials is also revealed for partial coherent light fields. Our results on the GH shifts provide a direction on the applications for partial coherent light sources in ENZ metamaterials.

  18. Metasurfaces and epsilon-near-zero modes in semiconductors (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Brener, Igal; Campione, Salvatore; Marquier, Francois

    2015-09-01

    Thin layers of semiconductors where the permittivity crosses zero, support a particular polariton mode called epsilon-near-zero (ENZ) mode. This zero crossing can be obtained near optical phonon resonances in dielectrics or the plasma frequency in doped semiconductors. The coupling of metamaterial resonators to these ENZ modes leads to particularly large Rabi splittings. ENZ layers can be added to metamaterial-based strongly coupled systems to increase this coupling even further. I will discuss several examples of these coupled systems that include metasurfaces, phonons, intersubband transitions and ENZ modes.

  19. CONFIRMING FUNDAMENTAL PROPERTIES OF THE EXOPLANET HOST STAR {epsilon} ERIDANI USING THE NAVY OPTICAL INTERFEROMETER

    SciTech Connect

    Baines, Ellyn K.; Armstrong, J. Thomas E-mail: tarmstr@crater.nrl.navy.mil

    2012-01-10

    We measured the angular diameter of the exoplanet host star {epsilon} Eridani using the Navy Optical Interferometer. We determined its physical radius, effective temperature, and mass by combining our measurement with the star's parallax, photometry from the literature, and the Yonsei-Yale isochrones, respectively. We used the resulting stellar mass of 0.82 {+-} 0.05 M{sub Sun} plus the mass function from Benedict et al. to calculate the planet's mass, which is 1.53 {+-} 0.22 M{sub Jupiter}. Using our new effective temperature, we also estimated the extent of the habitable zone for the system.

  20. All-optical modulation in wavelength-sized epsilon-near-zero media.

    PubMed

    Ciattoni, Alessandro; Marini, Andrea; Rizza, Carlo

    2016-07-01

    We investigate the interaction of two pulses (pump and probe) scattered by a nonlinear epsilon-near-zero (ENZ) slab whose thickness is comparable with the ENZ wavelength. We show that when the probe has a narrow spectrum localized around the ENZ wavelength, its transmission is dramatically affected by the intensity of the pump. Conversely, if the probe is not in the ENZ regime, its propagation is not noticeably affected by the pump. Such all-optical modulation is due to the oversensitive character of the ENZ regime,