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Sample records for 14-3-3 protein epsilon

  1. Protein kinase C-alpha regulates insulin action and degradation by interacting with insulin receptor substrate-1 and 14-3-3 epsilon.

    PubMed

    Oriente, Francesco; Andreozzi, Francesco; Romano, Chiara; Perruolo, Giuseppe; Perfetti, Anna; Fiory, Francesca; Miele, Claudia; Beguinot, Francesco; Formisano, Pietro

    2005-12-09

    Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKCalpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKCalpha co-precipitation with IRS-1, but not with IRS-2, and with 14-3-3epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKCalpha activity, without altering IRS-1/PKCalpha co-precipitation. 14-3-3epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKCalpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKCalpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3epsilon depletion on insulin signaling. Moreover, PKCalpha inhibition was accompanied by a >2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKCalpha, and 14-3-3epsilon. The presence of 14-3-3epsilon in the complex is not necessary for IRS-1/PKCalpha interaction but modulates PKCalpha activity, thereby regulating insulin signaling and degradation.

  2. Identification of 14-3-3epsilon substrates from embryonic murine brain.

    PubMed

    Ballif, Bryan A; Cao, Zhongwei; Schwartz, Daniel; Carraway, Kermit L; Gygi, Steven P

    2006-09-01

    Mice deficient in 14-3-3epsilon exhibit abnormal neuronal migration and die perinatally. We report here the first large-scale analysis of 14-3-3 interacting partners from primary animal tissue, identifying from embryonic murine brain 163 14-3-3epsilon interacting proteins and 85 phosphorylation sites on these proteins. Phosphorylation of the deubiquitinating enzyme USP8 at serine 680 was found essential for its interaction with 14-3-3epsilon and for maintaining USP8 in the cytosol.

  3. 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.

  4. Yeast 14-3-3 proteins.

    PubMed

    van Heusden, G Paul H; Steensma, H Yde

    2006-02-01

    14-3-3 proteins form a family of highly conserved proteins which are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. They interact with more than 200 different, mostly phosphorylated proteins. The molecular consequences of 14-3-3 binding are diverse: this binding may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization, to the interaction with other proteins or to shielding of binding sites. The binding partners, and hence the 14-3-3 proteins, are involved in almost every cellular process and 14-3-3 proteins have been linked to several diseases, such as cancer, Alzheimer's disease, the neurological Miller-Dieker and spinocerebellar ataxia type 1 diseases and bovine spongiform encephalopathy (BSE). The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both have two genes encoding 14-3-3 proteins, BMH1 and BMH2 and rad24 and rad25, respectively. In these yeasts, 14-3-3 proteins are essential in most laboratory strains. As in higher eukaryotes, yeast 14-3-3 proteins bind to numerous proteins involved in a variety of cellular processes. Recent genome-wide studies on yeast strains with impaired 14-3-3 function support the participation of 14-3-3 proteins in numerous yeast cellular processes. Given the high evolutionary conservation of the 14-3-3 proteins, the experimental accessibility and relative simplicity of yeasts make them excellent model organisms for elucidating the function of the 14-3-3 protein family.

  5. 14-3-3 proteins: regulators of numerous eukaryotic proteins.

    PubMed

    van Heusden, G Paul H

    2005-09-01

    14-3-3 proteins form a family of highly conserved proteins capable of binding to more than 200 different mostly phosphorylated proteins. They are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. 14-3-3 binding partners are involved in almost every cellular process and 14-3-3 proteins play a key role in these processes. 14-3-3 proteins interact with products encoded by oncogenes, with filament forming proteins involved in Alzheimer'ss disease and many other proteins related to human diseases. Disturbance of the interactions with 14-3-3 proteins may lead to diseases like cancer and the neurological Miller-Dieker disease. The molecular consequences of 14-3-3 binding are diverse and only partly understood. Binding of a protein to a 14-3-3 protein may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization or to the interaction with other proteins. Currently genome- and proteome-wide studies are contributing to a wider knowledge of this important family of proteins.

  6. Isolation and expression analysis of a homolog of the 14-3-3 epsilon gene in the diamondback moth, Plutella xylostella.

    PubMed

    Yoo, Ji Yeon; Hwang, Se Hui; Han, Yeon Soo; Cho, Saeyoull

    2011-02-01

    A full-length 14-3-3 gene homolog (also referred to as the Px14-3-3 epsilon "ε" or Px14-3-3ε gene) was cloned from cDNA of the diamondback moth, Plutella xylostella. The Px14-3-3 transcript is 789 nucleotides in length, and the predicted polypeptide is 263 amino acids in length, with a calculated molecular mass of 29.6 kDa. The Px14-3-3 gene contains the typical and predicted 14-3-3 domains and motifs. The amino acid sequence of the diamondback moth 14-3-3 gene is very similar to that of other insect epsilons (ε) but not to other insect zetas (ζ). In particular, the protein sequence of the Px14-3-3 gene shows high identity to the Bombyx mori epsilon (96.2%). Western blot analysis using an antibody against Px14-3-3ε verified the expression of 14-3-3ε in the larval, pupal, and adult stages. The Px14-3-3ε expression patterns in all the different tissue types were examined in the fourth instar larvae. Px14-3-3ε was detected in every tissue examined, including the body fat, hemocytes, brain, gut, and cuticle.

  7. An account of fungal 14-3-3 proteins.

    PubMed

    Kumar, Ravinder

    2017-02-24

    14-3-3s are a group of relatively low molecular weight, acidic, dimeric, protein(s) conserved from single-celled yeast to multicellular vertebrates including humans. Despite lacking catalytic activity, these proteins have been shown to be involved in multiple cellular processes. Apart from their role in normal cellular physiology, recently these proteins have been implicated in various medical consequences. In this present review, fungal 14-3-3 protein localization, interactions, transcription, regulation, their role in the diverse cellular process including DNA duplication, cell cycle, protein trafficking or secretion, apoptosis, autophagy, cell viability under stress, gene expression, spindle positioning, role in carbon metabolism have been discussed. In the end, I also highlighted various roles of yeasts 14-3-3 proteins in tabular form. Thus this review with primary emphasis on yeast will help in appreciating the significance of 14-3-3 proteins in cell physiology.

  8. 14-3-3 proteins are promising LRRK2 interactors.

    PubMed

    Rudenko, Iakov N; Cookson, Mark R

    2010-09-15

    Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common cause of familial PD (Parkinson's disease). Mutations that cause PD are found in either the GTPase or kinase domains of LRRK2 or an intervening sequence called the COR [C-terminus of ROC (Ras of complex proteins)] domain. As well as the two catalytic domains, LRRK2 possesses several protein-protein interaction domains, but their function and the proteins with which they interact are poorly understood. In this issue of the Biochemical Journal, Nichols et al. study the interaction of the N-terminal region of LRRK2 with 14-3-3 proteins, regulatory proteins that often bind to phosphorylated regions of components of cell signalling pathways. Using a combination of techniques, Nichols et al. have identified two residues (Ser910 and Ser935) that are critically responsible for 14-3-3 binding. The interaction of LRRK2 with 14-3-3 proteins can prevent dephosphorylation of Ser910/Ser935 and stabilize LRRK2 structure, perhaps by influencing the dimerization of LRRK2. The ability to interact with 14-3-3 correlates with the pattern of intracellular LRRK2 distribution. Collectively, these new results identify a potentially important regulatory mechanism of this complex protein and might provide ways to think about therapeutic opportunities for PD.

  9. 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

  10. Eimeria tenella: 14-3-3 protein interacts with telomerase.

    PubMed

    Zhao, Na; Gong, Pengtao; Cheng, Baiqi; Li, Jianhua; Yang, Zhengtao; Li, He; Yang, Ju; Zhang, Guocai; Zhang, Xichen

    2014-10-01

    Telomerase, consisting of telomerase RNA and telomerase reverse transcriptase (TERT), is responsible for the maintenance of the end of linear chromosomes. TERT, as the catalytic subunit of telomerase, plays a critical role in telomerase activity. Researches indicate TERT-associated proteins participate in the regulation of telomerase assembly, posttranslational modification, localization, and enzymatic function. Here, the telomerase RNA-binding domain of Eimeria tenella TERT (EtTRBD) was cloned into pGBKT7 and performed as the bait. α-Galactosidase assay showed that the bait plasmid did not activate Gal4 reporter gene. Further, we isolated an EtTRBD-associated protein, 14-3-3, by yeast two-hybrid screening using the constructed bait plasmid. To confirm the interaction, EtTRBD and 14-3-3 were expressed by prokaryotic and eukaryotic expression systems. Pull-down assays by purified proteins demonstrated a direct bind between EtTRBD and 14-3-3. Co-immunoprecipitation techniques successfully validated that 14-3-3 interacted with EtTRBD in 293T cells. The protein-protein interaction provides a starting point for more in-depth studies on telomerase and telomere regulation in E. tenella.

  11. Identification and characterization of protein 14-3-3 in carcinogenic liver fluke Opisthorchis viverrini.

    PubMed

    Kafle, Alok; Puchadapirom, Pranom; Plumworasawat, Sirikanya; Dontumprai, Rieofarng; Chan-On, Waraporn; Buates, Sureemas; Laha, Thewarach; Sripa, Banchob; Suttiprapa, Sutas

    2016-10-27

    Protein 14-3-3s are abundant phospho-serine/threonine binding proteins, which are highly conserved among eukaryotes. Members of this protein family mediate metabolism and signal transduction networks through binding to hundreds of other protein partners. Protein 14-3-3s have been studied in other species of parasitic helminthes, but little is known about this protein in the carcinogenic liver fluke Opisthorchis viverrini. In this study, we identified and characterized protein 14-3-3s of O. viverrini. Seven protein 14-3-3 encoded sequences were retrieved from the O. viverrini genome database. Multiple alignment and phylogenetic analysis were performed. Two isoforms (protein 14-3-3 zeta and protein 14-3-3 epsilon) that have been previously found in the excretory-secretory (ES) products of O. viverrini were produced as recombinant protein in E. coli and the proteins were then used to immunize mice to obtain specific antibodies. Western blot analysis showed that both proteins were detected in all obtainable developmental stages of O. viverrini and the ES products. Immunolocalization revealed that both isoforms were expressed throughout tissues and organs except the gut epithelium. The highest expression was observed in testes especially in developing spermatocytes, suggesting their role in spermatogenesis. Prominent expression was also detected on tegumental surface of the parasite and on epical surface of bile duct epithelium indicates their additional role in host-parasite interaction. These findings indicate that protein 14-3-3s play important role in the life cycle of the parasite and might be involved in the pathogenesis of O. viverrini infection.

  12. Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases

    PubMed Central

    Swatek, Kirby N.; Wilson, Rashaun S.; Ahsan, Nagib; Tritz, Rebecca L.; Thelen, Jay J.

    2014-01-01

    Plant 14-3-3 proteins are phosphorylated at multiple sites in vivo; however, the protein kinase(s) responsible are unknown. Of the 34 CPK (calcium-dependent protein kinase) paralogues in Arabidopsis thaliana, three (CPK1, CPK24 and CPK28) contain a canonical 14-3-3-binding motif. These three, in addition to CPK3, CPK6 and CPK8, were tested for activity against recombinant 14-3-3 proteins χ and ε. Using an MS-based quantitative assay we demonstrate phosphorylation of 14-3-3 χ and ε at a total of seven sites, one of which is an in vivo site discovered in Arabidopsis. CPK autophosphorylation was also comprehensively monitored by MS and revealed a total of 45 sites among the six CPKs analysed, most of which were located within the N-terminal variable and catalytic domains. Among these CPK autophosphorylation sites was Tyr463 within the calcium-binding EF-hand domain of CPK28. Of all CPKs assayed, CPK28, which contained an autophosphorylation site (Ser43) within a canonical 14-3-3-binding motif, showed the highest activity against 14-3-3 proteins. Phosphomimetic mutagenesis of Ser72 to aspartate on 14-3-3χ, which is adjacent to the 14-3-3-binding cleft and conserved among all 14-3-3 isoforms, prevented 14-3-3-mediated inhibition of phosphorylated nitrate reductase. PMID:24438037

  13. 14-3-3 Proteins: insights from genome-wide studies in yeast.

    PubMed

    van Heusden, G Paul H

    2009-11-01

    14-3-3 proteins form a family of highly conserved, acidic, dimeric proteins. These proteins have been identified in all eukaryotic species investigated, often in multiple isoforms, up to 13 in the plant Arabidopsis thaliana. Hundreds of proteins, from diverse eukaryotic organisms, implicated in numerous cellular processes, have been identified as binding partners of 14-3-3 proteins. Therefore, the major activity of 14-3-3 proteins seems to be its ability to bind other intracellular proteins. Binding to 14-3-3 proteins may result in a conformational change of the protein required for its full activity or for inhibition of its activity, in interaction between two binding partners or in a different subcellular localization. Most of these interactions take place after phosphorylation of the binding partners. These observations suggest a major role of 14-3-3 proteins in regulatory networks. Here, the information on 14-3-3 proteins gathered from several genome- and proteome-wide studies in the yeast Saccharomyces cerevisiae is reviewed. In particular, the protein kinases responsible for the phosphorylation of 14-3-3 binding partners, phosphorylation of 14-3-3 proteins themselves, the transcriptional regulation of the 14-3-3 genes, and the role of 14-3-3 proteins in transcription are addressed. These large scale studies may help understand the function of 14-3-3 proteins at a cellular level rather than at the level of a single process.

  14. Membrane proteins as 14-3-3 clients in functional regulation and intracellular transport.

    PubMed

    Smith, Andrew J; Daut, Jürgen; Schwappach, Blanche

    2011-06-01

    14-3-3 proteins regulate the function and subcellular sorting of membrane proteins. Often, 14-3-3 binding to client proteins requires phosphorylation of the client, but the relevant kinase is unknown in most cases. We summarize current progress in identifying kinases that target membrane proteins with 14-3-3 binding sites and discuss the molecular mechanisms of 14-3-3 action. One of the kinases involved is Akt/PKB, which has recently been shown to activate the 14-3-3-dependent switch in a number of client membrane proteins.

  15. Structural basis for protein–protein interactions in the 14-3-3 protein family

    PubMed Central

    Yang, Xiaowen; Lee, Wen Hwa; Sobott, Frank; Papagrigoriou, Evangelos; Robinson, Carol V.; Grossmann, J. Günter; Sundström, Michael; Doyle, Declan A.; Elkins, Jonathan M.

    2006-01-01

    The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein–protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners. PMID:17085597

  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. Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes

    PubMed Central

    2004-01-01

    14-3-3 proteins exert an extraordinarily widespread influence on cellular processes in all eukaryotes. They operate by binding to specific phosphorylated sites on diverse target proteins, thereby forcing conformational changes or influencing interactions between their targets and other molecules. In these ways, 14-3-3s ‘finish the job’ when phosphorylation alone lacks the power to drive changes in the activities of intracellular proteins. By interacting dynamically with phosphorylated proteins, 14-3-3s often trigger events that promote cell survival – in situations from preventing metabolic imbalances caused by sudden darkness in leaves to mammalian cell-survival responses to growth factors. Recent work linking specific 14-3-3 isoforms to genetic disorders and cancers, and the cellular effects of 14-3-3 agonists and antagonists, indicate that the cellular complement of 14-3-3 proteins may integrate the specificity and strength of signalling through to different cellular responses. PMID:15167810

  18. Light modulated activity of root alkaline/neutral invertase involves the interaction with 14-3-3 proteins.

    PubMed

    Gao, Jing; van Kleeff, Paula J M; Oecking, Claudia; Li, Ka Wan; Erban, Alexander; Kopka, Joachim; Hincha, Dirk K; de Boer, Albertus H

    2014-12-01

    Alkaline/neutral invertases (A/N-Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible breakdown of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At-A/N-InvG or AT1G35580). We demonstrate that Ser547 at the extreme C-terminus of the AtCINV1 protein is a substrate of calcium-dependent kinases (CPK3 and 21) and that phosphorylation creates a high-affinity binding site for 14-3-3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14-3-3 proteins enhances its activity. The analysis of three quadruple 14-3-3 mutants generated from six T-DNA insertion mutants of the non-epsilon family shows both specificity as well as redundancy for this function of 14-3-3 proteins. The strong reduction in hexose levels in the roots of one 14-3-3 quadruple mutant plant is in line with the activating function of 14-3-3 proteins. The physiological relevance of this mechanism that affects A/N-invertase activity is underscored by the light-induced activation and is another example of the central role of 14-3-3 proteins in mediating dark/light signaling. The nature of the light-induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca(++) changes that activate calcium-dependent kinases, await further study.

  19. 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

  20. Arabidopsis 14-3-3 Proteins: Fascinating and Less Fascinating Aspects

    PubMed Central

    Jaspert, Nina; Throm, Christian; Oecking, Claudia

    2011-01-01

    14-3-3 Dimers are well known to interact with diverse target proteins throughout eukaryotes. Most notably, association of 14-3-3s commonly requires phosphorylation of a serine or threonine residue within a specific sequence motif of the client protein. Studies with a focus on individual target proteins have unequivocally demonstrated 14-3-3s to be the crucial factors modifying the client’s activity state upon phosphorylation and, thus, finishing the job initiated by a kinase. In this respect, a recent in-depth analysis of the rice transcription factor FLOWERING LOCUS D1 (OsFD1) revealed 14-3-3s to be essential players in floral induction. Such fascinating discoveries, however, can often be ascribed to the random identification of 14-3-3 as an interaction partner of the favorite protein. In contrast, our understanding of 14-3-3 function in higher organisms is frustratingly limited, mainly due to an overwhelming spectrum of putative targets in combination with the existence of a multigene 14-3-3 family. In this review we will discuss our current understanding of the function of plant 14-3-3 proteins, taking into account recent surveys of the Arabidopsis 14-3-3 interactome. PMID:22639620

  1. Small-Molecule Stabilization of 14-3-3 Protein-Protein Interactions Stimulates Axon Regeneration.

    PubMed

    Kaplan, Andrew; Morquette, Barbara; Kroner, Antje; Leong, SooYuen; Madwar, Carolin; Sanz, Ricardo; Banerjee, Sara L; Antel, Jack; Bisson, Nicolas; David, Samuel; Fournier, Alyson E

    2017-03-08

    Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage.

  2. Mechanism of inhibition of protein kinase C by 14-3-3 isoforms. 14-3-3 isoforms do not have phospholipase A2 activity.

    PubMed Central

    Robinson, K; Jones, D; Patel, Y; Martin, H; Madrazo, J; Martin, S; Howell, S; Elmore, M; Finnen, M J; Aitken, A

    1994-01-01

    The ability of individual members of the 14-3-3 protein family to inhibit protein kinase C (PKC) has been studied by using a synthetic peptide based on the specific 80 kDa substrate for PKC (MARCKS protein) in two different assay systems. Recombinant 14-3-3 and isoforms renatured by a novel method after separation by reverse-phase h.p.l.c. were studied. The detailed effects of diacylglycerol and the phorbol ester phorbol 12-myristate 13-acetate on the inhibition were also investigated. This suggests that one of the sites of interaction of 14-3-3 may be the cysteine-rich (C1) domain in PKC. Since a region in secreted phospholipase A2 (PLA2) shares similarity with this domain, the ability of 14-3-3 to interact with mammalian PLA2 was studied. Cytosolic PLA2 has some similarity to the C2 region of PKC, and the effect of 14-3-3 on this class of PLA2 was also analysed. In contrast with a previous report, no PLA2 activity was found in brain 14-3-3, nor in any of the recombinant proteins tested. These include zeta 14-3-3 isoform, on which the original observation was made. Images Figure 2 PMID:8192676

  3. Fusicoccin, 14-3-3 Proteins, and Defense Responses in Tomato Plants1

    PubMed Central

    Roberts, Michael R.; Bowles, Dianna J.

    1999-01-01

    Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed. PMID:10198082

  4. Efficient nuclear export of p65-IkappaBalpha complexes requires 14-3-3 proteins.

    PubMed

    Aguilera, Cristina; Fernández-Majada, Vanessa; Inglés-Esteve, Julia; Rodilla, Verónica; Bigas, Anna; Espinosa, Lluís

    2006-09-01

    IkappaB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFkappaB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFkappaB signaling pathway by physically interacting with p65 and IkappaBalpha proteins. We identify two functional 14-3-3 binding domains in the p65 protein involving residues 38-44 and 278-283, and map the interaction region of IkappaBalpha in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IkappaBalpha results in a predominantly nuclear distribution of both proteins. TNFalpha treatment promotes recruitment of 14-3-3 and IkappaBalpha to NFkappaB-dependent promoters and enhances the binding of 14-3-3 to p65. Disrupting 14-3-3 activity by transfection with a dominant-negative 14-3-3 leads to the accumulation of nuclear p65-IkappaBalpha complexes and the constitutive association of p65 with the chromatin. In this situation, NFkappaB-dependent genes become unresponsive to TNFalpha stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IkappaBalpha-p65 complexes and are required for the appropriate regulation of NFkappaB signaling.

  5. 14-3-3 Protein isoforms and atypical patterns of the 14-3-3 assay in the diagnosis of Creutzfeldt-Jakob disease.

    PubMed

    Sánchez-Valle, Raquel; Saiz, Albert; Graus, Francesc

    2002-03-01

    A positive 14-3-3 assay is a criterion for probable Creutzfeldt-Jakob disease (CJD). Cerebrospinal fluid (CSF) 14-3-3 is usually detected by immunoblot using an antibody that recognizes all of the 14-3-3 isoforms. In a few cases, the antibody recognizes an inferior band and this pattern is associated with false positive results. We analyzed 43 CSF (26 CJD, 17 controls) samples using antibodies against specific isoforms (beta, epsilon, gamma, tau, xi) and compared the results with those obtained with the standard antibody. The anti-gamma and anti-beta antibody achieved similar results but the presence of atypical patterns made the standard antibody more accurate for the CJD diagnosis. To study the nature of the inferior band, CSF samples were probed with antibodies against light chain immunoglobulins, and immunoblots of human IgG with the standard antibody. The experiments suggested a cross-reaction of the anti-14-3-3 antibody with light chain immunoglobulins.

  6. Interaction network of the 14-3-3 protein in the ancient protozoan parasite Giardia duodenalis.

    PubMed

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Sayadi, Ahmed; Crescenzi, Marco; Pozio, Edoardo

    2012-05-04

    14-3-3s are phosphoserine/phosphotreonine binding proteins that play pivotal roles as regulators of multiple cellular processes in eukaryotes. The flagellated protozoan parasite Giardia duodenalis, the causing agent of giardiasis, is a valuable simplified eukaryotic model. A single 14-3-3 isoform (g14-3-3) is expressed in Giardia, and it is directly involved in the differentiation of the parasite into cyst. To define the overall functions of g14-3-3, the protein interactome has been investigated. A transgenic G. duodenalis strain was engineered to express a FLAG-tagged g14-3-3 under its own promoter. Affinity chromatography coupled with tandem mass spectrometry analysis have been used to purify and identify FLAG-g14-3-3-associated proteins from trophozoites and encysting parasites. A total of 314 putative g14-3-3 interaction partners were identified, including proteins involved in several pathways. Some interactions seemed to be peculiar of one specific stage, while others were shared among the different stages. Furthermore, the interaction of g14-3-3 with the giardial homologue of the CDC7 protein kinase (gCDC7) was characterized, leading to the identification of a multiprotein complex containing not only g14-3-3 and gCDC7 but also a newly identified and highly divergent homologue of DBF4, the putative regulatory subunit of gCDC7. The relevance of g14-3-3 interactions in G. duodenalis biology was discussed.

  7. 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

  8. 14-3-3 proteins: key regulators of cell division, signalling and apoptosis.

    PubMed

    van Hemert, M J; Steensma, H Y; van Heusden, G P

    2001-10-01

    The 14-3-3 proteins constitute a family of conserved proteins present in all eukaryotic organisms so far investigated. These proteins have attracted interest because they are involved in important cellular processes such as signal transduction, cell-cycle control, apoptosis, stress response and malignant transformation and because at least 100 different binding partners for the 14-3-3 proteins have been reported. Although the exact function of 14-3-3 proteins is still unknown, they are known to (1) act as adaptor molecules stimulating protein-protein interactions, (2) regulate the subcellular localisation of proteins and (3) activate or inhibit enzymes. In this review, we discuss the role of the 14-3-3 proteins in three cellular processes: cell cycle control, signal transduction and apoptosis. These processes are regulated by the 14-3-3 proteins at multiple steps. The 14-3-3 proteins have an overall inhibitory effect on cell cycle progression and apoptosis, whereas in signal transduction they may act as stimulatory or inhibitory factors. This article contains supplementary material which may be viewed at the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/Suppmat/23/v23_10.936.

  9. Regulation of the wheat MAP kinase phosphatase 1 by 14-3-3 proteins.

    PubMed

    Ghorbel, Mouna; Cotelle, Valérie; Ebel, Chantal; Zaidi, Ikram; Ormancey, Mélanie; Galaud, Jean-Philippe; Hanin, Moez

    2017-04-01

    Plant MAP kinase phosphatases (MKPs) are major regulators of MAPK signaling pathways and play crucial roles in controlling growth, development and stress responses. The presence of several functional domains in plant MKPs such as a dual specificity phosphatase catalytic domain, gelsolin, calmodulin-binding and serine-rich domains, suggests that MKPs can interact with distinct cellular partners, others than MAPKs. In this report, we identified a canonical mode I 14-3-3-binding motif (574KLPSLP579) located at the carboxy-terminal region of the wheat MKP, TMKP1. We found that this motif is well-conserved among other MKPs from monocots including Hordeum vulgare, Brachypodium distachyon and Aegilops taushii. Using co-immunoprecipitation assays, we provide evidence for interaction between TMKP1 and 14-3-3 proteins in wheat. Moreover, the phosphatase activity of TMKP1 is increased in a phospho-dependent manner by either Arabidopsis or yeast 14-3-3 isoforms. TMKP1 activation by 14-3-3 proteins is enhanced by Mn(2+), whereas in the presence of Ca(2+) ions, TMKP1 activation was limited to Arabidopsis 14-3-3φ (phi), an isoform harboring an EF-hand motif. Such findings strongly suggest that 14-3-3 proteins, in conjunction with specific divalent cations, may stimulate TMKP1 activity and point-out that 14-3-3 proteins bind and regulate the activity of a MKP in eukaryotes.

  10. CDPKs and 14-3-3 Proteins: Emerging Duo in Signaling.

    PubMed

    Ormancey, Mélanie; Thuleau, Patrice; Mazars, Christian; Cotelle, Valérie

    2017-03-01

    Calcium-dependent protein kinases (CDPKs) are Ca(2+)-sensors that play pivotal roles in plant development and stress responses. They have the unique ability to directly translate intracellular Ca(2+) signals into reversible phosphorylation events of diverse substrates which can mediate interactions with 14-3-3 proteins to modulate protein functions. Recent studies have revealed roles for the coordinated action of CDPKs and 14-3-3s in regulating diverse aspects of plant biology including metabolism, development, and stress responses. We review here the underlying interaction and cross-regulation of the two signaling proteins, and we discuss how this insight has led to the emerging concept of CDPK/14-3-3 signaling modules that could contribute to response specificity.

  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.

  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. The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

    PubMed

    Satoh, Jun-ichi; Onoue, Hiroyuki; Arima, Kunimasa; Yamamura, Takashi

    2005-10-01

    The 14-3-3 protein family consists of acidic 30-kDa proteins composed of 7 isoforms expressed abundantly in neurons and glial cells of the central nervous system (CNS). The 14-3-3 protein identified in the cerebrospinal fluid provides a surrogate marker for premortem diagnosis of Creutzfeldt-Jakob disease, although an active involvement of 14-3-3 in the pathogenesis of prion diseases remains unknown. By protein overlay and mass spectrometric analysis of protein extract of NTera2-derived differentiated neurons, we identified heat shock protein Hsp60 as a 14-3-3-interacting protein. The 14-3-3zeta and gamma isoforms interacted with Hsp60, suggesting that the interaction is not isoform-specific. Furthermore, the interaction was identified in SK-N-SH neuroblastoma, U-373MG astrocytoma, and HeLa cervical carcinoma cells. The cellular prion protein (PrPC) along with Hsp60 was coimmunoprecipitated with 14-3-3 in the human brain protein extract. By protein overlay, 14-3-3 interacted with both recombinant human Hsp60 and PrPC produced by Escherichia coli, indicating that the molecular interaction is phosphorylation-independent. The 14-3-3-binding domain was located in the N-terminal half (NTF) of Hsp60 spanning amino acid residues 27-287 and the NTF of PrPC spanning amino acid residues 23-137. By immunostaining, the 14-3-3 protein Hsp60 and PrPC were colocalized chiefly in the mitochondria of human neuronal progenitor cells in culture, and were coexpressed most prominently in neurons and reactive astrocytes in the human brain. These observations indicate that the 14-3-3 protein forms a molecular complex with Hsp60 and PrPC in the human CNS under physiological conditions and suggest that this complex might become disintegrated in the pathologic process of prion diseases.

  14. Loss of Par3 promotes lung adenocarcinoma metastasis through 14-3-3ζ protein.

    PubMed

    Song, Tong; Tian, Xia; Kai, Fan; Ke, Jiang; Wei, Zhai; Jing-Song, Li; Si-Hua, Wang; Jian-Jun, Wang

    2016-09-27

    Partitioning defective protein 3 (Par3) can activate the Tiam1/Rac pathway to inhibit invasion and metastasis in many cancers; however, the role of Par3 in lung adenocarcinoma remains unknown. Here we show that Par3 is downregulated in lung adenocarcinoma tissues and is associated with higher rates of lymph node metastasis and recurrence. Our functional study demonstrated that knock-down of Par3 promoted lung adenocarcinoma cell growth, cell migration, tumor formation, and metastasis, all of which were effectively inhibited when 14-3-3ζ was silenced. We found that Par3 binded with 14-3-3ζ protein and also showed that Par3 abrogated the binding of 14-3-3ζ to Tiam1, which was responsible for Rac1 activation. Knock-down of 14-3-3ζ inhibited Tiam1/Rac-GTP activation and blocked the invasive behavior of cells lacking Par3. These data suggest that loss of Par3 promotes metastatic behavior in lung adenocarcinoma cells through 14-3-3ζ protein.

  15. Loss of Par3 promotes lung adenocarcinoma metastasis through 14-3-3ζ protein

    PubMed Central

    Tong, Song; Xia, Tian; Fan, Kai; Jiang, Ke; Zhai, Wei; Li, Jing-Song; Wang, Si-Hua; Wang, Jian-Jun

    2016-01-01

    Partitioning defective protein 3 (Par3) can activate the Tiam1/Rac pathway to inhibit invasion and metastasis in many cancers; however, the role of Par3 in lung adenocarcinoma remains unknown. Here we show that Par3 is downregulated in lung adenocarcinoma tissues and is associated with higher rates of lymph node metastasis and recurrence. Our functional study demonstrated that knock-down of Par3 promoted lung adenocarcinoma cell growth, cell migration, tumor formation, and metastasis, all of which were effectively inhibited when 14-3-3ζ was silenced. We found that Par3 binded with 14-3-3ζ protein and also showed that Par3 abrogated the binding of 14-3-3ζ to Tiam1, which was responsible for Rac1 activation. Knock-down of 14-3-3ζ inhibited Tiam1/Rac-GTP activation and blocked the invasive behavior of cells lacking Par3. These data suggest that loss of Par3 promotes metastatic behavior in lung adenocarcinoma cells through 14-3-3ζ protein. PMID:27588399

  16. alpha-Synuclein shares physical and functional homology with 14-3-3 proteins.

    PubMed

    Ostrerova, N; Petrucelli, L; Farrer, M; Mehta, N; Choi, P; Hardy, J; Wolozin, B

    1999-07-15

    alpha-Synuclein has been implicated in the pathophysiology of many neurodegenerative diseases, including Parkinson's disease (PD) and Alzheimer's disease. Mutations in alpha-synuclein cause some cases of familial PD (Polymeropoulos et al., 1997; Kruger et al., 1998). In addition, many neurodegenerative diseases show accumulation of alpha-synuclein in dystrophic neurites and in Lewy bodies (Spillantini et al., 1998). Here, we show that alpha-synuclein shares physical and functional homology with 14-3-3 proteins, which are a family of ubiquitous cytoplasmic chaperones. Regions of alpha-synuclein and 14-3-3 proteins share over 40% homology. In addition, alpha-synuclein binds to 14-3-3 proteins, as well as some proteins known to associate with 14-3-3, including protein kinase C, BAD, and extracellular regulated kinase, but not Raf-1. We also show that overexpression of alpha-synuclein inhibits protein kinase C activity. The association of alpha-synuclein with BAD and inhibition of protein kinase C suggests that increased expression of alpha-synuclein could be harmful. Consistent with this hypothesis, we observed that overexpression of wild-type alpha-synuclein is toxic, and overexpression of alpha-synuclein containing the A53T or A30P mutations exhibits even greater toxicity. The activity and binding profile of alpha-synuclein suggests that it might act as a protein chaperone and that accumulation of alpha-synuclein could contribute to cell death in neurodegenerative diseases.

  17. Abscisic acid and 14-3-3 proteins control K channel activity in barley embryonic root.

    PubMed

    van den Wijngaard, Paul W J; Sinnige, Mark P; Roobeek, Ilja; Reumer, Annet; Schoonheim, Peter J; Mol, Jos N M; Wang, Mei; De Boer, Albertus H

    2005-01-01

    Germination of seeds proceeds in general in two phases, an initial imbibition phase and a subsequent growth phase. In grasses like barley, the latter phase is evident as the emergence of the embryonic root (radicle). The hormone abscisic acid (ABA) inhibits germination because it prevents the embryo from entering and completing the growth phase. Genetic and physiological studies have identified many steps in the ABA signal transduction cascade, but how it prevents radicle elongation is still not clear. For elongation growth to proceed, uptake of osmotically active substances (mainly K(+)) is essential. Therefore, we have addressed the question of how the activity of K(+) permeable ion channels in the plasma membrane of radicle cells is regulated under conditions of slow (+ABA) and rapid germination (+fusicoccin). We found that ABA arrests radicle growth, inhibits net K(+) uptake and reduces the activity of K(+) (in) channels as measured with the patch-clamp technique. In contrast, fusicoccin (FC), a well-known stimulator of germination, stimulates radicle growth, net K(+) uptake and reduces the activity of K(+) (out) channels. Both types of channels are under the control of 14-3-3 proteins, known as integral components of signal transduction pathways and instrumental in FC action. Intriguingly, 14-3-3 affected both channels in an opposite fashion: whereas K(+) (in) channel activity was fully dependent upon 14-3-3 proteins, K(+) (out) channel activity was reduced by 14-3-3 proteins by 60%. Together with previous data showing that 14-3-3 proteins control the activity of the plasma membrane H(+)-ATPase, this makes 14-3-3 a prime candidate for molecular master regulator of the cellular osmo-pump. Regulation of the osmo-pump activity by ABA and FC is an important mechanism in controlling the growth of the embryonic root during seed germination.

  18. 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

  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.

  20. 14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response.

    PubMed

    Mohammad, Duaa H; Yaffe, Michael B

    2009-09-02

    The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phosphorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G(2)/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.

  1. Regulation of transcription by Saccharomyces cerevisiae 14-3-3 proteins

    PubMed Central

    Bruckmann, Astrid; Steensma, H. Yde; Teixeira de Mattos, M. Joost; van Heusden, G. Paul H.

    2004-01-01

    14-3-3 proteins form a family of highly conserved eukaryotic proteins involved in a wide variety of cellular processes, including signalling, apoptosis, cell-cycle control and transcriptional regulation. More than 150 binding partners have been found for these proteins. The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2. A bmh1 bmh2 double mutant is unviable in most laboratory strains. Previously, we constructed a temperature-sensitive bmh2 mutant and showed that mutations in RTG3 and SIN4, both encoding transcriptional regulators, can suppress the temperature-sensitive phenotype of this mutant, suggesting an inhibitory role of the 14-3-3 proteins in Rtg3-dependent transcription [van Heusden and Steensma (2001) Yeast 18, 1479–1491]. In the present paper, we report a genome-wide transcription analysis of a temperature-sensitive bmh2 mutant. Steady-state mRNA levels of 60 open reading frames were increased more than 2.0-fold in the bmh2 mutant, whereas those of 78 open reading frames were decreased more than 2.0-fold. In agreement with our genetic experiments, six genes known to be regulated by Rtg3 showed elevated mRNA levels in the mutant. In addition, several genes with other cellular functions, including those involved in gluconeogenesis, ergosterol biosynthesis and stress response, had altered mRNA levels in the mutant. Our data show that the yeast 14-3-3 proteins negatively regulate Rtg3-dependent transcription, stimulate the transcription of genes involved in ergosterol metabolism and in stress response and are involved in transcription regulation of multiple other genes. PMID:15142031

  2. Regulation of transcription by Saccharomyces cerevisiae 14-3-3 proteins.

    PubMed

    Bruckmann, Astrid; Steensma, H Yde; Teixeira De Mattos, M Joost; Van Heusden, G Paul H

    2004-09-15

    14-3-3 proteins form a family of highly conserved eukaryotic proteins involved in a wide variety of cellular processes, including signalling, apoptosis, cell-cycle control and transcriptional regulation. More than 150 binding partners have been found for these proteins. The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2. A bmh1 bmh2 double mutant is unviable in most laboratory strains. Previously, we constructed a temperature-sensitive bmh2 mutant and showed that mutations in RTG3 and SIN4, both encoding transcriptional regulators, can suppress the temperature-sensitive phenotype of this mutant, suggesting an inhibitory role of the 14-3-3 proteins in Rtg3-dependent transcription [van Heusden and Steensma (2001) Yeast 18, 1479-1491]. In the present paper, we report a genome-wide transcription analysis of a temperature-sensitive bmh2 mutant. Steady-state mRNA levels of 60 open reading frames were increased more than 2.0-fold in the bmh2 mutant, whereas those of 78 open reading frames were decreased more than 2.0-fold. In agreement with our genetic experiments, six genes known to be regulated by Rtg3 showed elevated mRNA levels in the mutant. In addition, several genes with other cellular functions, including those involved in gluconeogenesis, ergosterol biosynthesis and stress response, had altered mRNA levels in the mutant. Our data show that the yeast 14-3-3 proteins negatively regulate Rtg3-dependent transcription, stimulate the transcription of genes involved in ergosterol metabolism and in stress response and are involved in transcription regulation of multiple other genes.

  3. 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.

  4. 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.

  5. Quantifying protein interaction dynamics by SWATH mass spectrometry: application to the 14-3-3 system.

    PubMed

    Collins, Ben C; Gillet, Ludovic C; Rosenberger, George; Röst, Hannes L; Vichalkovski, Anton; Gstaiger, Matthias; Aebersold, Ruedi

    2013-12-01

    Protein complexes and protein interaction networks are essential mediators of most biological functions. Complexes supporting transient functions such as signal transduction processes are frequently subject to dynamic remodeling. Currently, the majority of studies on the composition of protein complexes are carried out by affinity purification and mass spectrometry (AP-MS) and present a static view of the system. For a better understanding of inherently dynamic biological processes, methods to reliably quantify temporal changes of protein interaction networks are essential. Here we used affinity purification combined with sequential window acquisition of all theoretical spectra (AP-SWATH) mass spectrometry to study the dynamics of the 14-3-3β scaffold protein interactome after stimulation of the insulin-PI3K-AKT pathway. The consistent and reproducible quantification of 1,967 proteins across all stimulation time points provided insights into the 14-3-3β interactome and its dynamic changes following IGF1 stimulation. We therefore establish AP-SWATH as a tool to quantify dynamic changes in protein-complex interaction networks.

  6. Stimulation of 14-3-3 protein and its isoform on histamine secretion from permeabilized rat peritoneal mast cells.

    PubMed

    Fujii, Toshihiro; Ueeda, Takayuki

    2002-12-01

    The effect of the 14-3-3 protein, an adaptor protein of intracellular signal pathways, on histamine release from rat peritoneal mast cells was investigated. The exogenous 14-3-3 protein from bovine brain increased the Ca(2+)-dependent histamine release from permeabilized mast cells, but only slightly affected the non-permeabilized cells. Partial amino acid sequences showed that the bovine brain 14-3-3 protein contained 14-3-3beta, gamma and zeta isoforms, and that these recombinant isoforms were prepared. Among them, 14-3-3zeta was an active species while the 14-3-3beta and gamma were inactive for histamine release from the permeabilized mast cells. Approximately 15% of the histamine release was stimulated by 14-3-3zeta at 2.5 microM, and half-maximal stimulation occurred at 1 microM. Treatment of the mast cells with wortmannin or staurosporine completely inhibited the stimulatory effect on histamine release caused by Ca(2+) or Ca(2+)/14-3-3zeta, and genistein partially inhibited both stimulatory effects. PD 98059, however, had little effect on the histamine release. These results suggest the possibility that 14-3-3zeta is associated with signal transduction for degranulation of the mast cells.

  7. Identification of novel 14-3-3ζ interacting proteins by quantitative immunoprecipitation combined with knockdown (QUICK).

    PubMed

    Ge, Feng; Li, Wen-Liang; Bi, Li-Jun; Tao, Sheng-Ce; Zhang, Zhi-Ping; Zhang, Xian-En

    2010-11-05

    The family of 14-3-3 proteins has emerged as critical regulators of diverse cellular responses under both physiological and pathological conditions. To gain insight into the molecular action of 14-3-3ζ in multiple myeloma (MM), we performed a systematic proteomic analysis of 14-3-3ζ-associated proteins. This analysis, recently developed by Matthias Mann, termed quantitative immunoprecipitation combined with knockdown (QUICK), integrates RNAi, SILAC, immunoprecipitation, and quantitative MS technologies. Quantitative mass spectrometry analysis allowed us to distinguish 14-3-3ζ-interacting proteins from background proteins, resulting in the identification of 292 proteins in total with 95 novel interactions. Three 14-3-3ζ-interacting proteins-BAX, HSP70, and BAG3-were further confirmed by reciprocal coimmunoprecipitations and colocalization analysis. Our results therefore not only uncover a large number of novel 14-3-3ζ-associated proteins that possess a variety of cellular functions, but also provide new research directions for the study of the functions of 14-3-3ζ. This study also demonstrated that QUICK is a useful approach to detect specific protein-protein interactions with very high confidence and may have a wide range of applications in the investigation of protein complex interaction networks.

  8. Toxoplasma gondii: Effect of infection on expression of 14-3-3 proteins in human epithelial cells

    PubMed Central

    Monroy, Fernando P.

    2008-01-01

    14-3-3 proteins are expressed in most eukaryotes organisms and play varied and crucial roles in a wide range of regulatory processes. In mammalian cells, seven 14-3-3 isoforms have been identified. However; it is not known what effect infection has on 14-3-3 isoform expression. In this study human colonic carcinoma cell lines were infected with Toxoplasma gondii for 24 h and expression of 14-3-3 proteins was determined by RT-PCR. HT-29 cells only expressed 3 out of the 7 isoforms while 5 and all 7 isoforms were found in HCT-116 and Caco-2 cells respectively. Infection had little or no effect in the expression of 14-3-3γ, ε, σ, and ξ; but in HCT-116 cells induced expression of 14-3-3η and σ, while 14-3-3β, η, and ξ were induced in HT-29 cells. If 14-3-3 proteins are involved in cell survival and/or prevention of parasite replication, longer incubation times may be required as no differences in percentage of infection were found among the cell lines at 24 h post-infection. PMID:17825295

  9. IDENTIFICATION AND EXPRESSION ANALYSIS OF TWO 14-3-3 PROTEINS IN THE MOSQUITO Aedes aegypti, AN IMPORTANT ARBOVIRUSES VECTOR.

    PubMed

    Trujillo-Ocampo, Abel; Cázares-Raga, Febe Elena; Celestino-Montes, Antonio; Cortés-Martínez, Leticia; Rodríguez, Mario H; Hernández-Hernández, Fidel de la Cruz

    2016-11-01

    The 14-3-3 proteins are evolutionarily conserved acidic proteins that form a family with several isoforms in many cell types of plants and animals. In invertebrates, including dipteran and lepidopteran insects, only two isoforms have been reported. 14-3-3 proteins are scaffold molecules that form homo- or heterodimeric complexes, acting as molecular adaptors mediating phosphorylation-dependent interactions with signaling molecules involved in immunity, cell differentiation, cell cycle, proliferation, apoptosis, and cancer. Here, we describe the presence of two isoforms of 14-3-3 in the mosquito Aedes aegypti, the main vector of dengue, yellow fever, chikungunya, and zika viruses. Both isoforms have the conserved characteristics of the family: two protein signatures (PS1 and PS2), an annexin domain, three serine residues, targets for phosphorylation (positions 58, 184, and 233), necessary for their function, and nine alpha helix-forming segments. By sequence alignment and phylogenetic analysis, we found that the molecules correspond to Ɛ and ζ isoforms (Aeae14-3-3ε and Aeae14-3-3ζ). The messengers and protein products were present in all stages of the mosquito life cycle and all the tissues analyzed, with a small predominance of Aeae14-3-3ζ except in the midgut and ovaries of adult females. The 14-3-3 proteins in female midgut epithelial cells were located in the cytoplasm. Our results may provide insights to further investigate the functions of these proteins in mosquitoes.

  10. 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.

  11. Human 14-3-3 gamma protein results in abnormal cell proliferation in the developing eye of Drosophila melanogaster

    PubMed Central

    Hong, Sophia W; Qi, Wenqing; Brabant, Marc; Bosco, Giovanni; Martinez, Jesse D

    2008-01-01

    Background 14-3-3 proteins are a family of adaptor proteins that participate in a wide variety of cellular processes. Recent evidence indicates that the expression levels of these proteins are elevated in some human tumors providing circumstantial evidence for their involvement in human cancers. However, the mechanism through which these proteins act in tumorigenesis is uncertain. Results To determine whether elevated levels of 14-3-3 proteins may perturb cell growth we overexpressed human 14-3-3 gamma (h14-3-3 gamma) in Drosophila larvae using the heat shock promoter or the GMR-Gal4 driver and then examined the effect that this had on cell proliferation in the eye imaginal discs of third instar larvae. We found that induction of h14-3-3 gamma resulted in the abnormal appearance of replicating cells in the differentiating proneural photoreceptor cells of eye imaginal discs where h14-3-3 gamma was driven by the heat shock promoter. Similarly, we found that driving h14-3-3 gamma expression specifically in developing eye discs with the GMR-Gal4 driver resulted in increased numbers of replicative cells following the morphogenetic furrow. Interestingly, we found that the effects of overexpressing h1433 gamma on eye development were increased in a genetic background where String (cdc25) function was compromised. Conclusion Taken together our results indicate that h14-3-3 gamma can promote abnormal cell proliferation and may act through Cdc25. This has important implications for 14-3-3 gamma as an oncogene as it suggests that elevated levels of 14-3-3 may confer a growth advantage to cells that overexpress it. PMID:18194556

  12. 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.

  13. 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.

  14. Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates aberrant DNA methylation and gene expression

    PubMed Central

    Estève, Pierre-Olivier; Zhang, Guoqiang; Ponnaluri, V.K. Chaithanya; Deepti, Kanneganti; Chin, Hang Gyeong; Dai, Nan; Sagum, Cari; Black, Karynne; Corrêa, Ivan R.; Bedford, Mark T.; Cheng, Xiaodong; Pradhan, Sriharsa

    2016-01-01

    Mammalian DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for maintenance methylation. Phosphorylation of Ser143 (pSer143) stabilizes DNMT1 during DNA replication. Here, we show 14-3-3 is a reader protein of DNMT1pSer143. In mammalian cells 14-3-3 colocalizes and binds DNMT1pSer143 post-DNA replication. The level of DNMT1pSer143 increased with overexpression of 14-3-3 and decreased by its depletion. Binding of 14-3-3 proteins with DNMT1pSer143 resulted in inhibition of DNA methylation activity in vitro. In addition, overexpression of 14-3-3 in NIH3T3 cells led to decrease in DNMT1 specific activity resulting in hypomethylation of the genome that was rescued by transfection of DNMT1. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, overexpression of 14-3-3 also resulted in enhanced cell invasion. Analysis of TCGA breast cancer patient data showed significant correlation for DNA hypomethylation and reduced patient survival with increased 14-3-3 expressions. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNA methylation and altered gene expression that contributes to cell invasion. PMID:26553800

  15. 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.

  16. 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

  17. Evolution of signal multiplexing by 14-3-3-binding 2R-ohnologue protein families in the vertebrates

    PubMed Central

    Tinti, Michele; Johnson, Catherine; Toth, Rachel; Ferrier, David E. K.; MacKintosh, Carol

    2012-01-01

    14-3-3 proteins regulate cellular responses to stimuli by docking onto pairs of phosphorylated residues on target proteins. The present study shows that the human 14-3-3-binding phosphoproteome is highly enriched in 2R-ohnologues, which are proteins in families of two to four members that were generated by two rounds of whole genome duplication at the origin of the vertebrates. We identify 2R-ohnologue families whose members share a ‘lynchpin’, defined as a 14-3-3-binding phosphosite that is conserved across members of a given family, and aligns with a Ser/Thr residue in pro-orthologues from the invertebrate chordates. For example, the human receptor expression enhancing protein (REEP) 1–4 family has the commonest type of lynchpin motif in current datasets, with a phosphorylatable serine in the –2 position relative to the 14-3-3-binding phosphosite. In contrast, the second 14-3-3-binding sites of REEPs 1–4 differ and are phosphorylated by different kinases, and hence the REEPs display different affinities for 14-3-3 dimers. We suggest a conceptual model for intracellular regulation involving protein families whose evolution into signal multiplexing systems was facilitated by 14-3-3 dimer binding to lynchpins, which gave freedom for other regulatory sites to evolve. While increased signalling complexity was needed for vertebrate life, these systems also generate vulnerability to genetic disorders. PMID:22870394

  18. Cotton (Gossypium hirsutum) 14-3-3 proteins participate in regulation of fibre initiation and elongation by modulating brassinosteroid signalling.

    PubMed

    Zhou, Ying; Zhang, Ze-Ting; Li, Mo; Wei, Xin-Zheng; Li, Xiao-Jie; Li, Bing-Ying; Li, Xue-Bao

    2015-02-01

    Cotton (Gossypium hirsutum) fibre is an important natural raw material for textile industry in the world. Understanding the molecular mechanism of fibre development is important for the development of future cotton varieties with superior fibre quality. In this study, overexpression of Gh14-3-3L in cotton promoted fibre elongation, leading to an increase in mature fibre length. In contrast, suppression of expression of Gh14-3-3L, Gh14-3-3e and Gh14-3-3h in cotton slowed down fibre initiation and elongation. As a result, the mature fibres of the Gh14-3-3 RNAi transgenic plants were significantly shorter than those of wild type. This 'short fibre' phenotype of the 14-3-3 RNAi cotton could be partially rescued by application of 2,4-epibrassinolide (BL). Expression levels of the BR-related and fibre-related genes were altered in the Gh14-3-3 transgenic fibres. Furthermore, we identified Gh14-3-3 interacting proteins (including GhBZR1) in cotton. Site mutation assay revealed that Ser163 in GhBZR1 and Lys51/56/53 in Gh14-3-3L/e/h were required for Gh14-3-3-GhBZR1 interaction. Nuclear localization of GhBZR1 protein was induced by BR, and phosphorylation of GhBZR1 by GhBIN2 kinase was helpful for its binding to Gh14-3-3 proteins. Additionally, 14-3-3-regulated GhBZR1 protein may directly bind to GhXTH1 and GhEXP promoters to regulate gene expression for responding rapid fibre elongation. These results suggested that Gh14-3-3 proteins may be involved in regulating fibre initiation and elongation through their interacting with GhBZR1 to modulate BR signalling. Thus, our study provides the candidate intrinsic genes for improving fibre yield and quality by genetic manipulation.

  19. 14-3-3 Proteins are essential for regulation of RTG3-dependent transcription in Saccharomyces cerevisiae.

    PubMed

    van Heusden, G P; Steensma, H Y

    2001-12-01

    14-3-3 proteins comprise a family of highly conserved proteins that bind more than 60 different, mostly phosphorylated, proteins. The yeast Saccharomyces cerevisiae has two genes, BMH1 and BMH2, encoding 14-3-3 proteins. Disruption of both genes together is lethal. In this study we constructed a mutant with a single, temperature-sensitive bmh allele. Recessive mutations in SIN4 and RTG3 can suppress the temperature-sensitive phenotype of this mutant. These genes encode a global transcriptional regulator and a basic helix-loop-helix transcription factor, respectively. The yeast 14-3-3 proteins were shown to bind to the Rtg3 protein. Overexpression of RTG3 is lethal even in wild-type cells. These genetic and biochemical data are consistent with a model in which the 14-3-3 proteins are required to keep the Rtg3 protein in an inactive state, which is (one of) the essential function(s) of the 14-3-3 proteins.

  20. Association of GABA(B) receptors and members of the 14-3-3 family of signaling proteins.

    PubMed

    Couve, A; Kittler, J T; Uren, J M; Calver, A R; Pangalos, M N; Walsh, F S; Moss, S J

    2001-02-01

    Two GABA(B) receptors, GABA(B)R1 and GABA(B)R2, have been cloned recently. Unlike other G protein-coupled receptors, the formation of a heterodimer between GABA(B)R1 and GABA(B)R2 is required for functional expression. We have used the yeast two hybrid system to identify proteins that interact with the C-terminus of GABA(B)R1. We report a direct association between GABA(B) receptors and two members of the 14-3-3 protein family, 14-3-3eta and 14-3-3zeta. We demonstrate that the C-terminus of GABA(B)R1 associates with 14-3-3zeta in rat brain preparations and tissue cultured cells, that they codistribute after rat brain fractionation, colocalize in neurons, and that the binding site overlaps partially with the coiled-coil domain of GABA(B)R1. Furthermore we show a reduced interaction between the C-terminal domains of GABA(B)R1 and GABA(B)R2 in the presence of 14-3-3. The results strongly suggest that GABA(B)R1 and 14-3-3 associate in the nervous system and begin to reveal the signaling complexities of the GABA(B)R1/GABA(B)R2 receptor heterodimer.

  1. Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice

    PubMed Central

    Bai, Ming-Yi; Zhang, Li-Ying; Gampala, Srinivas S.; Zhu, Sheng-Wei; Song, Wen-Yuan; Chong, Kang; Wang, Zhi-Yong

    2007-01-01

    Brassinosteroids (BR) are essential growth hormones found throughout the plant kingdom. BR bind to the receptor kinase BRI1 on the cell surface to activate a signal transduction pathway that regulates nuclear gene expression and plant growth. To understand the downstream BR signaling mechanism in rice, we studied the function of OsBZR1 using reverse genetic approaches and identified OsBZR1-interacting proteins. Suppressing OsBZR1 expression by RNAi resulted in dwarfism, erect leaves, reduced BR sensitivity, and altered BR-responsive gene expression in transgenic rice plants, demonstrating an essential role of OsBZR1 in BR responses in rice. Moreover, a yeast two-hybrid screen identified 14-3-3 proteins as OsBZR1-interacting proteins. Mutation of a putative 14-3-3-binding site of OsBZR1 abolished its interaction with the 14-3-3 proteins in yeast and in vivo. Such mutant OsBZR1 proteins suppressed the phenotypes of the Arabidopsis bri1–5 mutant and showed an increased nuclear distribution compared with the wild-type protein, suggesting that 14-3-3 proteins directly inhibit OsBZR1 function at least in part by reducing its nuclear localization. These results demonstrate a conserved function of OsBZR1 and an important role of 14-3-3 proteins in brassinosteroid signal transduction in rice. PMID:17699623

  2. Neuroprotective Effect of TAT-14-3-3ε Fusion Protein against Cerebral Ischemia/Reperfusion Injury in Rats

    PubMed Central

    Liu, Xiaoyan; Hu, Wenhui; Wang, Yinye

    2014-01-01

    Stroke is the major cause of death and disability worldwide, and the thrombolytic therapy currently available was unsatisfactory. 14-3-3ε is a well characterized member of 14-3-3 family, and has been reported to protect neurons against apoptosis in cerebral ischemia. However, it cannot transverse blood brain barrier (BBB) due to its large size. A protein transduction domain (PTD) of HIV TAT protein, is capable of delivering a large variety of proteins into the brain. In this study, we generated a fusion protein TAT-14-3-3ε, and evaluated its potential neuroprotective effect in rat focal ischemia/reperfusion (I/R) model. Western blot analysis validated the efficient transduction of TAT-14-3-3ε fusion protein into brain via a route of intravenous injection. TAT-14-3-3ε pre-treatment 2 h before ischemia significantly reduced cerebral infarction volume and improved neurologic score, while post-treatment 2 h after ischemia was less effective. Importantly, pre- or post-ischemic treatment with TAT-14-3-3ε significantly increased the number of surviving neurons as determined by Nissl staining, and attenuated I/R-induced neuronal apoptosis as showed by the decrease in apoptotic cell numbers and the inhibition of caspase-3 activity. Moreover, the introduction of 14-3-3ε into brain by TAT-mediated delivering reduced the formation of autophagosome, attenuated LC3B-II upregulation and reversed p62 downregulation induced by ischemic injury. Such inhibition of autophagy was reversed by treatment with an autophagy inducer rapamycin (RAP), which also attenuated the neuroprotective effect of TAT-14-3-3ε. Conversely, autophagy inhibitor 3-methyladenine (3-MA) inhibited I/R-induced the increase in autophagic activity, and attenuated I/R-induced brain infarct. These results suggest that TAT-14-3-3ε can be efficiently transduced into brain and exert significantly protective effect against brain ischemic injury through inhibiting neuronal apoptosis and autophagic activation. PMID

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

    PubMed

    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.

  4. Interaction of 14-3-3 proteins with the estrogen receptor alpha F domain provides a drug target interface.

    PubMed

    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

    2013-05-28

    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-T(594)) for high-affinity interaction. We confirm that T(594) is a distinct ERα phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T(594)-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.

  5. Antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using fully automated two-dimensional chip gel electrophoresis.

    PubMed

    Iwamoto, M; Miura, Y; Tsumoto, H; Tanaka, Y; Morisawa, H; Endo, T; Toda, T

    2014-12-01

    We here described the antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using the fully automated two-dimensional chip gel electrophoresis system (Auto2D). This system was easy and convenient to use, and the resolution obtained was more sensitive and higher than that of conventional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). We separated and identified five isoforms of the 14-3-3 protein (beta/alpha, gamma, epsilon, zeta/delta, and eta) using the Auto2D system. We then examined the antioxidant effects of carnitine supplementation on the protein profiles of the cytosolic fraction in the aged rat hippocampus, demonstrating that carnitine supplementation suppressed the oxidation of methionine residues in these isoforms. Since methionine residues are easily oxidized to methionine sulfoxide, the convenient and high-resolution 2-D PAGE system can be available to analyze methionine oxidation avoiding artifactual oxidation. We showed here that the Auto2D system was a very useful tool for studying antioxidant effects through proteomic analysis of protein oxidation.

  6. Alternative application of Tau protein in Creutzfeldt-Jakob disease diagnosis: Improvement for weakly positive 14-3-3 protein in the laboratory

    PubMed Central

    Hyeon, Jae Wook; Kim, Su Yeon; Lee, Jeongmin; Park, Jun Sun; Hwang, Kyu Jam; Lee, Sol Moe; An, SeongSoo A.; Lee, Myung Koo; Ju, Young Ran

    2015-01-01

    The 14-3-3 protein has been used as a biomarker for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). However, weakly positive 14-3-3 leads to false positive results and an incorrect diagnosis. We attempted to use quantitative data for tau protein to provide an accurate diagnosis based on weak 14-3-3 protein. Sixty-two patients with sCJD, including pathologically confirmed, clinically definite, and probable cases, and 89 non-CJD patients were investigated based on a Korean population. Among them, 20 sCJD and 14 non-CJD showed weakly positive 14-3-3. The total tau (t-tau) and phosphorylated tau (p-tau) protein levels were measured by ELISA, and the p-tau to t-tau ratio (p/t ratio) was calculated. The combined use of the 14-3-3 protein assay, t-tau levels, and p/t ratio improved the specificity of diagnosis compared with the use of the 14-3-3 protein assay alone (47% for 14-3-3 alone; 85.94% for 14-3-3 combined with t-tau; 90.62% for 14-3-3 combined with the p/t ratio). In addition, 18 of 20 sCJD and 12 of 14 non-CJD who were weakly positive for 14-3-3 were positive for the p/t ratio and negative for the p/t ratio, respectively. When used in combination with the 14-3-3 protein, the tau protein is useful as a biomarker for the precise diagnosis of sCJD. PMID:26507666

  7. Alternative application of Tau protein in Creutzfeldt-Jakob disease diagnosis: Improvement for weakly positive 14-3-3 protein in the laboratory.

    PubMed

    Hyeon, Jae Wook; Kim, Su Yeon; Lee, Jeongmin; Park, Jun Sun; Hwang, Kyu Jam; Lee, Sol Moe; An, SeongSoo A; Lee, Myung Koo; Ju, Young Ran

    2015-10-28

    The 14-3-3 protein has been used as a biomarker for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). However, weakly positive 14-3-3 leads to false positive results and an incorrect diagnosis. We attempted to use quantitative data for tau protein to provide an accurate diagnosis based on weak 14-3-3 protein. Sixty-two patients with sCJD, including pathologically confirmed, clinically definite, and probable cases, and 89 non-CJD patients were investigated based on a Korean population. Among them, 20 sCJD and 14 non-CJD showed weakly positive 14-3-3. The total tau (t-tau) and phosphorylated tau (p-tau) protein levels were measured by ELISA, and the p-tau to t-tau ratio (p/t ratio) was calculated. The combined use of the 14-3-3 protein assay, t-tau levels, and p/t ratio improved the specificity of diagnosis compared with the use of the 14-3-3 protein assay alone (47% for 14-3-3 alone; 85.94% for 14-3-3 combined with t-tau; 90.62% for 14-3-3 combined with the p/t ratio). In addition, 18 of 20 sCJD and 12 of 14 non-CJD who were weakly positive for 14-3-3 were positive for the p/t ratio and negative for the p/t ratio, respectively. When used in combination with the 14-3-3 protein, the tau protein is useful as a biomarker for the precise diagnosis of sCJD.

  8. 14-3-3 and its binding partners are regulators of protein–protein interactions during spermatogenesis

    PubMed Central

    Sun, Shengyi; Wong, Elissa W P; Li, Michelle W M; Lee, Will M; Cheng, C Yan

    2009-01-01

    During spermatogenesis, spermiation takes place at the adluminal edge of the seminiferous epithelium at stage VIII of the epithelial cycle during which fully developed spermatids (i.e. spermatozoa) detach from the epithelium in adult rat testes. This event coincides with the migration of preleptotene/leptotene spermatocytes across the blood–testis barrier from the basal to the apical (or adluminal) compartment. At stage XIV of the epithelial cycle, Pachytene spermatocytes (diploid, 2n) differentiate into diplotene spermatocytes (tetraploid, 4n) in the apical compartment of the epithelium, which begin meiosis I to be followed by meiosis II to form spermatids (haploid, 1n) at stage XIVof the epithelial cycle. These spermatids, in turn, undergo extensive morphological changes and traverse the seminiferous epithelium until they differentiate into elongated spermatids. Thus, there are extensive changes at the Sertoli–Sertoli and Sertoli–germ cell interface via protein ‘coupling’ and ‘uncoupling’ between cell adhesion protein complexes, as well as changes in interactions between integral membrane proteins and their peripheral adaptors, regulatory protein kinases and phosphatases, and the cytoskeletal proteins. These precisely coordinated protein–protein interactions affect cell adhesion and cell movement. In this review, we focus on the 14-3-3 protein family, whose members have different binding partners in the seminiferous epithelium. Recent studies have illustrated that 14-3-3 affects protein–protein interactions in the seminiferous epithelium, and regulates cell adhesion possibly via its effects on intracellular protein trafficking and cell-polarity proteins. This review provides a summary on the latest findings regarding the role of 14-3-3 family of proteins and their potential implications on spermatogenesis. We also highlight research areas that deserve attentions by investigators. PMID:19366886

  9. Dexamethasone downregulated the expression of CSF 14-3-3β protein in mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection.

    PubMed

    Tsai, Hung-Chin; Lee, Bi-Yao; Yen, Chuan-Min; Wann, Shue-Ren; Lee, Susan Shin-Jung; Chen, Yao-Shen; Tai, Ming-Hong

    2014-03-01

    Angiostrongylus cantonensis is the main causative agent of human eosinophilic meningitis in Southeast Asia and the Pacific Islands. A previous study demonstrated that the 14-3-3β protein is a neuropathological marker in monitoring neuronal damage in meningitis. Steroids are commonly used in patients with eosinophilic meningitis caused by A. cantonensis infection. However, the mechanism by which steroids act in eosinophilic meningitis is unknown. We hypothesized that the beneficial effect of steroids on eosinophilic meningitis is partially mediated by the down-regulation of 14-3-3β protein expression in the cerebrospinal fluid (CSF). In this animal study, we determined the dynamic changes of 14-3-3β protein in mice with eosinophilic meningitis. The 14-3-3β protein in serum and CSF was increased in week 2 and 3 after infections. Dexamethasone administration significantly decreased the amounts of CSF 14-3-3β protein. By developing an in-house ELISA to measure 14-3-3β protein, it was found that the amounts of 14-3-3β protein in the CSF and serum increased over a three-week period after infection. There was a remarkable reduction of 14-3-3β protein in the CSF after 2 weeks of dexamethasone treatment. In conclusion, the administration of corticosteroids in mice with eosinophilic meningitis decreased the expression of 14-3-3β protein in the CSF.

  10. Identification of a 14-3-3 protein from Lentinus edodes that interacts with CAP (adenylyl cyclase-associated protein), and conservation of this interaction in fission yeast.

    PubMed

    Zhou, G L; Yamamoto, T; Ozoe, F; Yano, D; Tanaka, K; Matsuda, H; Kawamukai, M

    2000-01-01

    We previously identified a gene encoding a CAP (adenylyl cyclase-associated protein) homologue from the edible Basidiomycete Lentinus edodes. To further discover the cellular functions of the CAP protein, we searched for CAP-interacting proteins using a yeast two-hybrid system. Among the candidates thus obtained, many clones encoded the C-terminal half of an L. edodes 14-3-3 homologue (designated cip3). Southern blot analysis indicated that L. edodes contains only one 14-3-3 gene. Overexpression of the L. edodes 14-3-3 protein in the fission yeast Schizosaccharomyces pombe rad24 null cells complemented the loss of endogenous 14-3-3 protein functions in cell morphology and UV sensitivity, suggesting functional conservation of 14-3-3 proteins between L. edodes and S. pombe. The interaction between L. edodes CAP and 14-3-3 protein was restricted to the N-terminal domain of CAP and was confirmed by in vitro co-precipitation. Results from both the two-hybrid system and in vivo co-precipitation experiments showed the conservation of this interaction in S. pombe. The observation that a 14-3-3 protein interacts with the N-terminal portion of CAP but not with full-length CAP in L. edodes and S. pombe suggests that the C-terminal region of CAP may have a negative effect on the interaction between CAP and 14-3-3 proteins, and 14-3-3 proteins may play a role in regulation of CAP function.

  11. 14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility cofactor

    PubMed Central

    1996-01-01

    14-3-3 is a ubiquitous protein family that interacts with several signal transduction kinases. We show that 14-3-3 proteins associate with keratin intermediate filament polypeptides 8 and 18 (K8/18) that are expressed in simple-type epithelia. The association is stoichiometrically significant (> or = one 14-3-3 molecule/keratin tetramer), occurs preferentially with K18, and is phosphorylation- and cell cycle-dependent in that it occurs during S/G2/M phases of the cell cycle when keratins become hyperphosphorylated. Binding of phospho- K8/18 to 14-3-3 can be reconstituted in vitro using recombinant 14-3-3 or using total cellular cytosol. Phosphatase treatment results in dissociation of 14-3-3, and dephosphorylation of phospho-K8/18 prevents reconstitution of the binding. Three cellular keratin subpopulations were analyzed that showed parallel gradients of keratin phosphorylation and 14-3-3 binding. Incubation of 14-3-3 with keratins during or after in vitro filament assembly results in sequestering of additional soluble keratin, only in cases when the keratins were hyperphosphorylated. Our results demonstrate a stoichiometrically significant cell cycle- and phosphorylation-regulated binding of 14-3-3 proteins to K18 and in vitro evidence of a simple epithelial keratin sequestering role for 14-3-3 proteins. PMID:8609167

  12. Protein Phosphatase 2A Reactivates FOXO3a through a Dynamic Interplay with 14-3-3 and AKT

    PubMed Central

    Singh, Amrik; Ye, Min; Bucur, Octavian; Zhu, Shudong; Tanya Santos, Maria; Rabinovitz, Isaac; Wei, Wenyi; Gao, Daming; Hahn, William C.

    2010-01-01

    Forkhead box transcription factor FOXO3a, a key regulator of cell survival, is regulated by reversible phosphorylation and subcellular localization. Although the kinases regulating FOXO3a activity have been characterized, the role of protein phosphatases (PP) in the control of FOXO3a subcellular localization and function is unknown. In this study, we detected a robust interaction between FOXO3a and PP2A. We further demonstrate that 14-3-3, while not impeding the interaction between PP2A and FOXO3a, restrains its activity toward AKT phosphorylation sites T32/S253. Disruption of PP2A function revealed that after AKT inhibition, PP2A-mediated dephosphorylation of T32/S253 is required for dissociation of 14-3-3, nuclear translocation, and transcriptional activation of FOXO3a. Our findings reveal that distinct phosphatases dephosphorylate conserved AKT motifs within the FOXO family and that PP2A is entwined in a dynamic interplay with AKT and 14-3-3 to directly regulate FOXO3a subcellular localization and transcriptional activation. PMID:20110348

  13. 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

  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. Identification of cofilin and LIM-domain-containing protein kinase 1 as novel interaction partners of 14-3-3 zeta.

    PubMed Central

    Birkenfeld, Jörg; Betz, Heinrich; Roth, Dagmar

    2003-01-01

    Proteins of the 14-3-3 family have been implicated in various physiological processes, and are thought to function as adaptors in various signal transduction pathways. In addition, 14-3-3 proteins may contribute to the reorganization of the actin cytoskeleton by interacting with as yet unidentified actin-binding proteins. Here we show that the 14-3-3 zeta isoform interacts with both the actin-depolymerizing factor cofilin and its regulatory kinase, LIM (Lin-11/Isl-1/Mec-3)-domain-containing protein kinase 1 (LIMK1). In both yeast two-hybrid assays and glutathione S-transferase pull-down experiments, these proteins bound efficiently to 14-3-3 zeta. Deletion analysis revealed consensus 14-3-3 binding sites on both cofilin and LIMK1. Furthermore, the C-terminal region of 14-3-3 zeta inhibited the binding of cofilin to actin in co-sedimentation experiments. Upon co-transfection into COS-7 cells, 14-3-3 zeta-specific immunoreactivity was redistributed into characteristic LIMK1-induced actin aggregations. Our data are consistent with 14-3-3-protein-induced changes to the actin cytoskeleton resulting from interactions with cofilin and/or LIMK1. PMID:12323073

  16. 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.

  17. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    PubMed

    Fiorillo, Annarita; di Marino, Daniele; Bertuccini, Lucia; Via, Allegra; Pozio, Edoardo; Camerini, Serena; Ilari, Andrea; Lalle, Marco

    2014-01-01

    The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

  18. Deleting the 14-3-3 protein Bmh1 extends life span in Saccharomyces cerevisiae by increasing stress response.

    PubMed

    Wang, Chen; Skinner, Craig; Easlon, Erin; Lin, Su-Ju

    2009-12-01

    Enhanced stress response has been suggested to promote longevity in many species. Calorie restriction (CR) and conserved nutrient-sensing target of rapamycin (TOR) and protein kinase A (PKA) pathways have also been suggested to extend life span by increasing stress response, which protects cells from age-dependent accumulation of oxidative damages. Here we show that deleting the yeast 14-3-3 protein, Bmh1, extends chronological life span (CLS) by activating the stress response. 14-3-3 proteins are highly conserved chaperone-like proteins that play important roles in many cellular processes. bmh1Delta-induced heat resistance and CLS extension require the general stress-response transcription factors Msn2, Msn4, and Rim15. The bmh1Delta mutant also displays a decreased reactive oxygen species level and increased heat-shock-element-driven transcription activity. We also show that BMH1 genetically interacts with CR and conserved nutrient-sensing TOR- and PKA-signaling pathways to regulate life span. Interestingly, the level of phosphorylated Ser238 on Bmh1 increases during chronological aging, which is delayed by CR or by reduced TOR activities. In addition, we demonstrate that PKA can directly phosphorylate Ser238 on Bmh1. The status of Bmh1 phosphorylation is therefore likely to play important roles in life-span regulation. Together, our studies suggest that phosphorylated Bmh1 may cause inhibitory effects on downstream longevity factors, including stress-response proteins. Deleting Bmh1 may eliminate the inhibitory effects of Bmh1 on these longevity factors and therefore extends life span.

  19. Plasma Membrane CRPK1-Mediated Phosphorylation of 14-3-3 Proteins Induces Their Nuclear Import to Fine-Tune CBF Signaling during Cold Response.

    PubMed

    Liu, Ziyan; Jia, Yuxin; Ding, Yanglin; Shi, Yiting; Li, Zhen; Guo, Yan; Gong, Zhizhong; Yang, Shuhua

    2017-04-06

    In plant cells, changes in fluidity of the plasma membrane may serve as the primary sensor of cold stress; however, the precise mechanism and how the cell transduces and fine-tunes cold signals remain elusive. Here we show that the cold-activated plasma membrane protein cold-responsive protein kinase 1 (CRPK1) phosphorylates 14-3-3 proteins. The phosphorylated 14-3-3 proteins shuttle from the cytosol to the nucleus, where they interact with and destabilize the key cold-responsive C-repeat-binding factor (CBF) proteins. Consistent with this, the crpk1 and 14-3-3κλ mutants show enhanced freezing tolerance, and transgenic plants overexpressing 14-3-3λ show reduced freezing tolerance. Further study shows that CRPK1 is essential for the nuclear translocation of 14-3-3 proteins and for 14-3-3 function in freezing tolerance. Thus, our study reveals that the CRPK1-14-3-3 module transduces the cold signal from the plasma membrane to the nucleus to modulate CBF stability, which ensures a faithfully adjusted response to cold stress of plants.

  20. Structural characterization of a unique interface between carbohydrate response element-binding protein (ChREBP) and 14-3-3β protein.

    PubMed

    Ge, Qiang; Huang, Nian; Wynn, R Max; Li, Yang; Du, Xinlin; Miller, Bonnie; Zhang, Hong; Uyeda, Kosaku

    2012-12-07

    Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose-responsive ChREBP nuclear import and export. Here, we report the crystal structure of a complex of 14-3-3β bound to the N-terminal regulatory region of ChREBP at 2.4 Å resolution. The crystal structure revealed that the α2 helix of ChREBP (residues 117-137) adopts a well defined α-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein-binding modes. ChREBP α2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP α2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3β·ChREBP α2 complex.

  1. RGS3 interacts with 14-3-3 via the N-terminal region distinct from the RGS (regulator of G-protein signalling) domain.

    PubMed Central

    Niu, Jiaxin; Scheschonka, Astrid; Druey, Kirk M; Davis, Amanda; Reed, Eleanor; Kolenko, Vladimir; Bodnar, Richard; Voyno-Yasenetskaya, Tatyana; Du, Xiaoping; Kehrl, John; Dulin, Nickolai O

    2002-01-01

    RGS3 belongs to a family of the regulators of G-protein signalling (RGS), which bind and inhibit the G alpha subunits of heterotrimeric G-proteins via a homologous RGS domain. Increasing evidence suggests that RGS proteins can also interact with targets other than G-proteins. Employing yeast two-hybrid screening of a cDNA library, we identified an interaction between RGS3 and the phosphoserine-binding protein 14-3-3. This interaction was confirmed by in vitro binding and co-immunoprecipitation experiments. RGS3-deletion analysis revealed the presence of a single 14-3-3-binding site located outside of the RGS domain. Ser(264) was then identified as the 14-3-3-binding site of RGS3. The S(264)A mutation resulted in the loss of RGS3 binding to 14-3-3, without affecting its ability to bind G alpha(q). Signalling studies showed that the S(264)A mutant was more potent than the wild-type RGS3 in inhibition of G-protein-mediated signalling. Binding experiments revealed that RGS3 exists in two separate pools, either 14-3-3-bound or G-protein-bound, and that the 14-3-3-bound RGS3 is unable to interact with G-proteins. These data are consistent with the model wherein 14-3-3 serves as a scavenger of RGS3, regulating the amounts of RGS3 available for binding G-proteins. This study describes a new level in the regulation of G-protein signalling, in which the inhibitors of G-proteins, RGS proteins, can themselves be regulated by phosphorylation and binding 14-3-3. PMID:11985497

  2. Discovery of 14-3-3 protein-protein interaction inhibitors that sensitize multidrug-resistant cancer cells to doxorubicin and the Akt inhibitor GSK690693.

    PubMed

    Mori, Mattia; Vignaroli, Giulia; Cau, Ylenia; Dinić, Jelena; Hill, Richard; Rossi, Matteo; Colecchia, David; Pešić, Milica; Link, Wolfgang; Chiariello, Mario; Ottmann, Christian; Botta, Maurizio

    2014-05-01

    14-3-3 is a family of highly conserved adapter proteins that is attracting much interest among medicinal chemists. Small-molecule inhibitors of 14-3-3 protein-protein interactions (PPIs) are in high demand, both as tools to increase our understanding of 14-3-3 actions in human diseases and as leads to develop innovative therapeutic agents. Herein we present the discovery of novel 14-3-3 PPI inhibitors through a multidisciplinary strategy combining molecular modeling, organic synthesis, image-based high-content analysis of reporter cells, and in vitro assays using cancer cells. Notably, the two most active compounds promoted the translocation of c-Abl and FOXO pro-apoptotic factors into the nucleus and sensitized multidrug-resistant cancer cells to apoptotic inducers such as doxorubicin and the pan-Akt inhibitor GSK690693, thus becoming valuable lead candidates for further optimization. Our results emphasize the possible role of 14-3-3 PPI inhibitors in anticancer combination therapies.

  3. Engagement of beta2 integrins recruits 14-3-3 proteins to c-Cbl in human neutrophils.

    PubMed

    Melander, Fredrik; Andersson, Tommy; Dib, Karim

    2004-05-14

    We found that engagement of beta2 integrins on human neutrophils triggered both tyrosine and serine phosphorylation of c-Cbl. Pretreatment of the neutrophils with the broad range protein kinase C (PKC) inhibitor GF-109203X blocked the serine but not the tyrosine phosphorylation of c-Cbl. Moreover, the Src kinase inhibitor PP1 prevented the beta2 integrin-induced tyrosine phosphorylation of c-Cbl but not the simultaneous serine phosphorylation. These results indicate that Src family kinases and PKC can separately modulate the properties of c-Cbl. Indeed, tyrosine kinase-dependent phosphorylation of c-Cbl regulated the ubiquitin ligase activity of that protein, whereas PKC-dependent phosphorylation of c-Cbl had no such effect. Instead, c-Cbl that underwent PKC-induced serine phosphorylation associated with the scaffolding and anti-apoptotic 14-3-3 proteins. Consequently, c-Cbl can independently target proteins for degradation or intracellular localization and may initiate an anti-apoptotic signal in neutrophils.

  4. Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein

    PubMed Central

    Gayen, Nilanjan; Mroue, Kamal H.; Kar, Rajiv K.; Mandal, Atin K.; Bhunia, Anirban

    2015-01-01

    Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3ζ to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3ζ protein and determined the possible structural adaptation of this region. Biophysical elucidation of the interaction was carried out using phosphopeptide (residue number 615–630) in the presence of 14-3-3ζ protein. Using isothermal titration calorimetry (ITC), a high binding affinity with micro-molar range was found to exist between the peptide and 14-3-3ζ protein, whereas the non-phosphorylated peptide did not show any appreciable binding affinity. Further interaction details were investigated using several biophysical techniques such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, in addition to molecular modeling. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3ζ protein as well as structural insights responsible for phosphorylated S621-mediated 14-3-3ζ binding at an atomic resolution. PMID:26295714

  5. The Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 directly influence the DNA damage-dependent functions of Rad53

    PubMed Central

    Usui, Takehiko; Petrini, John H. J.

    2007-01-01

    In this study, we mutated autophosphorylation sites in Rad53 based on their conservation with previously identified autophosphorylation sites in the mammalian Rad53 ortholog, Chk2. As with wild-type Rad53, the autophosphorylation mutant, rad53-TA, undergoes Mec1/Tel1-dependent interactions with Rad9 and Dun1 in response to genotoxic stress. Whereas rad53-TA in vitro kinase activity is severely impaired, the rad53-TA strains are not completely deficient for cell-cycle checkpoint functions, indicating that the mutant kinase retains a basal level of function. We describe a genetic interaction among Rad53, Dun1, and the 14-3-3 proteins Bmh1 and Bmh2 and present evidence that 14-3-3 proteins directly facilitate Rad53 function in vivo. The data presented account for the previously observed checkpoint defects associated with 14-3-3 mutants in Saccharomyces pombe and Saccharomyces cerevisiae. The 14-3-3 functional interaction appears to modulate Rad53 activity, reminiscent of 14-3-3's effect on human Raf1 kinase and distinct from the indirect mode of regulation by 14-3-3 observed for Chk1 or Cdc25. PMID:17299042

  6. 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

  7. 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

    2016-05-19

    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.

  8. Proteomic identification of 14-3-3ϵ as a linker protein between pERK1/2 inhibition and BIM upregulation in human osteosarcoma cells.

    PubMed

    Kim, Kyung Ok; Hsu, Anny C; Lee, Heon Goo; Patel, Neel; Chandhanayingyong, Chandhanarat; Hickernell, Thomas; Lee, Francis Young-In

    2014-06-01

    Despite advancements in multimodality chemotherapy, conventional cytotoxic treatments still remain ineffective for a subset of patients with aggressive metastatic or multifocal osteosarcoma. It has been shown that pERK1/2 inhibition enhances chemosensitivity to doxorubicin and promotes osteosarcoma cell death in vivo and in vitro. One of the pro-apoptotic mechanisms is upregulation of Bim by pERK1/2 inhibitors. To this end, we examined proteomic changes of 143B human osteosarcoma cells with and without treatment of PD98059, pERK1/2 inhibitor. Specifically, we identified 14-3-3ϵ protein as a potential mediator of Bim expression in response to inhibition of pERK1/2. We hypothesized that 14-3-3ϵ mediates upregulation of Bim expression after pERK1/2 inhibition. We examined the expression of Bim after silencing 14-3-3ϵ using siRNA. The 14-3-3ϵ gene silencing resulted in downregulation of Bim expression after PD98059 treatment. These data indicate that 14-3-3ϵ is required for Bim expression and that it has an anti-cancer effect under pERK1/2 inhibition in 143B cells. By playing an essential role upstream of Bim, 14-3-3ϵ may potentially be a coadjuvant factor synergizing the effect of pERK1/2 inhibitors in addition to conventional cytotoxic agents for more effective osteosarcoma treatments.

  9. Structural Basis for the Interaction of a Human Small Heat Shock Protein with the 14-3-3 Universal Signaling Regulator.

    PubMed

    Sluchanko, Nikolai N; Beelen, Steven; Kulikova, Alexandra A; Weeks, Stephen D; Antson, Alfred A; Gusev, Nikolai B; Strelkov, Sergei V

    2017-02-07

    By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein, HSPB6, within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering, and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants.

  10. Structural basis for the interaction of a human small heat shock protein with the 14-3-3 universal signaling regulator

    PubMed Central

    Sluchanko, Nikolai N.; Beelen, Steven; Kulikova, Alexandra A.; Weeks, Stephen D.; Antson, Alfred A.; Gusev, Nikolai B.; Strelkov, Sergei V.

    2017-01-01

    Summary By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein HSPB6 within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first-ever atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants. PMID:28089448

  11. Organellar proteomics of human platelet dense granules reveals that 14-3-3zeta is a granule protein related to atherosclerosis.

    PubMed

    Hernandez-Ruiz, Laura; Valverde, Federico; Jimenez-Nuñez, Maria D; Ocaña, Esther; Sáez-Benito, Ana; Rodríguez-Martorell, Javier; Bohórquez, Juan-Carlos; Serrano, Aurelio; Ruiz, Felix A

    2007-11-01

    Dense granules, a type of platelet secretory organelle, are known to accumulate high concentrations of small molecules such as calcium, adenine nucleotides, serotonin, pyrophosphate, and polyphosphate. Protein composition of these granules has been obscure, however. In this paper, we use proteomics techniques to describe, for the first time, the soluble protein composition of platelet dense granules. We have isolated highly enriched human platelet dense granule fractions that have been analyzed using two proteomics methods. Using this approach, we have identified 40 proteins, and most of them, such as actin-associated proteins, glycolytic enzymes, and regulatory proteins, have not previously been related to the organelle. We have focused our efforts on studying 14-3-3zeta, a member of a conserved family of proteins that interact with hundreds of different proteins. We have demonstrated that 14-3-3zeta is localized mostly on dense granules and that it is secreted after platelet activation. As some proteins secreted from activated platelets could promote the development of atherosclerosis and thrombosis, we have studied the expression of 14-3-3zeta in sections of human abdominal aorta of patients with aneurysm, identifying it at the atherosclerotic plaques. Together, our results reveal new details of the composition of the platelet dense granule and suggest an extracellular function for 14-3-3zeta associated with atherosclerosis.

  12. Extract from mistletoe, Viscum album L., reduces Hsp27 and 14-3-3 protein expression and induces apoptosis in C6 rat glioma cells.

    PubMed

    Uçar, E Ö; Arda, N; Aitken, A

    2012-08-24

    Extracts of mistletoe (Viscum album) are intensively used in complementary medicine, but their mechanisms are not fully understood in most cases, and the effects on metabolism have not been investigated in detail. However, some biologically active natural products are well known to provoke unexpected cellular responses. They reduce overexpression of heat shock proteins (Hsps) in cancer cells. The aim of the current study was to determine whether methanolic extract of V. album, which possesses antioxidant activity, has an effect on expression levels of Hsp27 and 14-3-3 proteins in a C6 glioma cell line. For the first time, the apoptosis-inducing effect of this extract was also determined via caspase-3 activation in the cells. Overexpression of Hsps was induced by heat shock at 42°C for 1 h. Expression levels of Hsp27 and 14-3-3 proteins were determined using Western blot analysis. The apoptosis-inducing effect was also evaluated via caspase-3 activation in C6 glioma cells. Pretreatment of the cells with a nontoxic dose (100 μg/mL) of V. album extract before heat shock significantly reduced expression levels of Hsp27 (73%) and 14-3-3β (124%), 14-3-3γ (23%), and 14-3-3ζ (84%) proteins. Pretreatment with the extract before heat shock increased apoptosis via caspase-3 activation (60%) in C6 glioma cells. This result suggested that the methanolic extract of V. album downregulates expression of Hsp27 and 14-3-3 chaperone proteins and induces apoptosis, which warrants further exploration as a potential bioactive compound for cancer therapy.

  13. Induction of androgen formation in the male by a TAT-VDAC1 fusion peptide blocking 14-3-3ɛ protein adaptor and mitochondrial VDAC1 interactions.

    PubMed

    Aghazadeh, Yasaman; Martinez-Arguelles, Daniel B; Fan, Jinjiang; Culty, Martine; Papadopoulos, Vassilios

    2014-10-01

    Low testosterone (T), a major cause of male hypogonadism and infertility, is linked to mood changes, fatigue, osteoporosis, reduced bone-mass index, and aging. The treatment of choice, T replacement therapy, has been linked with increased risk for prostate cancer and luteinizing hormone (LH) suppression, and shown to lead to infertility, cardiovascular diseases, and obesity. Alternate methods to induce T with lower side effects are desirable. In search of the mechanisms regulating T synthesis in the testes, we identified the 14-3-3ɛ protein adaptor as a negative regulator of steroidogenesis. Steroidogenesis begins in mitochondria. 14-3-3ɛ interacts with the outer mitochondrial membrane voltage-dependent anion channel (VDAC1) protein, forming a scaffold that limits the availability of cholesterol for steroidogenesis. We report the development of a tool able to induce endogenous T formation. Peptides able to penetrate testes conjugated to 14-3-3ɛ site of interaction with VDAC1 blocked 14-3-3ɛ-VDAC1 interactions while at the same time increased VDAC1-translocator protein (18 kDa) interactions that induced steroid formation in rat testes, leading to increased serum T levels. These peptides rescued intratesticular and serum T formation in adult male rats treated with gonadotropin-releasing hormone antagonist, which dampened LH and T production.

  14. Meiotic failure in cyclin A1-deficient mouse spermatocytes triggers apoptosis through intrinsic and extrinsic signaling pathways and 14-3-3 proteins

    PubMed Central

    Panigrahi, Sunil K.; Manterola, Marcia; Wolgemuth, Debra J.

    2017-01-01

    Cyclin A1 (Ccna1), a member of the mammalian A type cyclins, is most abundantly expressed in spermatocytes and is essential for spermatogenesis in the mouse. Ccna1- deficient spermatocytes arrest at late meiotic prophase and undergo apoptosis. To further delineate the mechanisms and key factors involved in this process, we have examined changes in expression of genes involved in both intrinsic and extrinsic signaling pathways that trigger apoptosis in the mutant spermatocytes. Our results show that both pathways are involved, and that the factors involved in the intrinsic pathway were expressed earlier than those involved in the extrinsic pathway. We have also begun to identify in vivo Ccna1-interacting proteins, using an unbiased biochemical approach, and identified 14-3-3, a key regulator of apoptosis, as a Ccna1-interacting protein. Expression levels of 14-3-3 proteins remain unchanged between wild type and mutant testes but there were differences in the subcellular distribution. In wild type control, 14-3-3 is detected in both cytosolic and nuclear fractions whereas it is restricted to the cytoplasm in mutant testes. This differential distribution of 14-3-3 may contribute to the induction of apoptosis in Ccna1-deficient spermatocytes. These results provide insight into the apoptotic mechanisms and pathways that are triggered when progression through the meiotic cell cycle is defective in male gametogenesis. PMID:28301569

  15. Induction of Androgen Formation in the Male by a TAT-VDAC1 Fusion Peptide Blocking 14-3-3ɛ Protein Adaptor and Mitochondrial VDAC1 Interactions

    PubMed Central

    Aghazadeh, Yasaman; Martinez-Arguelles, Daniel B; Fan, Jinjiang; Culty, Martine; Papadopoulos, Vassilios

    2014-01-01

    Low testosterone (T), a major cause of male hypogonadism and infertility, is linked to mood changes, fatigue, osteoporosis, reduced bone-mass index, and aging. The treatment of choice, T replacement therapy, has been linked with increased risk for prostate cancer and luteinizing hormone (LH) suppression, and shown to lead to infertility, cardiovascular diseases, and obesity. Alternate methods to induce T with lower side effects are desirable. In search of the mechanisms regulating T synthesis in the testes, we identified the 14-3-3ɛ protein adaptor as a negative regulator of steroidogenesis. Steroidogenesis begins in mitochondria. 14-3-3ɛ interacts with the outer mitochondrial membrane voltage-dependent anion channel (VDAC1) protein, forming a scaffold that limits the availability of cholesterol for steroidogenesis. We report the development of a tool able to induce endogenous T formation. Peptides able to penetrate testes conjugated to 14-3-3ɛ site of interaction with VDAC1 blocked 14-3-3ɛ-VDAC1 interactions while at the same time increased VDAC1-translocator protein (18 kDa) interactions that induced steroid formation in rat testes, leading to increased serum T levels. These peptides rescued intratesticular and serum T formation in adult male rats treated with gonadotropin-releasing hormone antagonist, which dampened LH and T production. PMID:24947306

  16. The Saccharomyces cerevisiae 14-3-3 protein Bmh2 is required for regulation of the phosphorylation status of Fin1, a novel intermediate filament protein.

    PubMed Central

    Mayordomo, Isabel; Sanz, Pascual

    2002-01-01

    In order to identify proteins that interact with Bmh2, a yeast member of the 14-3-3 protein family, we performed a two-hybrid screening using LexA-Bmh2 as bait. We identified Fin1, a novel intermediate filament protein, as the protein that showed the highest degree of interaction. We also identified components of the vesicular transport machinery such as Gic2 and Msb3, proteins involved in transcriptional regulation such as Mbf1, Gcr2 and Reg2, and a variety of other different proteins (Ppt1, Lre1, Rps0A and Ylr177w). We studied the interaction between Bmh2 and Fin1 in more detail and found that Bmh2 only interacted with phosphorylated forms of Fin1. In addition, we showed that Glc7, the catalytic subunit of the protein phosphatase 1 complex, was also able to interact with Fin1. PMID:11931638

  17. Higher order Arabidopsis 14-3-3 mutants show 14-3-3 involvement in primary root growth both under control and abiotic stress conditions

    PubMed Central

    van Kleeff, P. J. M.; Jaspert, N.; Li, K. W.; Rauch, S.; Oecking, C.; de Boer, A. H.

    2014-01-01

    Arabidopsis 14-3-3 proteins are a family of conserved proteins that interact with numerous partner proteins in a phospho-specific manner, and can affect the target proteins in a number of ways; e.g. modification of enzymatic activity. We isolated T-DNA insertion lines in six 14-3-3 genes within the non-epsilon group that phylogenetically group in three closely related gene pairs. In total, 6 single, 3 double, 12 triple, and 3 quadruple mutants were generated. The mutants were phenotyped for primary root growth on control plates: single and double mutants were indistinguishable from WT, whereas six triples and all quadruples showed a shorter primary root. In addition, length of the first epidermal cell with a visible root hair bulge (LEH) was used to determine primary root elongation on medium containing mannitol and 1-aminocyclopropane-1-carboxylic acid (ACC). This analysis showed clear differences depending on the stress and 14-3-3 gene combinations. Next to the phenotypic growth analyses, a 14-3-3 pull-down assay on roots treated with and without mannitol showed that mannitol stress strongly affects the 14-3-3 interactome. In conclusion, we show gene specificity and functional redundancy among 14-3-3 proteins in primary root elongation under control and under abiotic stress conditions and changes in the 14-3-3 interactome during the onset of stress adaptation. PMID:25189593

  18. Protein kinase CK2 interacts at the neuromuscular synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 proteins and phosphorylates the latter two.

    PubMed

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-09-11

    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.

  19. 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.

  20. 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

    2016-01-21

    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.

  1. 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

  2. 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

  3. Nucleocytoplasmic Shuttling of the Golgi Phosphatidylinositol 4-Kinase Pik1 Is Regulated by 14-3-3 Proteins and Coordinates Golgi Function with Cell Growth

    PubMed Central

    Demmel, Lars; Beck, Mike; Klose, Christian; Schlaitz, Anne-Lore; Gloor, Yvonne; Hsu, Peggy P.; Havlis, Jan; Shevchenko, Andrej; Krause, Eberhard; Kalaidzidis, Yannis

    2008-01-01

    The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p–14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling. PMID:18172025

  4. 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

  5. Molecular cloning of cDNA for the zeta isoform of the 14-3-3 protein: homologous sequences in the 3'-untranslated region of frog and human zeta isoforms.

    PubMed

    Miura, I; Nakajima, T; Ohtani, H; Kashiwagi, A; Nakamura, M

    1997-10-01

    14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that possess diverse biochemical activities such as regulation of gene transcription, cell proliferation and activation of protein kinase C. At least 7 subtypes (alpha to theta) of 14-3-3 protein are known, but the zeta subtype of this protein has been cloned only in mammals. We cloned the zeta subtype of 14-3-3 protein (14-3-3 zeta) from the frog, Rana rugosa. The sequence encoded 245 amino acids that share 92% identity with rat and bovine 14-3-3 zeta s, and 92% with human phospholipase A2 (PLA2; 14-3-3 zeta). Northern blot analysis revealed a single band of about 1.8 kb in tadpoles at stage 25. The 14-3-3 zeta mRNA level was high in the brain, lung, spleen and kidney, and low in the heart and testis, as opposed to the mRNA level, which was only faintly detected in the liver, pancreas, ovary and muscle. Furthermore, high similarity in the 3'-untranslated region (3'-UTR) was observed between frog and human 14-3-3 zeta cDNA. The results suggest that 14-3-3 zeta is highly conserved throughout eukaryotic evolution, and that the homologous sequence in the 3'-UTR of 14-3-3 zeta cDNA may be conserved in frogs and humans.

  6. 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.

  7. Two genes encoding GF14 (14-3-3) proteins in Zea mays. Structure, expression, and potential regulation by the G-box binding complex.

    PubMed Central

    de Vetten, N C; Ferl, R J

    1994-01-01

    Two maize (Zea mays) genes, designated GRF1 and GRF2, have been isolated and characterized. The proteins encoded by these genes, called GF14 proteins, participate in protein/DNA complexes and show more than 60% identity with a highly conserved, widely distributed protein family, collectively referred to as 14-3-3 proteins. Members of the 14-3-3 protein family have been reported to activate Tyr and Trp hydroxylases, modulate protein kinase C activity, and activate ADP-ribosyltransferase. The mRNAs of the GRF genes are encoded by six exons interrupted by five introns. The transcriptional units of the GRF genes were found to be very similar, with complete conservation of the intron positions. In addition, the length and nucleotide sequences of the two genes' introns were highly conserved. The 5' flanking sequences of the two GRF genes were compared and regions of homology and divergence identified. This comparison revealed the presence of a conserved G-box element in the 5' flanking region of both genes. Electrophoretic mobility shift assays of maize protein extract with the GRF G-box indicates that GBF binds to this G-box site in the 5' up stream region of GRF. Antibody supershifts indicate that GF14 protein is associated with the G-box-binding complex that interacts with the GRF upstream region. PMID:7846163

  8. The 14-3-3s

    PubMed Central

    Ferl, Robert J; Manak, Michael S; Reyes, Matthew F

    2002-01-01

    Multiple members of the 14-3-3 protein family have been found in all eukaryotes so far investigated, yet they are apparently absent from prokaryotes. The major native forms of 14-3-3s are homo- and hetero-dimers, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client. As a result, 14-3-3s are involved in a vast array of processes such as the response to stress, cell-cycle control, and apoptosis, serving as adapters, activators, and repressors. There are currently 133 full-length sequences available in GenBank for this highly conserved protein family. A phylogenetic tree based on the conserved middle core region of the protein sequences shows that, in plants, the 14-3-3 family can be divided into two clearly defined groups. The core region encodes an amphipathic groove that binds the multitude of client proteins that have conserved 14-3-3-recognition sequences. The amino and carboxyl termini of 14-3-3 proteins are much more divergent than the core region and may interact with isoform-specific client proteins and/or confer specialized subcellular and tissue localization. PMID:12184815

  9. The Pseudomonas syringae effector HopQ1 promotes bacterial virulence and interacts with tomato 14-3-3 proteins in a phosphorylation-dependent manner.

    PubMed

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

    2013-04-01

    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.

  10. 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.

  11. ­­Presence and distribution of 14-3-3 proteins in human ocular surface tissues

    PubMed Central

    Shankardas, Jwalitha; Senchyna, Michelle

    2008-01-01

    Purpose 14–3-3 is a highly conserved, ubiquitously expressed family of proteins. At least seven mammalian isoforms (β, ε, γ, η, θ, σ, and ζ) are known. These proteins associate with over 200 different target molecules and activate several downstream signaling cascades involved in the regulation of metabolism, cell cycle, apoptosis, protein trafficking, transcription, stress responses, and malignant transformations. We are interested in the role of these proteins in the mechanisms regulating homeostasis and the pathologies of the human ocular surface. Therefore, our purpose is to determine the expression of the 14–3-3 proteins in the human cornea, the conjunctiva, and the primary cells comprising these tissues. Methods Using immunofluorescence, we determined the expression of 14–3-3 β, ε, γ, η, θ, σ, and ζ in paraffin sections of the human cornea and conjunctiva. Using indirect immunofluorescence and western blot analysis, we also determined the expression of these isoforms in primary corneal epithelial cells, keratocytes, endothelial cells, and primary conjunctival epithelial cells. The expressions of these isoforms in primary epithelial and endothelial cells were compared with the same expressions in several corneal cell lines. Western blot analysis was used to determine the presence of 14–3-3 isoforms in the culture medium from corneal epithelial cells, cell lines, and the tear fluid. Results All the 14–3-3 isoforms were expressed in the corneal and conjunctival epithelia as well as primary epithelial cells and cell lines. Expression of 14–3-3 σ was confined to epithelial cells and was secreted into the culture medium of primary cells and cell lines. We also report for the first time that two of the secreted isoforms, 14–3-3 γ and ζ, are also present in the human tear fluid. Conclusions We have determined that all the mammalian 14–3-3 isoforms are expressed in the human cornea, conjunctiva, and the component cells and that the 14

  12. Unveiling equal importance of two 14-3-3 proteins for morphogenesis, conidiation, stress tolerance and virulence of an insect pathogen.

    PubMed

    Liu, Qian; Li, Jin-Gen; Ying, Sheng-Hua; Wang, Juan-Juan; Sun, Wen-Liang; Tian, Chao-Guang; Feng, Ming-Guang

    2015-04-01

    Two conserved 14-3-3 proteins orthologous to Saccharomyces cerevisiae Bmh1/2 are poorly understood in filamentous fungi. Here we show that Bmh1 and Bmh2 contribute equally to the fundamental biology and physiology of Beauveria bassiana by targeting many sets of proteins/enzymes. Single Bmh deletion caused similar upregulation of another. Excellent knockdown (∼91%) expressions of Bmh1 in ΔBmh2 and Bmh2 in ΔBmh1 resulted in equally more severe multiphenotypic defects than the single deletions, including G2 /M transition, blastospore size, carbon/nitrogen utilization, conidiation, germination and conidial tolerances to high osmolarity, oxidation, cell wall stress, high temperature and UV-B irradiation. All the deletion and deletion/knockdown mutants showed similar defects in blastospore yield and density, hyphal septation and cell size, hyphal responses to most chemical stresses and virulence. All the defects were evident with altered transcripts of phenotype-related genes and well restored by each Bmh complementation. Our Bmh1- and Bmh2-specific transcriptomes generated under osmotic and oxidative stresses revealed up to 6% genes differentially expressed by at least twofold in the fungal genome. Many of those were greatly depressed or co-depressed in ΔBmh1 and ΔBmh2. Our findings provide a thorough insight into the functions and complementary effects of the two 14-3-3 proteins in the filamentous entomopathogen.

  13. Crystal structures of a yeast 14-3-3 protein from Lachancea thermotolerans in the unliganded form and bound to a human lipid kinase PI4KB-derived peptide reveal high evolutionary conservation.

    PubMed

    Eisenreichova, Andrea; Klima, Martin; Boura, Evzen

    2016-11-01

    14-3-3 proteins bind phosphorylated binding partners to regulate several of their properties, including enzymatic activity, stability and subcellular localization. Here, two crystal structures are presented: the crystal structures of the 14-3-3 protein (also known as Bmh1) from the yeast Lachancea thermotolerans in the unliganded form and bound to a phosphopeptide derived from human PI4KB (phosphatidylinositol 4-kinase B). The structures demonstrate the high evolutionary conservation of ligand recognition by 14-3-3 proteins. The structural analysis suggests that ligand recognition by 14-3-3 proteins evolved very early in the evolution of eukaryotes and remained conserved, underlying the importance of 14-3-3 proteins in physiology.

  14. Osmoregulation in Lilium pollen grains occurs via modulation of the plasma membrane H+ ATPase activity by 14-3-3 proteins.

    PubMed

    Pertl, Heidi; Pöckl, Magdalena; Blaschke, Christian; Obermeyer, Gerhard

    2010-12-01

    To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment. An acidification and alkalinization of the external medium was monitored after hyper- and hypoosmotic treatments, respectively, and pH changes were blocked by vanadate, indicating a putative role of the PM H(+) ATPase. Indeed, an increase in PM-associated 14-3-3 proteins and an increase in PM H(+) ATPase activity were detected in PGs challenged by hyperosmolar medium. We therefore suggest that in PGs the PM H(+) ATPase via modulation of its activity by 14-3-3 proteins is involved in the regulation of turgor pressure.

  15. Osmoregulation in Lilium Pollen Grains Occurs via Modulation of the Plasma Membrane H+ ATPase Activity by 14-3-3 Proteins1[C][W][OA

    PubMed Central

    Pertl, Heidi; Pöckl, Magdalena; Blaschke, Christian; Obermeyer, Gerhard

    2010-01-01

    To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment. An acidification and alkalinization of the external medium was monitored after hyper- and hypoosmotic treatments, respectively, and pH changes were blocked by vanadate, indicating a putative role of the PM H+ ATPase. Indeed, an increase in PM-associated 14-3-3 proteins and an increase in PM H+ ATPase activity were detected in PGs challenged by hyperosmolar medium. We therefore suggest that in PGs the PM H+ ATPase via modulation of its activity by 14-3-3 proteins is involved in the regulation of turgor pressure. PMID:20974894

  16. 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

  17. 14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase.

    PubMed

    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(680)SP, by phosphatase treatment, and by competition with the Ser(680)-phosphorylated RSYS(680)SP peptide. Metabolic labeling with [(32)P]orthophosphate and immunoblotting using antibody against the phosphorylated 14-3-3-binding motif showed that Ser(680) is a major phosphorylation site in UBPY. These results indicated that 14-3-3s bind to the region surrounding Ser(680) in a phosphorylation-dependent manner. The mutation at Ser(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 14-3-3epsilon inhibited the UBPY activity in vitro. Finally, UBPY was dephosphorylated at Ser(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.

  18. 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

  19. Two cytosolic puromycin-sensitive aminopeptidase isozymes in chicken brain: molecular homology to brain-specific 14-3-3 protein.

    PubMed

    Hui, K S; Saito, M; Hui, M; Saito, M; Lajtha, A; Yamamoto, K; Osawa, T

    1993-05-01

    Two puromycin-sensitive aminopeptidase isozymes (PSA-I and PSA-II) were isolated from chicken brain cytosol by ammonium sulfate fractionation followed by column chromatography on Cellex D and AH-Sepharose 4B and separated on Bio-Gel HTP. Each was purified to homogeneity on Sephadex G-200, Arg-Tyr-AH-Sepharose, Bio-Gel HTP, and preparative gel electrophoresis. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PSA-I appeared to be a monomer with a molecular mass of 105 kDa, and PSA-II to be composed of two subunits of 25 kDa and 100 kDa. The tryptic maps of 100 kDa and 105 kDa protein in HPLC are different in peak frequency, height, and composition. The internal peptide sequence of PSA-I has a considerable homology to PSA-II. Both isozymes have repeated copies of common peptide segments and have no significant sequence homology to other peptidases and proteinases. These thio and Co(2+)-activated isozymes have a neutral pH optimum and are inhibited by puromycin and bestatin. PSA-II is more sensitive to trypsin and heat treatment, has a lower Km to Met-enkephalin, and is more active on Arg BNA and Pro BNA. Our results suggest that PSA-I and PSA-II derive from translation of two RNAs of a new gene family related to the brain-specific 14-3-3 protein.

  20. Self-association of the spindle pole body-related intermediate filament protein Fin1p and its phosphorylation-dependent interaction with 14-3-3 proteins in yeast.

    PubMed

    van Hemert, Martijn J; Deelder, André M; Molenaar, Chris; Steensma, H Yde; van Heusden, G Paul H

    2003-04-25

    The Fin1 protein of the yeast Saccharomyces cerevisiae forms filaments between the spindle pole bodies of dividing cells. In the two-hybrid system it binds to 14-3-3 proteins, which are highly conserved proteins involved in many cellular processes and which are capable of binding to more than 120 different proteins. Here, we describe the interaction of the Fin1 protein with the 14-3-3 proteins Bmh1p and Bmh2p in more detail. Purified Fin1p interacts with recombinant yeast 14-3-3 proteins. This interaction is strongly reduced after dephosphorylation of Fin1p. Surface plasmon resonance analysis showed that Fin1p has a higher affinity for Bmh2p than for Bmh1p (K(D) 289 versus 585 nm). Sequences in both the central and C-terminal part of Fin1p are required for the interaction with Bmh2p in the two-hybrid system. In yeast strains lacking 14-3-3 proteins Fin1 filament formation was observed, indicating that the 14-3-3 proteins are not required for this process. Fin1 also interacts with itself in the two-hybrid system. For this interaction sequences at the C terminus, containing one of two putative coiled-coil regions, are sufficient. Fin1p-Fin1p interactions were demonstrated in vivo by fluorescent resonance energy transfer between cyan fluorescent protein-labeled Fin1p and yellow fluorescent protein-labeled Fin1p.

  1. 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

  2. The 14-3-3 protein Bmh1 functions in the spindle position checkpoint by breaking Bfa1 asymmetry at yeast centrosomes.

    PubMed

    Caydasi, Ayse Koca; Micoogullari, Yagmur; Kurtulmus, Bahtiyar; Palani, Saravanan; Pereira, Gislene

    2014-07-15

    In addition to their well-known role in microtubule organization, centrosomes function as signaling platforms and regulate cell cycle events. An important example of such a function is the spindle position checkpoint (SPOC) of budding yeast. SPOC is a surveillance mechanism that ensures alignment of the mitotic spindle along the cell polarity axis. Upon spindle misalignment, phosphorylation of the SPOC component Bfa1 by Kin4 kinase engages the SPOC by changing the centrosome localization of Bfa1 from asymmetric (one centrosome) to symmetric (both centrosomes). Here we show that, unexpectedly, Kin4 alone is unable to break Bfa1 asymmetry at yeast centrosomes. Instead, phosphorylation of Bfa1 by Kin4 creates a docking site on Bfa1 for the 14-3-3 family protein Bmh1, which in turn weakens Bfa1-centrosome association and promotes symmetric Bfa1 localization. Consistently, BMH1-null cells are SPOC deficient. Our work thus identifies Bmh1 as a new SPOC component and refines the molecular mechanism that breaks Bfa1 centrosome asymmetry upon SPOC activation.

  3. The 14-3-3 protein Bmh1 functions in the spindle position checkpoint by breaking Bfa1 asymmetry at yeast centrosomes

    PubMed Central

    Caydasi, Ayse Koca; Micoogullari, Yagmur; Kurtulmus, Bahtiyar; Palani, Saravanan; Pereira, Gislene

    2014-01-01

    In addition to their well-known role in microtubule organization, centrosomes function as signaling platforms and regulate cell cycle events. An important example of such a function is the spindle position checkpoint (SPOC) of budding yeast. SPOC is a surveillance mechanism that ensures alignment of the mitotic spindle along the cell polarity axis. Upon spindle misalignment, phosphorylation of the SPOC component Bfa1 by Kin4 kinase engages the SPOC by changing the centrosome localization of Bfa1 from asymmetric (one centrosome) to symmetric (both centrosomes). Here we show that, unexpectedly, Kin4 alone is unable to break Bfa1 asymmetry at yeast centrosomes. Instead, phosphorylation of Bfa1 by Kin4 creates a docking site on Bfa1 for the 14-3-3 family protein Bmh1, which in turn weakens Bfa1–centrosome association and promotes symmetric Bfa1 localization. Consistently, BMH1-null cells are SPOC deficient. Our work thus identifies Bmh1 as a new SPOC component and refines the molecular mechanism that breaks Bfa1 centrosome asymmetry upon SPOC activation. PMID:24850890

  4. A20 zinc finger protein inhibits TNF-induced apoptosis and stress response early in the signaling cascades and independently of binding to TRAF2 or 14-3-3 proteins.

    PubMed

    Lademann, U; Kallunki, T; Jäättelä, M

    2001-03-01

    A20 zinc finger protein is a negative regulator of tumor necrosis factor (TNF)-induced signaling pathways leading to apoptosis, stress response and inflammation. A20 has been shown to bind to TNF-receptor-associated factor 2 (TRAF2) and 14-3-3 chaperone proteins. Our data indicate that the zinc finger domain of A20 is sufficient and that neither TRAF2 nor 14-3-3 binding is necessary for the inhibitory effects of A20. Mutations in the 14-3-3 binding site of A20 did, however, result in a partial cleavage of A20 protein suggesting that 14-3-3 chaperone proteins may stabilize A20. Furthermore, we show that A20 acts early in TNF-induced signaling cascades blocking both TNF-induced rapid activation of c-Jun N-terminal kinase and processing of the receptor-associated caspase-8. Taken together our data indicate that the zinc finger domain of A20 contains all necessary functional domains required for the inhibition of TNF signaling and that A20 may function at the level of the receptor signaling complex.

  5. Up-regulation and interaction of the plasma membrane H(+)-ATPase and the 14-3-3 protein are involved in the regulation of citrate exudation from the broad bean (Vicia faba L.) under Al stress.

    PubMed

    Chen, Qi; Guo, Chuan-Long; Wang, Ping; Chen, Xuan-Qin; Wu, Kong-Huan; Li, Kui-Zhi; Yu, Yong-Xiong; Chen, Li-Mei

    2013-09-01

    Our previous study showed that citrate excretion coupled with a concomitant release of protons was involved in aluminum (Al) resistance in the broad bean. Furthermore, genes encoding plasma membrane (PM) H(+)-ATPase (vha2) and the 14-3-3 protein (vf14-3-3b) were up-regulated by Al in Al-resistant (YD) broad bean roots. In this study, the roles of PM H(+)-ATPase (E.C. 3.6.3.6) and the 14-3-3 protein in the regulation of citrate secretion were further investigated in Al-resistant (YD) and Al-sensitive (AD) broad bean cultivars under Al stress. The results showed that greater citrate exudation was positively correlated with higher activities of PM H(+)-ATPase in roots of YD than AD. Real-time RT-PCR analysis revealed that vha2 was clearly up-regulated by Al in YD but not in AD roots, whereas the transcription levels of vf14-3-3b were elevated in a time-dependent manner in both YD and AD roots. Immunoprecipitation and Western analysis suggested that phosphorylation and interaction with the vf14-3-3b protein of the VHA2 were enhanced in YD roots but not in AD roots with increasing Al treatment time. Fusicoccin or adenosine 5'-monophosphate increased or decreased the interaction between the phosphorylated VHA2 and the vf14-3-3b protein, followed by an enhancement or reduction of the PM H(+)-ATPase activity and citrate exudation in both cultivars under Al stress conditions, respectively. Taken together, these results suggested that Al enhanced the expression and interaction of the PM H(+)-ATPase and the 14-3-3 protein, which thereby led to higher activity of the PM H(+)-ATPase and more citrate exudation from YD plants.

  6. 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.

  7. Human 14-3-3 Paralogs Differences Uncovered by Cross-Talk of Phosphorylation and Lysine Acetylation

    PubMed Central

    Uhart, Marina; Bustos, Diego M.

    2013-01-01

    The 14-3-3 protein family interacts with more than 700 different proteins in mammals, in part as a result of its specific phospho-serine/phospho-threonine binding activity. Upon binding to 14-3-3, the stability, subcellular localization and/or catalytic activity of the ligands are modified. Seven paralogs are strictly conserved in mammalian species. Although initially thought as redundant, the number of studies showing specialization is growing. We created a protein-protein interaction network for 14-3-3, kinases and their substrates signaling in human cells. We included information of phosphorylation, acetylation and other PTM sites, obtaining a complete representation of the 14-3-3 binding partners and their modifications. Using a computational system approach we found that networks of each 14-3-3 isoform are statistically different. It was remarkable to find that Tyr was the most phosphorylatable amino acid in domains of 14-3-3 epsilon partners. This, together with the over-representation of SH3 and Tyr_Kinase domains, suggest that epsilon could be involved in growth factors receptors signaling pathways particularly. We also found that within zeta’s network, the number of acetylated partners (and the number of modify lysines) is significantly higher compared with each of the other isoforms. Our results imply previously unreported hidden differences of the 14-3-3 isoforms interaction networks. The phosphoproteome and lysine acetylome within each network revealed post-transcriptional regulation intertwining phosphorylation and lysine acetylation. A global understanding of these networks will contribute to predict what could occur when regulatory circuits become dysfunctional or are modified in response to external stimuli. PMID:23418452

  8. Plant 14-3-3s: omnipotent metabolic phosphopartners?

    PubMed

    Sehnke, P C; Ferl, R J

    2000-10-31

    The accurate regulation of metabolism is crucial to the existence all organisms. The inappropriate activation of metabolic enzymes can waste precious energy; likewise, the failure to activate metabolic enzymes can disrupt homeostasis and lead to suboptimal cellular (and organismic) responses. Plants use several means to control their metabolic proteins, including a two-step process of protein phosphorylation and subsequent binding by phosphospecific binding proteins termed 14-3-3 proteins. Sehnke and Ferl discuss how 14-3-3 proteins regulate the activity of nitrate reductase and the H(+)-ATPase pump in plants, and compare the functions of 14-3-3 proteins in plants and animals.

  9. Phosphorylation of HopQ1, a Type III Effector from Pseudomonas syringae, Creates a Binding Site for Host 14-3-3 Proteins1[C][W][OA

    PubMed Central

    Giska, Fabian; Lichocka, Małgorzata; Piechocki, Marcin; Dadlez, Michał; Schmelzer, Elmon; Hennig, Jacek; Krzymowska, Magdalena

    2013-01-01

    HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector. PMID:23396834

  10. 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.

  11. GARNL1, a major RalGAP α subunit in skeletal muscle, regulates insulin-stimulated RalA activation and GLUT4 trafficking via interaction with 14-3-3 proteins.

    PubMed

    Chen, Qiaoli; Quan, Chao; Xie, Bingxian; Chen, Liang; Zhou, Shuilian; Toth, Rachel; Campbell, David G; Lu, Shuangshuang; Shirakawa, Ryutaro; Horiuchi, Hisanori; Li, Chaojun; Yang, Zhongzhou; MacKintosh, Carol; Wang, Hong Yu; Chen, Shuai

    2014-08-01

    Insulin and muscle contraction each stimulate translocation of the glucose transporter GLUT4 to the plasma membrane in skeletal muscle, an important process regulating whole-body glucose homeostasis. RalA mediates insulin-stimulated GLUT4 translocation; however, it is unclear how this small GTPase is regulated in skeletal muscle in response to insulin. Here, we identified GARNL1/RalGAPα1, a major α subunit of the Ral-GTPase activating protein in skeletal muscle, as a protein whose phosphorylation and binding to the regulatory 14-3-3 proteins is stimulated by insulin and also by muscle contraction. The insulin-stimulated interaction with 14-3-3 involved PKB/Akt-mediated phosphorylation of Thr(735) on GARNL1/RalGAPα1. Knockdown of GARNL1/RalGAPα1 increased, while overexpression of GARNL1/RalGAPα1(Thr735Ala) mutant protein decreased, the RalA activation and the RalA-dependent GLUT4 translocation in response to insulin in muscle cells. These findings show that GARNL1/RalGAPα1 is the missing link that connects the insulin-PKB/Akt signaling pathway with the activation of the RalA small GTPase in muscle cells. GARNL1/RalGAPα1 and its phosphorylation and/or binding to 14-3-3s are critical for GLUT4 trafficking through RalA in muscle cells.

  12. 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

  13. 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

    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

    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.

  14. The Acid-sensitive, Anesthetic-activated Potassium Leak Channel, KCNK3, Is Regulated by 14-3-3β-dependent, Protein Kinase C (PKC)-mediated Endocytic Trafficking*

    PubMed Central

    Gabriel, Luke; Lvov, Anatoli; Orthodoxou, Demetra; Rittenhouse, Ann R.; Kobertz, William R.; Melikian, Haley E.

    2012-01-01

    The acid-sensitive neuronal potassium leak channel, KCNK3, is vital for setting the resting membrane potential and is the primary target for volatile anesthetics. Recent reports demonstrate that KCNK3 activity is down-regulated by PKC; however, the mechanisms responsible for PKC-induced KCNK3 down-regulation are undefined. Here, we report that endocytic trafficking dynamically regulates KCNK3 activity. Phorbol esters and Group I metabotropic glutamate receptor (mGluR) activation acutely decreased both native and recombinant KCNK3 currents with concomitant KCNK3 surface losses in cerebellar granule neurons and cell lines. PKC-mediated KCNK3 internalization required the presence of both 14-3-3β and a novel potassium channel endocytic motif, because depleting either 14-3-3β protein levels or ablating the endocytic motif completely abrogated PKC-regulated KCNK3 trafficking. These results demonstrate that neuronal potassium leak channels are not static membrane residents but are subject to 14-3-3β-dependent regulated trafficking, providing a straightforward mechanism to modulate neuronal excitability and synaptic plasticity by Group I mGluRs. PMID:22846993

  15. 14-3-3 Protects against stress-induced apoptosis

    PubMed Central

    Clapp, C; Portt, L; Khoury, C; Sheibani, S; Norman, G; Ebner, P; Eid, R; Vali, H; Mandato, C A; Madeo, F; Greenwood, M T

    2012-01-01

    Expression of human Bax, a cardinal regulator of mitochondrial membrane permeabilization, causes death in yeast. We screened a human cDNA library for suppressors of Bax-mediated yeast death and identified human 14-3-3β/α, a protein whose paralogs have numerous chaperone-like functions. Here, we show that, yeast cells expressing human 14-3-3β/α are able to complement deletion of the endogenous yeast 14-3-3 and confer resistance to a variety of different stresses including cadmium and cycloheximide. The expression of 14-3-3β/α also conferred resistance to death induced by the target of rapamycin inhibitor rapamycin and by starvation for the amino acid leucine, conditions that induce autophagy. Cell death in response to these autophagic stimuli was also observed in the macroautophagic-deficient atg1Δ and atg7Δ mutants. Furthermore, 14-3-3β/α retained its ability to protect against the autophagic stimuli in these autophagic-deficient mutants arguing against so called ‘autophagic death'. In line, analysis of cell death markers including the accumulation of reactive oxygen species, membrane integrity and cell surface exposure of phosphatidylserine indicated that 14-3-3β/α serves as a specific inhibitor of apoptosis. Finally, we demonstrate functional conservation of these phenotypes using the yeast homolog of 14-3-3: Bmh1. In sum, cell death in response to multiple stresses can be counteracted by 14-3-3 proteins. PMID:22785534

  16. The Arabidopsis SERK1 protein interacts with the AAA-ATPase AtCDC48, the 14-3-3 protein GF14lambda and the PP2C phosphatase KAPP.

    PubMed

    Rienties, Ingrid M; Vink, Josefien; Borst, Jan Willem; Russinova, Eugenia; de Vries, Sacco C

    2005-06-01

    Leucine-rich repeat (LRR)-containing transmembrane receptor-like kinases (RLKs) are important components of plant signal transduction. The Arabidopsis thaliana somatic embryogenesis receptor-like kinase 1 (AtSERK1) is an LRR-RLK proposed to participate in a signal transduction cascade involved in embryo development. By yeast two-hybrid screening we identified AtCDC48, a homologue of the mammalian AAA-ATPase p97 and GF14lambda, a member of the Arabidopsis family of 14-3-3 proteins as AtSERK1 interactors. In vitro, the AtSERK1 kinase domain is able to transphosphorylate and bind both AtCDC48 and GF14lambda. In yeast, AtCDC48 interacts with GF14lambda and with the PP2C phosphatase KAPP. In plant protoplasts AtSERK1 interacts with GF14lambda.

  17. 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

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

    2015-10-01

    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.

  18. 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

  19. Differences in Spatial Expression between 14-3-3 Isoforms in Germinating Barley Embryos1

    PubMed Central

    Testerink, Christa; van der Meulen, René M.; Oppedijk, Berry J.; de Boer, Albertus H.; Heimovaara-Dijkstra, Sjoukje; Kijne, Jan W.; Wang, Mei

    1999-01-01

    The family of 14-3-3 proteins is ubiquitous in eukaryotes and has been shown to exert an array of functions. We were interested in the possible role of 14-3-3 proteins in seed germination. Therefore, we studied the expression of 14-3-3 mRNA and protein in barley (Hordeum distichum L.) embryos during germination. With the use of specific cDNA probes and antibodies, we could detect individual expression of three 14-3-3 isoforms, 14-3-3A, 14-3-3B, and 14-3-3C. Each homolog was found to be expressed in barley embryos. Whereas protein levels of all three isoforms were constant during germination, mRNA expression was found to be induced upon imbibition of the grains. The induction of 14-3-3A gene expression during germination was different from that of 14-3-3B and 14-3-3C. In situ immunolocalization analysis showed similar spatial expression for 14-3-3A and 14-3-3B, while 14-3-3C expression was markedly different. Whereas 14-3-3A and 14-3-3B were expressed throughout the embryo, 14-3-3C expression was tissue specific, with the strongest expression observed in the scutellum and the L2 layer of the shoot apical meristem. These results show that 14-3-3 homologs are differently regulated in barley embryos, and provide a first step in acquiring more knowledge about the role of 14-3-3 proteins in the germination process. PMID:10482663

  20. 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...

  1. 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.

  2. Royal Jelly-Mediated Prolongevity and Stress Resistance in Caenorhabditis elegans Is Possibly Modulated by the Interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 Proteins.

    PubMed

    Wang, Xiaoxia; Cook, Lauren F; Grasso, Lindsay M; Cao, Min; Dong, Yuqing

    2015-07-01

    Recent studies suggest that royal jelly (RJ) and its related substances may have antiaging properties. However, the molecular mechanisms underlying the beneficial effects remain elusive. We report that the effects of RJ and enzyme-treated RJ (eRJ) on life span and health span in Caenorhabditis elegans (C elegans) are modulated by the sophisticated interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 proteins. Dietary supplementation with RJ or eRJ increased C. elegans life span in a dose-dependent manner. The RJ and eRJ consumption increased the tolerance of C elegans to oxidative stress, ultraviolet irradiation, and heat shock stress. Our genetic analyses showed that RJ/eRJ-mediated life-span extension requires insulin/IGF-1 signaling and the activities of DAF-16, SIR-2.1, HCF-1, and FTT-2, a 14-3-3 protein. Earlier studies reported that DAF-16/FOXO, SIR-2.1/SIRT1, FTT-2, and HCF-1 have extensive interplays in worms and mammals. Our present findings suggest that RJ/eRJ-mediated promotion of longevity and stress resistance in C elegans is dependent on these conserved interplays. From an evolutionary point of view, this study not only provides new insights into the molecular mechanisms of RJ's action on health span promotion in C elegans, but also has imperative implications in using RJ/eRJ as nutraceuticals to delay aging and age-related disorders.

  3. 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.

  4. A novel interaction partner for the C-terminus of Arabidopsis thaliana plasma membrane H+-ATPase (AHA1 isoform): site and mechanism of action on H+-ATPase activity differ from those of 14-3-3 proteins.

    PubMed

    Morandini, Piero; Valera, Marco; Albumi, Cristina; Bonza, Maria Cristina; Giacometti, Sonia; Ravera, Giuseppe; Murgia, Irene; Soave, Carlo; De Michelis, Maria Ida

    2002-08-01

    Using the two-hybrid technique we identified a novel protein whose N-terminal 88 amino acids (aa) interact with the C-terminal regulatory domain of the plasma membrane (PM) H+-ATPase from Arabidopsis thaliana (aa 847-949 of isoform AHA1). The corresponding gene has been named Ppi1 for Proton pump interactor 1. The encoded protein is 612 aa long and rich in charged and polar residues, except for the extreme C-terminus, where it presents a hydrophobic stretch of 24 aa. Several genes in the A. thaliana genome and many ESTs from different plant species share significant similarity (50-70% at the aa level over stretches of 200-600 aa) to Ppi1. The PPI1 N-terminus, expressed in bacteria as a fusion protein with either GST or a His-tag, binds the PM H+-ATPase in overlay experiments. The same fusion proteins and the entire coding region fused to GST stimulate H+-ATPase activity. The effect of the His-tagged peptide is synergistic with that of fusicoccin (FC) and of tryptic removal of a C-terminal 10 kDa fragment. The His-tagged peptide binds also the trypsinised H+-ATPase. Altogether these results indicate that PPI1 N-terminus is able to modulate the PM H+-ATPase activity by binding to a site different from the 14-3-3 binding site and is located upstream of the trypsin cleavage site.

  5. 14-3-3 inhibition promotes dopaminergic neuron loss and 14-3-3θ overexpression promotes recovery in the MPTP mouse model of Parkinson's disease

    PubMed Central

    Ding, Huiping; Underwood, Rachel; Lavalley, Nicholas; Yacoubian, Talene A.

    2015-01-01

    14-3-3s are a highly conserved protein family that plays important roles in cell survival and interact with several proteins implicated in Parkinson's disease (PD). Disruption of 14-3-3 expression and function has been implicated in the pathogenesis of PD. We have previously shown that increasing the expression level of 14-3-3θ is protective against rotenone and 1-methyl-4-phenylpyridinium (MPP+) in cultured cells. Here, we extend our studies to examine the effects of 14-3-3s in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. We first investigated whether targeted nigral 14-3-3θ overexpression mediated by adeno-associated virus offers neuroprotection against MPTP-induced toxicity. 14-3-3θ overexpression using this approach did not reduce MPTP-induced dopaminergic cell loss in the substantia nigra nor the depletion of dopamine and its metabolites in the striatum at three weeks after MPTP administration. However, 14-3-3θ-overexpressing mice showed a later partial recovery in striatal dopamine metabolites at eight weeks after MPTP administration compared to controls, suggesting that 14-3-3θ overexpression may help in the functional recovery of those dopaminergic neurons that survive. Conversely, we investigated whether disrupting 14-3-3 function in transgenic mice expressing the pan 14-3-3 inhibitor difopein exacerbates MPTP-induced toxicity. We found that difopein expression promoted dopaminergic cell loss in response to MPTP treatment. Together, these findings suggest that 14-3-3θ overexpression promotes recovery of dopamine metabolites whereas 14-3-3 inhibition exacerbates neuron loss in the MPTP mouse model of PD. PMID:26314634

  6. The Double-Edged Sword of Prostate Cancer: 14-3-3(sigma)

    DTIC Science & Technology

    2005-12-01

    are highly conserved (1,2). The 14-3-3 proteins play important roles in many biological activities by directly binding to and altering the...apoptotic cell death. 15. SUBJECT TERMS No subject terms provided. 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18 . NUMBER OF...as Adriamycin and mitoxantrone (unpublished observations). 14-3-3σ is a member of a highly conserved family of 14-3-3 proteins that are present in

  7. 14-3-3 phosphoprotein interaction networks – does isoform diversity present functional interaction specification?

    PubMed Central

    Paul, Anna-Lisa; Denison, Fiona C.; Schultz, Eric R.; Zupanska, Agata K.; Ferl, Robert J.

    2012-01-01

    The 14-3-3 proteins have emerged as major phosphoprotein interaction proteins and thereby constitute a key node in the Arabidopsis Interactome Map, a node through which a large number of important signals pass. Throughout their history of discovery and description, the 14-3-3s have been described as protein families and there has been some evidence that the different 14-3-3 family members within any organism might carry isoform-specific functions. However, there has also been evidence for redundancy of 14-3-3 function, suggesting that the perceived 14-3-3 diversity may be the accumulation of neutral mutations over evolutionary time and as some 14-3-3 genes develop tissue or organ-specific expression. This situation has led to a currently unresolved question – does 14-3-3 isoform sequence diversity indicate functional diversity at the biochemical or cellular level? We discuss here some of the key observations on both sides of the resulting debate, and present a set of contrastable observations to address the theory functional diversity does exist among 14-3-3 isoforms. The resulting model suggests strongly that there are indeed functional specificities in the 14-3-3s of Arabidopsis. The model further suggests that 14-3-3 diversity and specificity should enter into the discussion of 14-3-3 roles in signal transduction and be directly approached in 14-3-3 experimentation. It is hoped that future studies involving 14-3-3s will continue to address specificity in experimental design and analysis. PMID:22934100

  8. Induction of expression of a 14-3-3 gene in response to copper exposure in the marine alga, Fucus vesiculosus.

    PubMed

    Owen, Jennifer R; Morris, Ceri A; Nicolaus, Beate; Harwood, John L; Kille, Peter

    2012-01-01

    The macro-alga Fucus vesiculosus has a broad global and estuarine distribution and exhibits exceptional resistance to toxic metals, the molecular basis of which is poorly understood. To address this issue a cDNA library was constructed from an environmental isolate of F. vesiculosus growing in an area with chronic copper pollution. Characterisation of this library led to the identification of a cDNA encoding a protein known to be synthesised in response to toxicity, a full length 14-3-3 exhibiting a 71% identity to human/mouse epsilon isoform, 70-71% identity to yeast BMH1/2 and 95 and 71% identity to the Ectocarpus siliculosus 14-3-3 isoforms 1 and 2 respectively. Preliminary characterisation of the expression profile of the 14-3-3 indicated concentration- and time-dependent inductions on acute exposure of F. vesiculosus of copper (3-30 μg/l). Higher concentrations of copper (≥150 μg/l) did not elicit significant induction of the 14-3-3 gene compared with the control even though levels of both intracellular copper and the expression of a cytosolic metal chaperone, metallothionein, continued to rise. Analysis of gene expression within environmental isolates demonstrated up-regulation of the 14-3-3 gene associated with the known copper pollution gradient. Here we report for the first time, identification of a gene encoding a putative 14-3-3 protein in a multicellular alga and provide preliminary evidence to link the induction of this 14-3-3 gene to copper exposure in this alga. Interestingly, the threshold exposure profile may be associated with a decrease in the organism's ability to control copper influx so that it perceives copper as a toxic response.

  9. Protein kinase C epsilon in cell division: control of abscission.

    PubMed

    Saurin, Adrian T; Brownlow, Nicola; Parker, Peter J

    2009-02-15

    Cell division requires the separation and partitioning of sister chromatids to opposite ends of the cell before an actomyosin ring contracts the membrane in between during cytokinesis. The final irreversible step occurs during abscission when the ring breaks down and the membrane is sealed in its place. The physical mechanics of contraction depend on RhoA, which is stimulated by a centralspindlin complex around the cell equator. However exactly how these events are reversed to allow actomyosin breakdown and abscission were not well understood. Here we will discuss new findings that implicate Protein Kinase C epsilon (PKCepsilon) as a regulator of RhoA signalling required for abscission.

  10. Modulation of 14-3-3/phosphotarget interaction by physiological concentrations of phosphate and glycerophosphates.

    PubMed

    Sluchanko, Nikolai N; Chebotareva, Natalia A; Gusev, Nikolai B

    2013-01-01

    Molecular mechanisms governing selective binding of a huge number of various phosphorylated protein partners to 14-3-3 remain obscure. Phosphate can bind to 14-3-3 and therefore being present at high intracellular concentration, which undergoes significant changes under physiological conditions, phosphate can theoretically regulate interaction of 14-3-3 with phosphorylated targets. In order to check this hypothesis we analyzed effect of phosphate and other natural abundant anions on interaction of 14-3-3 with phosphorylated human small heat shock protein HspB6 (Hsp20) participating in regulation of different intracellular processes. Inorganic phosphate, glycerol-1-phosphate and glycerol-2-phosphate at physiologically relevant concentrations (5-15 mM) significantly destabilized complexes formed by 14-3-3ζ and phosphorylated HspB6 (pHspB6), presumably, via direct interaction with the substrate-binding site of 14-3-3. Phosphate also destabilized complexes between pHspB6 and 14-3-3γ or the monomeric mutant form of 14-3-3ζ. Inorganic sulfate and pyrophosphate were less effective in modulation of 14-3-3 interaction with its target protein. The inhibitory effect of all anions on pHspB6/14-3-3 interaction was concentration-dependent. It is hypothesized that physiological changes in phosphate anions concentration can modulate affinity and specificity of interaction of 14-3-3 with its multiple targets and therefore the actual phosphointeractome of 14-3-3.

  11. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination.

    PubMed

    Lam, Tonika; Thomas, Lisa M; White, Clayton A; Li, Guideng; Pone, Egest J; Xu, Zhenming; Casali, Paolo

    2013-01-01

    Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

  12. Molecular characterization of cotton 14-3-3L gene preferentially expressed during fiber elongation.

    PubMed

    Shi, Haiyan; Wang, Xiulan; Li, Dengdi; Tang, Wenkai; Wang, Hong; Xu, Wenliang; Li, Xuebao

    2007-02-01

    The 14-3-3 protein, highly conserved in all eukaryotic cells, is an important regulatory protein. It plays an important role in the growth, amplification, apoptosis, signal transduction, and other crucial life activities of cells. A cDNA encoding a putative 14-3-3 protein was isolated from cotton fiber cDNA library. The cDNA, designated as Gh14-3-3L (Gossypium hirsutum 14-3-3-like), is 1,029 bp in length (including a 762 bp long open reading frame and 5'-/3'-untranslated regions) and deduced a protein with 253 amino acids. The Gh14-3-3L shares higher homology with the known plant 14-3-3 proteins, and possesses the basic structure of 14-3-3 proteins: one dimeric domain, one phosphoralated-serine rich motif, four CC domains, and one EF Hand motif. Northern blotting analysis showed that Gh14-3-3L was predominantly expressed during early fiber development, and reached to the peak of expression in 10 days post anthers (DPA) fiber cells, suggesting that the gene may be involved in regulating fiber elongation. The gene is also expressed at higher level in both ovule and petal, but displays lower or undetectable level of activity in other tissues of cotton.

  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. 14-3-3 Mediates Histone Cross-Talk during Transcription Elongation in Drosophila

    PubMed Central

    Karam, Caline S.; Kellner, Wendy A.; Takenaka, Naomi; Clemmons, Alexa W.; Corces, Victor G.

    2010-01-01

    Post-translational modifications of histone proteins modulate the binding of transcription regulators to chromatin. Studies in Drosophila have shown that the phosphorylation of histone H3 at Ser10 (H3S10ph) by JIL-1 is required specifically during early transcription elongation. 14-3-3 proteins bind H3 only when phosphorylated, providing mechanistic insights into the role of H3S10ph in transcription. Findings presented here show that 14-3-3 functions downstream of H3S10ph during transcription elongation. 14-3-3 proteins localize to active genes in a JIL-1–dependent manner. In the absence of 14-3-3, levels of actively elongating RNA polymerase II are severely diminished. 14-3-3 proteins interact with Elongator protein 3 (Elp3), an acetyltransferase that functions during transcription elongation. JIL-1 and 14-3-3 are required for Elp3 binding to chromatin, and in the absence of either protein, levels of H3K9 acetylation are significantly reduced. These results suggest that 14-3-3 proteins mediate cross-talk between histone phosphorylation and acetylation at a critical step in transcription elongation. PMID:20532201

  15. Nucleotide sequence and expression of the 14-3-3 from the halotolerant alga Dunaliella salina.

    PubMed

    Wang, Tian-yun; Jing, Chang-Qin; Dong, Wei-Hua; Zhang, Jun-He; Zhang, Yu

    2010-02-01

    Previously we reported the nucleotide sequence of a 14-3-3 cDNA cloned from the unicellular green alga Dunaliella salina, however, the nucleotide sequence of this gene have not been reported so far. In the present study, the cloning and characterization of the nucleotide sequence, the gene copy and expression were undertaken. The coding sequence of the gene was found to be interrupted by five introns of 132, 266, 153, 152 and 625 bp, respectively. Introns 3-5 were found in conserved positions as compared to the Chlamydomonas reinhardtii 14-3-3 gene. D. salina 14-3-3 cDNA was inserted into the prokaryotic expression plasmid pET-28 and transformed into E. coli BL21, and the recombinant expressed 14-3-3 protein was purified from E. coli and immunized the rabbit. Indirect ELISA coated with 14-3-3 illustrated that the rabbit antisera titration was 1:1.00E + 06. Western blotting assays confirmed that prepared rabbit antibodies could recognize the recombinant 14-3-3 protein. Southern blotting results showed that there was only one copy of the 14-3-3 present in the genome of D. salina and 14-3-3 expression did not change throughout the Dnualiella cell cycle.

  16. 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

  17. Drosophila 14-3-3/PAR-5 is an essential mediator of PAR-1 function in axis formation.

    PubMed

    Benton, Richard; Palacios, Isabel M; St Johnston, Daniel

    2002-11-01

    PAR-1 kinases are required to determine the anterior-posterior (A-P) axis in C. elegans and Drosophila, but little is known about their molecular function. We identified 14-3-3 proteins as Drosophila PAR-1 interactors and show that PAR-1 binds a domain of 14-3-3 distinct from the phosphoserine binding pocket. PAR-1 kinases phosphorylate proteins to generate 14-3-3 binding sites and may therefore directly deliver 14-3-3 to these targets. 14-3-3 mutants display identical phenotypes to par-1 mutants in oocyte determination and the polarization of the A-P axis. Together, these results indicate that PAR-1's function is mediated by the binding of 14-3-3 to its substrates. The C. elegans 14-3-3 protein, PAR-5, is also required for A-P polarization, suggesting that this is a conserved mechanism by which PAR-1 establishes cellular asymmetries.

  18. 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.

  19. 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

  20. 14-3-3ε Is Required for Germ Cell Migration in Drosophila

    PubMed Central

    Tsigkari, K. Kirki; Acevedo, Summer F.; Skoulakis, Efthimios M. C.

    2012-01-01

    Although 14-3-3 proteins participate in multiple biological processes, isoform-specific specialized functions, as well as functional redundancy are emerging with tissue and developmental stage-specificity. Accordingly, the two 14-3-3ε proteins in Drosophila exhibit functional specificity and redundancy. Homozygotes for loss of function alleles of D14-3-3ε contain significantly fewer germ line cells (pole cells) in their gonads, a phenotype not shared by mutants in the other 14-3-3 gene leo. We show that although D14-3-3ε is enriched within pole cells it is required in mesodermal somatic gonad precursor cells which guide pole cells in their migration through the mesoderm and coalesce with them to form the embryonic gonad. Loss of D14-3-3ε results in defective pole cell migration, reduced pole cell number. We present evidence that D14-3-3ε loss results in reduction or loss of the transcription factor Zfh-1, one of the main regulatory molecules of the pole cell migration, from the somatic gonad precursor cells. PMID:22666326

  1. Transgenic Overexpression of 14-3-3 Zeta Protects Hippocampus against Endoplasmic Reticulum Stress and Status Epilepticus In Vivo

    PubMed Central

    Brennan, Gary P.; Jimenez-Mateos, Eva M.; McKiernan, Ross C.; Engel, Tobias; Tzivion, Guri; Henshall, David C.

    2013-01-01

    14-3-3 proteins are ubiquitous molecular chaperones that are abundantly expressed in the brain where they regulate cell functions including metabolism, the cell cycle and apoptosis. Brain levels of several 14-3-3 isoforms are altered in diseases of the nervous system, including epilepsy. The 14-3-3 zeta (ζ) isoform has been linked to endoplasmic reticulum (ER) function in neurons, with reduced levels provoking ER stress and increasing vulnerability to excitotoxic injury. Here we report that transgenic overexpression of 14-3-3ζ in mice results in selective changes to the unfolded protein response pathway in the hippocampus, including down-regulation of glucose-regulated proteins 78 and 94, activating transcription factors 4 and 6, and Xbp1 splicing. No differences were found between wild-type mice and transgenic mice for levels of other 14-3-3 isoforms or various other 14-3-3 binding proteins. 14-3-3ζ overexpressing mice were potently protected against cell death caused by intracerebroventricular injection of the ER stressor tunicamycin. 14-3-3ζ overexpressing mice were also potently protected against neuronal death caused by prolonged seizures. These studies demonstrate that increased 14-3-3ζ levels protect against ER stress and seizure-damage despite down-regulation of the unfolded protein response. Delivery of 14-3-3ζ may protect against pathologic changes resulting from prolonged or repeated seizures or where injuries provoke ER stress. PMID:23359526

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

    PubMed Central

    Marcos, Caroline Maria; da Silva, Julhiany de Fátima; de Oliveira, Haroldo Cesar; 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

  3. 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.

  4. PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch.

    PubMed

    Espejo, Alexsandra B; Gao, Guozhen; Black, Karynne; Gayatri, Sitaram; Veland, Nicolas; Kim, Jeesun; Chen, Taiping; Sudol, Marius; Walker, Cheryl; Bedford, Mark T

    2017-02-10

    PRMT5 is the primary enzyme responsible for the deposition of the symmetric dimethylarginine in mammalian cells. In an effort to understand how PRMT5 is regulated, we identified a threonine phosphorylation site within a C-terminal tail motif, which is targeted by the Akt/serum- and glucocorticoid-inducible kinases. While investigating the function of this posttranslational modification, we serendipitously discovered that its free C-terminal tail binds PDZ domains (when unphosphorylated) and 14-3-3 proteins (when phosphorylated). In essence, a phosphorylation event within the last few residues of the C-terminal tail generates a posttranslational modification-dependent PDZ/14-3-3 interaction "switch." The C-terminal motif of PRMT5 is required for plasma membrane association, and loss of this switching capacity is not compatible with life. This signaling phenomenon was recently reported for the HPV E6 oncoprotein but has not yet been observed for mammalian proteins. To investigate the prevalence of PDZ/14-3-3 switching in signal transduction, we built a protein domain microarray that harbors PDZ domains and 14-3-3 proteins. We have used this microarray to interrogate the C-terminal tails of a small group of candidate proteins and identified ERBB4, PGHS2, and IRK1 (as well as E6 and PRMT5) as conforming to this signaling mode, suggesting that PDZ/14-3-3 switching may be a broad biological paradigm.

  5. 14-3-3ζ Orchestrates Mammary Tumor Onset and Progression via miR221-Mediated Cell Proliferation

    PubMed Central

    Wyszomierski, Shannon L.; Wang, Qingfei; Li, Ping; Sahin, Ozgur; Xiao, Yi; Zhang, Siyuan; Xiong, Yan; Yang, Jun; Wang, Hai; Guo, Hua; Zhang, Jitao D.; Medina, Daniel; Muller, William J.; Yu, Dihua

    2013-01-01

    14-3-3ζ is overexpressed in over 40% of breast cancers but its pathophysiological relevance to tumorigenesis has not been established. Here we show that 14-3-3ζ overexpression is sufficient to induce tumorigenesis in a transgenic mouse model of breast cancer. MMTV-LTR promoter driven HA-14-3-3ζ transgenic mice (MMTV-HA-14-3-3ζ) developed mammary tumors whereas control mice did not. Whey acidic protein promoter driven HA-14-3-3ζ transgenic mice (WAP-HA-14-3-3ζ) developed hyperplastic lesions and showed increased susceptibility to carcinogen-induced tumorigenesis. When crossed with MMTV-neu transgenic mice, 14-3-3ζ.neu transgenic mice exhibited accelerated mammary tumorigenesis and metastasis compared to MMTV-neu mice. Mechanistically, 14-3-3ζ overexpression enhanced MAPK/c-Jun signaling leading to increased miR-221 transcription, which inhibited p27 CDKI translation, and consequently, promoted cell proliferation. Importantly, this 14-3-3ζ/miR-221/p27/proliferation axis is also functioning in patients' breast tumors and associates with high grade cancers. Taken together, our findings show that 14-3-3ζ overexpression has a causal role in mammary tumorigenesis and progression, acting through miR-221 in cooperation with known oncogenic events to drive neoplastic cell proliferation. PMID:24197133

  6. 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

  7. 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

  8. Neurodevelopmental and neuropsychiatric behaviour defects arise from 14-3-3ζ deficiency.

    PubMed

    Cheah, P S; Ramshaw, H S; Thomas, P Q; Toyo-Oka, K; Xu, X; Martin, S; Coyle, P; Guthridge, M A; Stomski, F; van den Buuse, M; Wynshaw-Boris, A; Lopez, A F; Schwarz, Q P

    2012-04-01

    Complex neuropsychiatric disorders are believed to arise from multiple synergistic deficiencies within connected biological networks controlling neuronal migration, axonal pathfinding and synapse formation. Here, we show that deletion of 14-3-3ζ causes neurodevelopmental anomalies similar to those seen in neuropsychiatric disorders such as schizophrenia, autism spectrum disorder and bipolar disorder. 14-3-3ζ-deficient mice displayed striking behavioural and cognitive deficiencies including a reduced capacity to learn and remember, hyperactivity and disrupted sensorimotor gating. These deficits are accompanied by subtle developmental abnormalities of the hippocampus that are underpinned by aberrant neuronal migration. Significantly, 14-3-3ζ-deficient mice exhibited abnormal mossy fibre navigation and glutamatergic synapse formation. The molecular basis of these defects involves the schizophrenia risk factor, DISC1, which interacts isoform specifically with 14-3-3ζ. Our data provide the first evidence of a direct role for 14-3-3ζ deficiency in the aetiology of neurodevelopmental disorders and identifies 14-3-3ζ as a central risk factor in the schizophrenia protein interaction network.

  9. Aberrant overexpression of an epithelial marker, 14-3-3σ, in a subset of hematological malignancies

    PubMed Central

    Motokura, Toru; Nakamura, Yukari; Sato, Hiroyuki

    2007-01-01

    Background 14-3-3σ is a p53-mediated cell-cycle inhibitor in epithelial cells. The expression of 14-3-3σ is frequently altered in cancers of epithelial origin associated with altered DNA methylation. Since its involvement in a non-epithelial tumor is unknown, we examined 14-3-3σ expression in patients with haematological malignancies. Methods We analyzed 41 hematopoietic cell lines and 129 patients with a variety of hematological malignancies for 14-3-3σ expression with real-time RT-PCR. We also examined protein levels by Western blot analysis and DNA methylation status of the 14-3-3σ gene by methylation-specific PCR analysis of bisulfite-treated DNA. In addition, mutations of p53 gene were identified by RT-PCR-SSCP analysis and the expression levels of 14-3-3σ were compared with those of other cell-cycle inhibitor genes, CDKN2A and ARF. Results The expression levels of 14-3-3σ mRNA in almost all cell lines were low and comparable to those in normal hematopoietic cells except for 2 B-cell lines. On the contrary, 14-3-3σ mRNA was aberrantly overexpressed frequently in mature lymphoid malignancies (30 of 93, 32.3%) and rarely in acute leukemia (3 of 35, 8.6%). 14-3-3σ protein was readily detectable and roughly reflected the mRNA level. In contrast to epithelial tumors, methylation status of the 14-3-3σ gene was not associated with expression in hematological malignancies. Mutations of p53 were identified in 12 patients and associated with lower expression of 14-3-3σ. The expression levels of 14-3-3σ, CDKN2A and ARF were not correlated with but rather reciprocal to one another, suggesting that simultaneous overexpression of any two of them is incompatible with tumor growth. Conclusion 14-3-3σ, an epithelial cell marker, was overexpressed significantly in a subset of mature lymphoid malignancies. This is the first report of aberrant 14-3-3σ expression in non-epithelial tumors in vivo. Since the significance of 14-3-3σ overexpression is unknown even in

  10. 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.

  11. 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

  12. Pear 14-3-3a gene (Pp14-3-3a) is regulated during fruit ripening and senescense, and involved in response to salicylic acid and ethylene signalling.

    PubMed

    Shi, Haiyan; Zhang, Yuxing

    2014-12-01

    14-3-3 proteins play important roles in regulating plant development and phytohormone (abscisic acid, gibberellin and brassinosteroids) signalling. However, their regulation in fruit ripening and senescense, and response to salicylic acid and ethylene signalling are yet to be illustrated. One cDNA encoding putative 14-3-3 protein was isolated from pear (Pyrus pyrifolia) and designated Pp14-3-3a. Phylogenetic analysis clearly demonstrated that Pp14-3-3a belonged to ε-like group of 14-3-3 superfamilies. Real-time quantitative PCR analysis indicated that the expression of Pp14-3-3a gene was developmentally regulated in the fruit. Further study demonstrated that Pp14-3-3a expression was inhibited by salicylic acid and induced by ethylene precursor 1-aminocyclopropane-1-carboxylic acid in pear fruit. These data suggested that Pp14-3-3a might be involved in response to salicylic acid and ethylene signalling during fruit ripening and senescence of pear.

  13. 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

  14. 14-3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity.

    PubMed

    Light, Yvonne; Paterson, Hugh; Marais, Richard

    2002-07-01

    We have investigated the role that S259 phosphorylation, S621 phosphorylation, and 14-3-3 binding play in regulating Raf-1 activity. We show that 14-3-3 binding, rather than Raf-1 phosphorylation, is required for the correct regulation of kinase activity. Phosphorylation of S621 is not required for activity, but 14-3-3 binding is essential. When 14-3-3 binding to conserved region 2 (CR2) was disrupted, Raf-1 basal kinase activity was elevated and it could be further activated by (V12,G37)Ras, (V23)TC21, and (V38)R-Ras. Disruption of 14-3-3 binding at CR2 did not recover binding of Raf-1 to (V12,G37)Ras but allowed more efficient recruitment of Raf-1 to the plasma membrane and stimulated its phosphorylation on S338. Finally, (V12)Ras, but not (V12,G37)Ras, displaced 14-3-3 from full-length Raf-1 and the Raf-1 bound to Ras. GTP was still phosphorylated on S259. Our data suggest that stable association of Raf-1 with the plasma membrane requires Ras-mediated displacement of 14-3-3 from CR2. Small G proteins that cannot displace 14-3-3 fail to recruit Raf-1 to the membrane efficiently and so fail to stimulate kinase activity.

  15. Impairment of GABAB receptor dimer by endogenous 14-3-3ζ in chronic pain conditions

    PubMed Central

    Laffray, Sophie; Bouali-Benazzouz, Rabia; Papon, Marie-Amélie; Favereaux, Alexandre; Jiang, Yang; Holm, Tina; Spriet, Corentin; Desbarats, Pascal; Fossat, Pascal; Le Feuvre, Yves; Decossas, Marion; Héliot, Laurent; Langel, Ulo; Nagy, Frédéric; Landry, Marc

    2012-01-01

    In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). However, the regulation of GABAB dimerization, and more generally of GPCR oligomerization, remains largely unknown. We propose a novel mechanism for inhibition of GPCR activity through de-dimerization in pathological conditions. We show here that 14-3-3ζ, a GABAB1-binding protein, dissociates the GABAB heterodimer, resulting in the impairment of GABAB signalling in spinal neurons. In the dorsal spinal cord of neuropathic rats, 14-3-3ζ is overexpressed and weakens GABAB inhibition. Using anti-14-3-3ζ siRNA or competing peptides disrupts 14-3-3ζ/GABAB1 interaction and restores functional GABAB heterodimers in the dorsal horn. Importantly, both strategies greatly enhance the anti-nociceptive effect of intrathecal Baclofen in neuropathic rats. Taken together, our data provide the first example of endogenous regulation of a GPCR oligomeric state and demonstrate its functional impact on the pathophysiological process of neuropathic pain sensitization. PMID:22692127

  16. Phosphorylation-related modification at the dimer interface of 14-3-3ω dramatically alters monomer interaction dynamics.

    PubMed

    Denison, Fiona C; Gökirmak, Tufan; Ferl, Robert J

    2014-01-01

    14-3-3 proteins are generally believed to function as dimers in a broad range of eukaryotic signaling pathways. The consequences of altering dimer stability are not fully understood. Phosphorylation at Ser58 in the dimer interface of mammalian 14-3-3 isoforms has been reported to destabilise dimers. An equivalent residue, Ser62, is present across most Arabidopsis isoforms but the effects of phosphorylation have not been studied in plants. Here, we assessed the effects of phosphorylation at the dimer interface of Arabidopsis 14-3-3ω. Protein kinase A phosphorylated 14-3-3ω at Ser62 and also at a previously unreported residue, Ser67, resulting in a monomer-sized band on native-PAGE. Phosphorylation at Ser62 alone, or with additional Ser67 phosphorylation, was investigated using phosphomimetic versions of 14-3-3ω. In electrophoretic and chromatographic analyses, these mutants showed mobilities intermediate between dimers and monomers. Mobility was increased by detergents, by reducing protein concentration, or by increasing pH or temperature. Urea gradient gels showed complex structural transitions associated with alterations of dimer stability, including a previously unreported 14-3-3 aggregation phenomenon. Overall, our analyses showed that dimer interface modifications such as phosphorylation reduce dimer stability, dramatically affecting the monomer-dimer equilibrium and denaturation trajectory. These findings may have dramatic implications for 14-3-3 structure and function in vivo.

  17. [Inhibitory effect of 14-3-3ζ on the proliferation of HL-60 cells and HL-60/VCR cells].

    PubMed

    Liang, Rong; Chen, Xie-Qun; Wang, Zhe; Xiong, Hua; Bai, Qing-Xian; Gao, Guang-Xun; Dong, Bao-Xia; Zhu, Hua-Feng

    2013-08-01

    This study was aimed to investigate the expression and role of 14-3-3ζ in the AML cell lines: sensitive HL-60 and drug-resistant HL-60/VCR cells. Semi-quantitative RT-PCR and Western blot were respectively used to examine the expression of mdr1 mRNA and Pgp in AML cell lines to validate the results of microarray. Western blot was performed to investigate the expression of Pgp, 14-3-3ζ, and anti-apoptosis protein BCL-2, MCL-1 proteins. Immunofluorescence assay was used to detect the subcellular location of 14-3-3ζ protein in HL-60 and HL-60/VCR cells by laser scanning confocal microscopy. Transduction with siRNA was used to silence 14-3-3ζ in AML cell lines. Cell count method and flow cytometry of cell cycle were used to analyze the changes of growth of AML cells. The results found that mdr1 mRNA and Pgp did not expressed in HL-60 cells, but significantly overexpressed in HL-60/VCR cells. Except 14-3-3σ, the expression of other subtypes of 14-3-3 was higher in HL-60/VCR cells than that in HL-60 cells, especially 14-3-3ζ. The higher expression of 14-3-3ζ, BCL-2, MCL-1 protein was observed in HL-60/VCR cells than that in HL-60 cells. These results were same results from gene chip. It was also noticed that 14-3-3ζ was located in the cytoplasma and nuclei of AML cell lines, especially over-expressed in HL-60/VCR cells. Furthermore, suppression of 14-3-3ζ by RNA interference resulted in inhibition of the proliferation of AML cells with decreased protein expression of BCL-2 and MCL-1, especially in HL-60/VCR cells. It is concluded that 14-3-3ζ plays an important role in proliferation of AML cells and associates with BCL-2 and MCL-1 expression. These results suggested that development of therapy targeting 14-3-3ζ may provide novel, effective strategies for refractory and relapsed AML.

  18. 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.

  19. Cannabinoid receptor activation inhibits cell cycle progression by modulating 14-3-3β.

    PubMed

    Jung, Hye-Won; Park, Inae; Ghil, Sungho

    2014-09-01

    Cannabinoids display various pharmacological activities, including tumor regression, anti-inflammatory and neuroprotective effects. To investigate the molecular mechanisms underlying the pharmacological effects of cannabinoids, we used a yeast two-hybrid system to screen a mouse brain cDNA library for proteins interacting with type 1 cannabinoid receptor (CB1R). Using the intracellular loop 3 of CB1R as bait, we identified 14-3-3β as an interacting partner of CB1R and confirmed their interaction using affinity-binding assays. 14-3-3β has been reported to induce a cell cycle delay at the G2/M phase. We tested the effects of cannabinoids on cell cycle progression in HeLa cells synchronized using a double-thymidine block-and-release protocol and found an increase in the population of G2/M phase cells. We further found that CB1R activation augmented the interaction of 14-3-3β with Wee1 and Cdc25B, and promoted phosphorylation of Cdc2 at Tyr-15. These results suggest that cannabinoids induce cell cycle delay at the G2/M phase by activating 14-3-3β.

  20. Genome-Wide Identification, Phylogeny, and Expression Analyses of the 14-3-3 Family Reveal Their Involvement in the Development, Ripening, and Abiotic Stress Response in Banana

    PubMed Central

    Li, Meiying; Ren, Licheng; Xu, Biyu; Yang, Xiaoliang; Xia, Qiyu; He, Pingping; Xiao, Susheng; Guo, Anping; Hu, Wei; Jin, Zhiqiang

    2016-01-01

    Plant 14-3-3 proteins act as critical components of various cellular signaling processes and play an important role in regulating multiple physiological processes. However, less information is known about the 14-3-3 gene family in banana. In this study, 25 14-3-3 genes were identified from the banana genome. Based on the evolutionary analysis, banana 14-3-3 proteins were clustered into ε and non-ε groups. Conserved motif analysis showed that all identified banana 14-3-3 genes had the typical 14-3-3 motif. The gene structure of banana 14-3-3 genes showed distinct class-specific divergence between the ε group and the non-ε group. Most banana 14-3-3 genes showed strong transcript accumulation changes during fruit development and postharvest ripening in two banana varieties, indicating that they might be involved in regulating fruit development and ripening. Moreover, some 14-3-3 genes also showed great changes after osmotic, cold, and salt treatments in two banana varieties, suggested their potential role in regulating banana response to abiotic stress. Taken together, this systemic analysis reveals the involvement of banana 14-3-3 genes in fruit development, postharvest ripening, and response to abiotic stress and provides useful information for understanding the functions of 14-3-3 genes in banana. PMID:27713761

  1. 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function

    PubMed Central

    Schoenwaelder, Simone M.; Darbousset, Roxane; Cranmer, Susan L.; Ramshaw, Hayley S.; Orive, Stephanie L.; Sturgeon, Sharelle; Yuan, Yuping; Yao, Yu; Krycer, James R.; Woodcock, Joanna; Maclean, Jessica; Pitson, Stuart; Zheng, Zhaohua; Henstridge, Darren C.; van der Wal, Dianne; Gardiner, Elizabeth E.; Berndt, Michael C.; Andrews, Robert K.; James, David E.; Lopez, Angel F.; Jackson, Shaun P.

    2016-01-01

    The 14-3-3 family of adaptor proteins regulate diverse cellular functions including cell proliferation, metabolism, adhesion and apoptosis. Platelets express numerous 14-3-3 isoforms, including 14-3-3ζ, which has previously been implicated in regulating GPIbα function. Here we show an important role for 14-3-3ζ in regulating arterial thrombosis. Interestingly, this thrombosis defect is not related to alterations in von Willebrand factor (VWF)–GPIb adhesive function or platelet activation, but instead associated with reduced platelet phosphatidylserine (PS) exposure and procoagulant function. Decreased PS exposure in 14-3-3ζ-deficient platelets is associated with more sustained levels of metabolic ATP and increased mitochondrial respiratory reserve, independent of alterations in cytosolic calcium flux. Reduced platelet PS exposure in 14-3-3ζ-deficient mice does not increase bleeding risk, but results in decreased thrombin generation and protection from pulmonary embolism, leading to prolonged survival. Our studies define an important role for 14-3-3ζ in regulating platelet bioenergetics, leading to decreased platelet PS exposure and procoagulant function. PMID:27670677

  2. Pharmacological inhibition of epsilon-protein kinase C attenuates cardiac fibrosis and dysfunction in hypertension-induced heart failure.

    PubMed

    Inagaki, Koichi; Koyanagi, Tomoyoshi; Berry, Natalia C; Sun, Lihan; Mochly-Rosen, Daria

    2008-06-01

    Studies on genetically manipulated mice suggest a role for epsilon-protein kinase C (epsilonPKC) in cardiac hypertrophy and in heart failure. The potential clinical relevance of these findings was tested here using a pharmacological inhibitor of epsilonPKC activity during the progression to heart failure in hypertensive Dahl rats. Dahl rats, fed an 8% high-salt diet from the age of 6 weeks, exhibited compensatory cardiac hypertrophy by 11 weeks, followed by heart failure at approximately 17 weeks and death by the age of approximately 20 weeks (123+/-3 days). Sustained treatment between weeks 11 and 17 with the selective epsilonPKC inhibitor epsilonV1-2 or with an angiotensin II receptor blocker olmesartan prolonged animal survival by approximately 5 weeks (epsilonV1-2: 154+/-7 days; olmesartan: 149+/-5 days). These treatments resulted in improved fractional shortening (epsilonV1-2: 58+/-2%; olmesartan: 53+/-2%; saline: 41+/-6%) and decreased cardiac parenchymal fibrosis when measured at 17 weeks without lowering blood pressure at any time during the treatment. Combined treatment with epsilonV1-2, together with olmesartan, prolonged animal survival by 5 weeks (37 days) relative to olmesartan alone (from 160+/-5 to 197+/-14 days, respectively) and by approximately 11 weeks (74 days) on average relative to saline-treated animals, suggesting that the pathway inhibited by epsilonPKC inhibition is not identical to the olmesartan-induced effect. These data suggest that an epsilonPKC-selective inhibitor such as epsilonV1-2 may have a potential in augmenting current therapeutic strategies for the treatment of heart failure in humans.

  3. 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

  4. 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.

  5. 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    PubMed Central

    Bolton, Diane L; Barnitz, Robert A; Sakai, Keiko; Lenardo, Michael J

    2008-01-01

    Background Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4+ T cell depletion in HIV-1-infected individuals remains unclear. The HIV-1 Vpr accessory protein causes cell death, likely through a mechanism related to its ability to arrest cells in the G2,M phase. Recent evidence implicated the scaffold protein, 14-3-3, in Vpr cell cycle blockade. Results We found that in human T cells, 14-3-3 plays an active role in mediating Vpr-induced cell cycle arrest and reveal a dramatic increase in the amount of Cdk1, Cdc25C, and CyclinB1 bound to 14-3-3 θ during Vprv-induced G2,M arrest. By contrast, a cell-cycle-arrest-dead Vpr mutant failed to augment 14-3-3 θ association with Cdk1 and CyclinB1. Moreover, G2,M arrest caused by HIV-1 infection strongly correlated with a disruption in 14-3-3 θ binding to centrosomal proteins, Plk1 and centrin. Finally, Vpr caused elevated levels of CyclinB1, Plk1, and Cdk1 in a complex with the nuclear transport and spindle assembly protein, importin β. Conclusion Thus, our data reveal a new facet of Vpr-induced cell cycle arrest involving previously unrecognized abnormal rearrangements of multiprotein assemblies containing key cell cycle regulatory proteins. Reviewers This article was reviewed by David Kaplan, Nathaniel R. Landau and Yan Zhou. PMID:18445273

  6. miR-137 modulates coelomocyte apoptosis by targeting 14-3-3ζ in the sea cucumber Apostichopus japonicus.

    PubMed

    Lv, Miao; Chen, Huahui; Shao, Yina; Li, Chenghua; Xu, Wei; Zhang, Weiwei; Zhao, Xuelin; Duan, Xuemei

    2017-02-01

    MicroRNAs (miRNAs) have emerged as key regulators in the host immune response and play a pivotal role in host-pathogen interactions by suppressing the transcriptional and post-transcriptional expression of target genes. miR-137, a well-documented tumor repressor, was previously found by high-throughput sequencing to be differentially expressed in diseased specimens of the sea cucumber Apostichopus japonicus. In this study, we identified 14-3-3ζ protein (Aj14-3-3ζ) as a novel target of miR-137 using isobaric tags for relative and absolute quantification (iTRAQ) and transcriptome screening. Expression analysis indicated that consistently depressed expression profiles of miR-137 and Aj14-3-3ζ were detected in both LPS-exposed primary coelomocytes and Vibrio splendidus-challenged sea cucumbers, suggesting a positive regulatory interaction. Consistently, miR-137 overexpression or inhibition in vitro and in vivo showed no effect on Aj14-3-3ζ mRNA levels, but the concentration of Aj14-3-3ζ protein was induced or repressed, respectively. Moreover, siRNA-mediated Aj14-3-3ζ knockdown in vivo decreased both mRNA and protein expression levels of Aj14-3-3ζ and significantly promoted coelomocyte apoptosis as assessed by flow cytometry, consistent with miR-137 inhibition. Overall, these results enhance our understanding of miR-137 regulatory roles in sea cucumber pathogenesis.

  7. The Peripheral Binding of 14-3-3γ to Membranes Involves Isoform-Specific Histidine Residues

    PubMed Central

    Ying, Ming; Halskau, Øyvind; Baumann, Anne; Rodriguez-Larrea, David; Costas, Miguel; Underhaug, Jarl; Sanchez-Ruiz, Jose M.; Martinez, Aurora

    2012-01-01

    Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states. PMID:23189152

  8. Rac1 activation driven by 14-3-3ζ dimerization promotes prostate cancer cell-matrix interactions, motility and transendothelial migration.

    PubMed

    Goc, Anna; Abdalla, Maha; Al-Azayzih, Ahmad; Somanath, Payaningal R

    2012-01-01

    14-3-3 proteins are ubiquitously expressed dimeric adaptor proteins that have emerged as key mediators of many cell signaling pathways in multiple cell types. Its effects are mainly mediated by binding to selective phosphoserine/threonine proteins. The importance of 14-3-3 proteins in cancer have only started to become apparent and its exact role in cancer progression as well as the mechanisms by which 14-3-3 proteins mediate cancer cell function remain unknown. While protein 14-3-3σ is widely accepted as a tumor suppressor, 14-3-3ζ, β and γ isoforms have been shown to have tumor promoting effects. Despite the importance of 14-3-3 family in mediating various cell processes, the exact role and mechanism of 14-3-3ζ remain unexplored. In the current study, we investigated the role of protein 14-3-3ζ in prostate cancer cell motility and transendothelial migration using biochemical, molecular biology and electric cell-substrate impedance sensing approaches as well as cell based functional assays. Our study indicated that expression with wild-type protein 14-3-3ζ significantly enhanced Rac activity in PC3 cells. In contrast, expression of dimer-resistant mutant of protein 14-3-3ζ (DM-14-3-3) inhibited Rac activity and associated phosphorylation of p21 activated kinase-1 and 2. Expression with wild-type 14-3-3ζ or constitutively active Rac1 enhanced extracellular matrix recognition, lamellipodia formation, cell migration and trans-endothelial migration by PC3 cells. In contrast, expression with DM 14-3-3ζ or DN-Rac1 in PC3 cells significantly inhibited these cell functions. Our results demonstrate for the first time that 14-3-3ζ enhances prostate cancer cell-matrix interactions, motility and transendothelial migration in vitro via activation of Rac1-GTPase and is an important target for therapeutic interventions for prostate cancer.

  9. Rethinking the role of phosducin: Light-regulated binding of phosducin to 14-3-3 in rod inner segments

    PubMed Central

    Nakano, Koichi; Chen, Jing; Tarr, George E.; Yoshida, Tatsuro; Flynn, Julia M.; Bitensky, Mark W.

    2001-01-01

    Phosducin (Pd), a small protein found abundantly in photoreceptors, is widely assumed to regulate light sensitivity in the rod outer segment through interaction with the heterotrimeric G protein transducin. But, based on histochemistry and Western blot analysis, Pd is found almost entirely in the inner segment in both light and dark, most abundantly near the rod synapse. We report a second small protein, 14-3-3, in the rod with a similar distribution. By immunoprecipitation, phospho-Pd is found to interact with 14-3-3 in material from dark-adapted retina, and this interaction is markedly diminished by light, which dephosphorylates Pd. Conversely, unphosphorylated Pd binds to inner segment G protein(s) in the light. From these results and reported functions of 14-3-3, we have constructed a hypothesis for the regulation of light sensitivity at the level of rod synapse. By dissociating the Pd/14-3-3 complex, light enables both proteins to function in this role. PMID:11287646

  10. Transcriptional regulation of YWHAZ, the gene encoding 14-3-3ζ.

    PubMed

    Kasinski, Andrea; Dong, Xueyuan; Khuri, Fadlo R; Boss, Jeremy; Fu, Haian

    2014-01-01

    Aberrant expression of oncogenic 14-3-3 proteins is correlated with poor survival of cancer patients. While the underlying mechanism of the abnormal expression in tumors remains elusive for the six oncogenic 14-3-3 isoforms; the potential involvement of a transcriptional component has been suggested. Unfortunately, little experimental data has been reported to support this hypothesis. In this study we describe the genetic structure of YWHAZ, the gene encoding 14-3-3ζ, including the identification of previously unreported transcript variants. In total, five transcript variants were revealed and their expressions confirmed in a panel of cell lines. Expressed sequence tag (EST) database mining and in vitro rapid-amplification of cDNA ends (RACE) confirmed that one variant, 1c, represents >80% of the expressed transcripts, which is also the most efficiently translated. An analysis of the proximal promoter of this variant revealed a functional Cyclic-AMP Response Element (CRE). Factors that bound to the CRE element were recognized through fractionation and subsequent EMSAs. This analysis identified CREB and ATF-1 as the trans-interacting factors. Cell-based assays confirm that ATF-1, and to a lesser extent CREB, bind the endogenous YWHAZ promoter especially under TNF-α stimulating conditions. In support of a role of ATF-1 in the regulation of YWHAZ, silencing of ATF-1 resulted in a marked reduction in two of the five YWHAZ transcripts. These data suggest a novel mechanism for the transcriptional regulation of a major pro-survival gene, YWHAZ, by ATF-1.

  11. 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.

  12. Molecular cloning and characterization of a novel human protein phosphatase 2C cDNA (PP2C epsilon*).

    PubMed

    Jin, Feng; Ji, Chaoneng; Liu, Lingfeng; Dai, Jianfeng; Gu, Shaohua; Sun, Xianfei; Xie, Yi; Mao, Yumin

    2004-09-01

    We have isolated a novel cDNA from the human fetal brain cDNA library with homology to the Mg2+ -dependent serine/threonine protein phosphatase 2C (PP2C) family. The cDNA is 3055 bp in length, and the predicted coding region encodes a 360-amino-acid protein, which shows 99% identity to the PP2C epsilon from rat and mouse. Then we term it human PP2C epsilon gene. The gene is mapped to chromosome 3q26.1 and contains 4 exons. RT-PCR analysis shows that the PP2C epsilon is widely expressed in human tissues and the expression levels in heart, placenta, lung, liver, kidney, and pancreas are relatively high.

  13. On the role of residue phosphorylation in 14-3-3 partners: AANAT as a case study.

    PubMed

    Masone, Diego; Uhart, Marina; Bustos, Diego M

    2017-04-07

    Twenty years ago, a novel concept in protein structural biology was discovered: the intrinsically disordered regions (IDRs). These regions remain largely unstructured under native conditions and the more are studied, more properties are attributed to them. Possibly, one of the most important is their ability to conform a new type of protein-protein interaction. Besides the classical domain-to-domain interactions, IDRs follow a 'fly-casting' model including 'induced folding'. Unfortunately, it is only possible to experimentally explore initial and final states. However, the complete movie of conformational changes of protein regions and their characterization can be addressed by in silico experiments. Here, we simulate the binding of two proteins to describe how the phosphorylation of a single residue modulates the entire process. 14-3-3 protein family is considered a master regulator of phosphorylated proteins and from a modern point-of-view, protein phosphorylation is a three component system, with writers (kinases), erasers (phosphatases) and readers. This later biological role is attributed to the 14-3-3 protein family. Our molecular dynamics results show that phosphorylation of the key residue Thr31 in a partner of 14-3-3, the aralkylamine N-acetyltransferase, releases the fly-casting mechanism during binding. On the other hand, the non-phosphorylation of the same residue traps the proteins, systematically and repeatedly driving the simulations into wrong protein-protein conformations.

  14. On the role of residue phosphorylation in 14-3-3 partners: AANAT as a case study

    PubMed Central

    Masone, Diego; Uhart, Marina; Bustos, Diego M.

    2017-01-01

    Twenty years ago, a novel concept in protein structural biology was discovered: the intrinsically disordered regions (IDRs). These regions remain largely unstructured under native conditions and the more are studied, more properties are attributed to them. Possibly, one of the most important is their ability to conform a new type of protein-protein interaction. Besides the classical domain-to-domain interactions, IDRs follow a ‘fly-casting’ model including ‘induced folding’. Unfortunately, it is only possible to experimentally explore initial and final states. However, the complete movie of conformational changes of protein regions and their characterization can be addressed by in silico experiments. Here, we simulate the binding of two proteins to describe how the phosphorylation of a single residue modulates the entire process. 14-3-3 protein family is considered a master regulator of phosphorylated proteins and from a modern point-of-view, protein phosphorylation is a three component system, with writers (kinases), erasers (phosphatases) and readers. This later biological role is attributed to the 14-3-3 protein family. Our molecular dynamics results show that phosphorylation of the key residue Thr31 in a partner of 14-3-3, the aralkylamine N-acetyltransferase, releases the fly-casting mechanism during binding. On the other hand, the non-phosphorylation of the same residue traps the proteins, systematically and repeatedly driving the simulations into wrong protein-protein conformations. PMID:28387381

  15. 14-3-3ε Plays a Role in Cardiac Ventricular Compaction by Regulating the Cardiomyocyte Cell Cycle

    PubMed Central

    Kosaka, Yasuhiro; Cieslik, Katarzyna A.; Li, Ling; Lezin, George; Maguire, Colin T.; Saijoh, Yukio; Toyo-oka, Kazuhito; Gambello, Michael J.; Vatta, Matteo; Wynshaw-Boris, Anthony; Baldini, Antonio; Yost, H. Joseph

    2012-01-01

    Trabecular myocardium accounts for the majority of the ventricles during early cardiogenesis, but compact myocardium is the primary component at later developmental stages. Elucidation of the genes regulating compact myocardium development is essential to increase our understanding of left ventricular noncompaction (LVNC), a cardiomyopathy characterized by increased ratios of trabecular to compact myocardium. 14-3-3ε is an adapter protein expressed in the lateral plate mesoderm, but its in vivo cardiac functions remain to be defined. Here we show that 14-3-3ε is expressed in the developing mouse heart as well as in cardiomyocytes. 14-3-3ε deletion did not appear to induce compensation by other 14-3-3 isoforms but led to ventricular noncompaction, with features similar to LVNC, resulting from a selective reduction in compact myocardium thickness. Abnormal compaction derived from a 50% decrease in cardiac proliferation as a result of a reduced number of cardiomyocytes in G2/M and the accumulation of cardiomyocytes in the G0/G1 phase of the cell cycle. These defects originated from downregulation of cyclin E1 and upregulation of p27Kip1, possibly through both transcriptional and posttranslational mechanisms. Our work shows that 14-3-3ε regulates cardiogenesis and growth of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via both cyclin E1 and p27Kip1. These data are consistent with the long-held view that human LVNC may result from compaction arrest, and they implicate 14-3-3ε as a new candidate gene in congenital human cardiomyopathies. PMID:23071090

  16. Overexpression of 14-3-3σ counteracts tumorigenicity by positively regulating p73 in vivo

    PubMed Central

    GENG, CUIZHI; SANG, MEIXIANG; YANG, RUILING; GAO, WEI; ZHOU, TAO; WANG, SHIJIE

    2011-01-01

    14-3-3σ, one of the 14-3-3 family members, was initially identified as a human mammary epithelium-specific marker 1. The expression of 14-3-3σ is directly regulated by p53. It has been demonstrated that 14-3-3σ stabilizes p53 and enhances its transcriptional activity through the interaction with p53, suggesting that 14-3-3σ has a positive feedback effect on p53. Our previous study showed that 14-3-3σ is a direct transcriptional target of p73 and enhances the p73-mediated transcriptional as well as pro-apoptotic activity in vitro. In the present study, we explored the tumor-suppressive effect of 14-3-3σ by establishing a breast cancer xenograft nude mouse model with an inducible expression of 14-3-3σ or with an inducible expression of p53/p73 plus 14-3-3σ with ADR treatment. Tumor formation was then assayed. Moreover, 66 primary breast cancer specimens and paired tumor-free breast specimens obtained from the female patients were examined. Results showed that the expression of p73 and 14-3-3σ in breast cancer specimens was significantly lower than the tumor-free breast specimens and that 14-3-3σ expression was positively correlated with the expression of p73. Furthermore, overexpression of 14-3-3σ counteracts tumorigenicity by positively regulating p73 in p53-mutated or -deficient cancers in vivo. Therefore, our results may lead to the use of 14-3-3σ in the therapeutic application for the p53-mutated and p73-expressed breast cancer patients. PMID:22848285

  17. IFNγ-induced suppression of β-catenin signaling: evidence for roles of Akt and 14.3.3ζ

    PubMed Central

    Nava, Porfirio; Kamekura, Ryuta; Quirós, Miguel; Medina-Contreras, Oscar; Hamilton, Ross W.; Kolegraff, Keli N.; Koch, Stefan; Candelario, Aurora; Romo-Parra, Hector; Laur, Oskar; Hilgarth, Roland S.; Denning, Timothy L.; Parkos, Charles A.; Nusrat, Asma

    2014-01-01

    The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. β-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/β-catenin signaling. Here we show that inhibition of Akt/β-catenin–mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated β-catenin 552 (pβ-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ. Augmented β-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/β-catenin from the nucleus, thereby inhibiting β-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation. PMID:25079689

  18. Deletion of the cruciform binding domain in CBP/14-3-3 displays reduced origin binding and initiation of DNA replication in budding yeast

    PubMed Central

    Yahyaoui, Wafaa; Callejo, Mario; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2007-01-01

    Background Initiation of eukaryotic DNA replication involves many protein-protein and protein-DNA interactions. We have previously shown that 14-3-3 proteins bind cruciform DNA and associate with mammalian and yeast replication origins in a cell cycle dependent manner. Results By expressing the human 14-3-3ε, as the sole member of 14-3-3 proteins family in Saccharomyces cerevisiae, we show that 14-3-3ε complements the S. cerevisiae Bmh1/Bmh2 double knockout, conserves its cruciform binding activity, and associates in vivo with the yeast replication origins ARS307. Deletion of the α5-helix, the potential cruciform binding domain of 14-3-3, decreased the cruciform binding activity of the protein as well as its association with the yeast replication origins ARS307 and ARS1. Furthermore, the mutant cells had a reduced ability to stably maintain plasmids bearing one or multiple origins. Conclusion 14-3-3, a cruciform DNA binding protein, associates with yeast origins of replication and functions as an initiator of DNA replication, presumably through binding to cruciform DNA forming at yeast replicators. PMID:17430600

  19. Engineering of poly(epsilon-caprolactone) microcarriers to modulate protein encapsulation capability and release kinetic.

    PubMed

    Coccoli, Valentina; Luciani, Alessia; Orsi, Silvia; Guarino, Vincenzo; Causa, Filippo; Netti, Paolo Antonio

    2008-04-01

    Drug delivery applications using biodegradable polymeric microspheres are becoming an important means of delivering therapeutic agents. The aim of this work was to modulate the microporosity of poly(epsilon-caprolactone) (PCL) microcarriers to control protein loading capability and release profile. PCL microparticles loaded with BSA (bovine serum albumin) have been de novo synthesized with double emulsion solvent evaporation technique transferred and adapted for different polymer concentrations (1.7 and 3% w/v) and stabilizer present in the inner aqueous phase (0.05, 0.5 and 1% w/v). SEM (scanning electron microscope) and CLSM (confocal laser scanning microscope) analysis map the drug distribution in homogeneously distributed cavities inside the microspheres with dimensions that can be modulated by varying double emulsion process parameters. The inner structure of BSA-loaded microspheres is greatly affected by the surfactant concentration in the internal aqueous phase, while a slight influence of polymer concentration in the oil phase was observed. The surfactant concentration mainly determines microspheres morphology, as well as drug release kinetics, as confirmed by our in-vitro BSA release study. Moreover, the entrapped protein remained unaltered during the protein encapsulation process, retaining its bio-activity and structure, as shown through a dedicated gel chromatographic analytical method.

  20. Epsilon-Q: An Automated Analyzer Interface for Mass Spectral Library Search and Label-Free Protein Quantification.

    PubMed

    Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Paik, Young-Ki

    2017-04-04

    Mass spectrometry (MS) is a widely used proteome analysis tool for biomedical science. In an MS-based bottom-up proteomic approach to protein identification, sequence database (DB) searching has been routinely used because of its simplicity and convenience. However, searching a sequence DB with multiple variable modification options can increase processing time, false-positive errors in large and complicated MS data sets. Spectral library searching is an alternative solution, avoiding the limitations of sequence DB searching and allowing the detection of more peptides with high sensitivity. Unfortunately, this technique has less proteome coverage, resulting in limitations in the detection of novel and whole peptide sequences in biological samples. To solve these problems, we previously developed the "Combo-Spec Search" method, which uses manually multiple references and simulated spectral library searching to analyze whole proteomes in a biological sample. In this study, we have developed a new analytical interface tool called "Epsilon-Q" to enhance the functions of both the Combo-Spec Search method and label-free protein quantification. Epsilon-Q performs automatically multiple spectral library searching, class-specific false-discovery rate control, and result integration. It has a user-friendly graphical interface and demonstrates good performance in identifying and quantifying proteins by supporting standard MS data formats and spectrum-to-spectrum matching powered by SpectraST. Furthermore, when the Epsilon-Q interface is combined with the Combo-Spec search method, called the Epsilon-Q system, it shows a synergistic function by outperforming other sequence DB search engines for identifying and quantifying low-abundance proteins in biological samples. The Epsilon-Q system can be a versatile tool for comparative proteome analysis based on multiple spectral libraries and label-free quantification.

  1. 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.

  2. Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

    PubMed Central

    Andersen, J N; Elson, A; Lammers, R; Rømer, J; Clausen, J T; Møller, K B; Møller, N P

    2001-01-01

    To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function. PMID:11237862

  3. 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

  4. 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

  5. Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex.

    PubMed

    Diviani, Dario; Abuin, Liliane; Cotecchia, Susanna; Pansier, Laetitia

    2004-07-21

    A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.

  6. Origin and evolution of a new exon of 14-3-3ξ in bees and phylogenetic analysis.

    PubMed

    Zhan, Leilei; Jiang, Chao

    2013-04-01

    Mutually exclusive splicing, one type of alternative splicing, involves selection of alternatively spliced exons arranged in tandem and creates protein products with substitution of one segment of the amino acid sequence for another. Previous studies revealed that exon 5 of 14-3-3ξ from Apis mellifera (western honeybee) had three mutually exclusive exons, while orthologous exon of Nasonia vitripennis (parasitic wasp) had only two, suggesting that cases of exon gain or loss might have happened during the evolution of hymenopteran species. In the current study, we annotated and analyzed the 14-3-3ξ genes from 20 hymenopteran species successfully, and the results of phylogenetic analysis revealed the presence of a new mutually exclusive exon in corbiculate bees. In addition, we found that duplication via staggered homologous recombination was responsible for the origin of the new exon.

  7. 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.

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

    PubMed Central

    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

    2016-01-01

    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

  9. Subcellular Targeting of p33ING1b by Phosphorylation-Dependent 14-3-3 Binding Regulates p21WAF1 Expression

    PubMed Central

    Gong, Wei; Russell, Michael; Suzuki, Keiko; Riabowol, Karl

    2006-01-01

    ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33ING1b splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33ING1b protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33ING1b increased levels of the p21Waf1 cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21Waf1 by p33ING1b, consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33ING1b by directing its subcellular localization. PMID:16581770

  10. 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

  11. Chemical Genetics of 14-3-3 Regulation and Role in Tumor Development

    DTIC Science & Technology

    2005-11-01

    from proteolysis (e.g. plant nitrate reductase) or from dephosphorylation (e.g. Raf and BAD). However, to date the most common mode of 14-3-3 function is...the general CRMI-mediated nuclear export I further studied the effects of haloprogin. This compound is a topical antifungal agent whose target is

  12. 14-3-3ζ and aPKC-ι synergistically facilitate epithelial-mesenchymal transition of cholangiocarcinoma via GSK-3β/snail signaling pathway

    PubMed Central

    He, Jun-chuang; Wang, Jian-ming; Schemmer, Peter; Ma, Chao-qun; Qian, Ya-wei; Yao, Wei; Zhang, Jian; Qi, Wei-peng; Fu, Yang; Feng, Wei; Yang, Tao

    2016-01-01

    Cholangiocarcinoma (CCA) invasion and metastasis are the primary causes of poor survival rates in patients. The epithelial-mesenchymal transition (EMT) is a crucial step in cancer invasion and metastasis. However, it is still unclear of the molecular mechanism. In this study, the expression of 14-3-3ζ and atypical protein kinase C-ι (aPKC-ι) was further detected in CCA tissues and cell lines. Meanwhile, we established the EMT model of CCA cells and investigated 14-3-3ζ and aPKC-ι co-regulatory effect on the EMT in vitro and in vivo. Further, we identified the downstream molecular glycogen synthase kinase 3 beta (GSK-3β)/Snail signalling pathway that contribute to regulating the EMT. Our data showed that the expression of 14-3-3ζ and aPKC-ι was synergistically increased in CCA tissues compared with adjacent noncancerous tissues and was intimately associated with differentiation and the tumour-node-metastasis (TNM) stage. Multivariate Cox regression analysis indicated that high 14-3-3ζ and aPKC-ι expression separately predicted a poor prognosis and were independent prognostic indicators in patients with CCA. The CO-IP experiment confirmed that the mutual binding relationship between 14-3-3ζ and aPKC-ι. Small interfering RNAs and siRNA rescue experiment demonstrated that 14-3-3ζ and aPKC-ι regulated each other. In addition, 14-3-3ζ and aPKC-ι pretreatment by si-RNA inhibit the phosphorylated GSK-3β and Snail expression during EMT. Meanwhile, silence of 14-3-3ζ or aPKC-ι suppressed CCA cells migration, metastasis and proliferation in vitro and in vivo. Our study demonstrates that 14-3-3ζ and aPKC-ι synergistically facilitate EMT of CCA via GSK-3β/Snail signalling pathway, and may be potential therapeutic target for CCA. PMID:27409422

  13. Controlled protein release from electrospun biodegradable fiber mesh composed of poly(epsilon-caprolactone) and poly(ethylene oxide).

    PubMed

    Kim, Taek Gyoung; Lee, Doo Sung; Park, Tae Gwan

    2007-06-29

    A blend mixture of poly(epsilon-caprolactone) (PCL) and poly(ethylene oxide) (PEO) was electrospun to produce fibrous meshes that could release a protein drug in a controlled manner. Various biodegradable polymers, such as poly(l-lactic acid) (PLLA), poly(epsilon-caprolactone) (PCL), and poly(d,l-lactic-co-glycolic acid) (PLGA) were dissolved, along with PEO and lysozyme, in a mixture of chloroform and dimethylsulfoxide (DMSO). The mixture was electrospun to produce lysozyme loaded fibrous meshes. Among the polymers, the PCL/PEO blend meshes showed good morphological stability upon incubation in the buffer solution, resulting in controlled release of lysozyme over an extended period with reduced initial bursts. With varying the PCL/PEO blending ratio, the release rate of lysozyme from the corresponding meshes could be readily modulated. The lysozyme release was facilitated by increasing the amount of PEO, indicating that entrapped lysozyme was mainly released out by controlled dissolution of PEO from the blend meshes. Lysozyme released from the electrospun fibers retained sufficient catalytic activity.

  14. Chemical Genetics of 14-3-3 Regulation and Role in Tumor Development

    DTIC Science & Technology

    2006-11-01

    antifungal chemical typically used in the treatment of fungal (Tinea) skin such as athlete’s foot, jock itch, ringworm and tinea versicolor...DeCaprio JA: Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site. Mol Cell Biol 1999, 19(6):4465- 4479. 12...epidermal growth factor receptor signaling pathways of the human prostate epithelial cell line M12. Oncogene 2004, 23(41):6881-6889. 15. Lopez-Girona

  15. 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.

  16. Direct interaction of Syk and Lyn protein tyrosine kinases in rat basophilic leukemia cells activated via type I Fc epsilon receptors.

    PubMed

    Amoui, M; Dráberová, L; Tolar, P; Dráber, P

    1997-01-01

    Activation of rat mast cells through the receptor with high affinity for IgE (Fc epsilonRI) requires a complex set of interactions involving transmembrane subunits of the Fc epsilonRI and two classes of nonreceptor protein tyrosine kinase (PTK). the Src family PTK p53/p56(lyn) (Lyn) and the Syk/ZAP-family PTK p72(syk) (Syk). Early activation events involve increased activity of Lyn and Syk kinases and their translocation into membrane domains containing aggregated Fc epsilonRI, but the molecular mechanisms responsible for these changes have remained largely unclear. To determine the role of Fc epsilonRI subunits in this process, we have analyzed Syk- and Lyn-associated proteins in activated rat basophilic leukemia (RBL) cells and their variants deficient in the expression of Fc epsilonRI beta or gamma subunits. Sepharose 4B gel chromatography of postnuclear supernatants from Nonidet-P40-solubilized antigen (Ag)- or pervanadate-activated RBL cells revealed extensive changes in the size of complexes formed by Lyn and Syk kinases and other cellular components. A fusion protein containing Src homology 2 (SH2) and SH3 domains of Lyn bound Syk from lysates of nonactivated RBL cells; an increased binding was observed when lysates from Ag- or pervanadate-activated cells were used. A similar amount of Syk was bound when lysates from pervanadate-activated variant cells deficient in the expression of Fc epsilonRI beta or gamma subunits were used, suggesting that Fc epsilonRI does not function as the only intermediate in the formation of the Syk-Lyn complexes. Further experiments have indicated that Syk-Lyn interactions occur in Ag-activated RBL cells under in vivo conditions and that these interactions could involve direct binding of the Lyn SH2 domain with phosphorylated tyrosine of Syk. The physical association of Lyn and Syk during mast-like cell activation supports the recently proposed functional cooperation of these two tyrosine kinases in Fc epsilonRI signaling.

  17. Internal Amino Acids Promote Gap1 Permease Ubiquitylation via TORC1/Npr1/14-3-3-Dependent Control of the Bul Arrestin-Like Adaptors

    PubMed Central

    Merhi, Ahmad

    2012-01-01

    Ubiquitylation of many plasma membrane proteins promotes their endocytosis followed by degradation in the lysosome. The yeast general amino acid permease, Gap1, is ubiquitylated and downregulated when a good nitrogen source like ammonium is provided to cells growing on a poor nitrogen source. This ubiquitylation requires the Rsp5 ubiquitin ligase and the redundant arrestin-like Bul1 and Bul2 adaptors. Previous studies have shown that Gap1 ubiquitylation involves the TORC1 kinase complex, which inhibits the Sit4 phosphatase. This causes inactivation of the protein kinase Npr1, which protects Gap1 against ubiquitylation. However, the mechanisms inducing Gap1 ubiquitylation after Npr1 inactivation remain unknown. We here show that on a poor nitrogen source, the Bul adaptors are phosphorylated in an Npr1-dependent manner and bound to 14-3-3 proteins that protect Gap1 against downregulation. After ammonium is added and converted to amino acids, the Bul proteins are dephosphorylated, dissociate from the 14-3-3 proteins, and undergo ubiquitylation. Furthermore, dephosphorylation of Bul requires the Sit4 phosphatase, which is essential to Gap1 downregulation. The data support the emerging concept that permease ubiquitylation results from activation of the arrestin-like adaptors of the Rsp5 ubiquitin ligase, this coinciding with their dephosphorylation, dissociation from the inhibitory 14-3-3 proteins, and ubiquitylation. PMID:22966204

  18. 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.

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

    PubMed

    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.

  20. Protein kinase C epsilon induces systolic cardiac failure marked by exhausted inotropic reserve and intact Frank-Starling mechanism.

    PubMed

    Montgomery, David E; Rundell, Veronica L M; Goldspink, Paul H; Urboniene, Dalia; Geenen, David L; de Tombe, Pieter P; Buttrick, Peter M

    2005-11-01

    Myofilament dysfunction is a common point of convergence for many forms of heart failure. Recently, we showed that cardiac overexpression of PKC epsilon initially depresses myofilament activity and then leads to a progression of changes characteristic of human heart failure. Here, we examined the effects of PKC epsilon on contractile reserve, Starling mechanism, and myofilament activation in this model of end-stage dilated cardiomyopathy. Pressure-volume loop analysis and echocardiography showed that the PKC epsilon mice have markedly compromised systolic function and increased end-diastolic volumes. Dobutamine challenge resulted in a small increase in contractility in PKC epsilon mice but failed to enhance cardiac output. The PKC epsilon mice showed a normal length-dependent tension development in skinned cardiac muscle preparations, although Frank-Starling mechanism appeared to be compromised in the intact animal. Simultaneous measurement of tension and ATPase demonstrated that the maximum tension and ATPase were markedly lower in the PKC epsilon mice at any length or Ca2+ concentration. However, the tension cost was also lower indicating less energy expenditure. We conclude 1) that prolonged overexpression of PKC epsilon ultimately leads to a dilated cardiomyopathy marked by exhausted contractile reserve, 2) that PKC epsilon does not compromise the Frank-Starling mechanism at the myofilament level, and 3) that the Starling curve excursion is limited by the inotropic state of the heart. These results reflect the significance of the primary myofilament contractilopathy induced by phosphorylation and imply a role for PKC epsilon-mediated phosphorylation in myofilament physiology and the pathophysiology of decompensated cardiac failure.

  1. MicroRNA-31 sensitizes human breast cells to apoptosis by direct targeting of protein kinase C epsilon (PKCepsilon).

    PubMed

    Körner, Cindy; Keklikoglou, Ioanna; Bender, Christian; Wörner, Angelika; Münstermann, Ewald; Wiemann, Stefan

    2013-03-22

    MicroRNAs post-transcriptionally regulate gene expression and thereby contribute to the modulation of numerous complex and disease-relevant cellular phenotypes, including cell proliferation, cell motility, apoptosis, and stress response. In breast cancer cell systems, miR-31 has been shown to inhibit cell migration, invasion, and metastasis. Here, we link enhanced expression of miR-31 to the inhibition of the oncogenic NF-κB pathway, thus supporting the tumor-suppressive function of this microRNA. We identified protein kinase C epsilon (PKCε encoded by the PRKCE gene) as a novel direct target of miR-31 and show that down-regulation of PKCε results in impaired NF-κB signaling, enhanced apoptosis, and increased sensitivity of MCF10A breast epithelial and MDA-MB-231 triple-negative breast cancer cells toward ionizing radiation as well as treatment with chemotherapeutics. Mechanistically, we attribute this sensitization to anti-cancer treatments to the PRKCE-mediated down-regulation of the anti-apoptotic factor BCL2. In clinical breast cancer samples, high BCL2 expression was associated with poor prognosis. Furthermore, we found an inverse correlation between miR-31 and BCL2 expression, highlighting the functional relevance of the indirect down-regulation of BCL2 via direct targeting of PRKCE by miR-31.

  2. Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation.

    PubMed

    Niebler, Stephan; Angele, Peter; Kujat, Richard; Bosserhoff, Anja K

    2015-01-01

    The transcription factor AP-2ε (activating enhancer-binding protein epsilon) is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2ε expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2ε mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angiopoietin-like 4 (Angptl4) strongly correlated with that of AP-2ε suggesting that hypoxia might represent an external regulator of AP-2ε expression in mammals. In order to show this, experiments directly targeting the activity of hypoxia-inducible factor-1 (HIF1), the complex mediating responses to oxygen deprivation, were performed. While the HIF1-activating compounds 2,2'-dipyridyl and desferrioxamine resulted in significantly enhanced mRNA concentration of AP-2ε, siRNA against HIF1α led to a significantly reduced expression rate of AP-2ε. Additionally, we detected a significant upregulation of the AP-2ε mRNA level after oxygen deprivation. In sum, these different experimental approaches revealed a novel role for the HIF1 complex in the regulation of the AP-2ε gene in cartilaginous cells and underlined the important role of hypoxia as an important external regulatory stimulus during chondrogenic differentiation modulating the expression of downstream transcription factors.

  3. Gabapentin Inhibits Protein Kinase C Epsilon Translocation in Cultured Sensory Neurons with Additive Effects When Coapplied with Paracetamol (Acetaminophen)

    PubMed Central

    2017-01-01

    Gabapentin is a well-established anticonvulsant drug which is also effective for the treatment of neuropathic pain. Although the exact mechanism leading to relief of allodynia and hyperalgesia caused by neuropathy is not known, the blocking effect of gabapentin on voltage-dependent calcium channels has been proposed to be involved. In order to further evaluate its analgesic mechanisms, we tested the efficacy of gabapentin on protein kinase C epsilon (PKCε) translocation in cultured peripheral neurons isolated from rat dorsal root ganglia (DRGs). We found that gabapentin significantly reduced PKCε translocation induced by the pronociceptive peptides bradykinin and prokineticin 2, involved in both inflammatory and chronic pain. We recently showed that paracetamol (acetaminophen), a very commonly used analgesic drug, also produces inhibition of PKCε. We tested the effect of the combined use of paracetamol and gabapentin, and we found that the inhibition of translocation adds up. Our study provides a novel mechanism of action for gabapentin in sensory neurons and suggests a mechanism of action for the combined use of paracetamol and gabapentin, which has recently been shown to be effective, with a cumulative behavior, in the control of postoperative pain in human patients. PMID:28299349

  4. Gabapentin Inhibits Protein Kinase C Epsilon Translocation in Cultured Sensory Neurons with Additive Effects When Coapplied with Paracetamol (Acetaminophen).

    PubMed

    Vellani, Vittorio; Giacomoni, Chiara

    2017-01-01

    Gabapentin is a well-established anticonvulsant drug which is also effective for the treatment of neuropathic pain. Although the exact mechanism leading to relief of allodynia and hyperalgesia caused by neuropathy is not known, the blocking effect of gabapentin on voltage-dependent calcium channels has been proposed to be involved. In order to further evaluate its analgesic mechanisms, we tested the efficacy of gabapentin on protein kinase C epsilon (PKCε) translocation in cultured peripheral neurons isolated from rat dorsal root ganglia (DRGs). We found that gabapentin significantly reduced PKCε translocation induced by the pronociceptive peptides bradykinin and prokineticin 2, involved in both inflammatory and chronic pain. We recently showed that paracetamol (acetaminophen), a very commonly used analgesic drug, also produces inhibition of PKCε. We tested the effect of the combined use of paracetamol and gabapentin, and we found that the inhibition of translocation adds up. Our study provides a novel mechanism of action for gabapentin in sensory neurons and suggests a mechanism of action for the combined use of paracetamol and gabapentin, which has recently been shown to be effective, with a cumulative behavior, in the control of postoperative pain in human patients.

  5. 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.

  6. The human cruciform-binding protein, CBP, is involved in DNA replication and associates in vivo with mammalian replication origins.

    PubMed

    Novac, Olivia; Alvarez, David; Pearson, Christopher E; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2002-03-29

    We previously identified and purified from human (HeLa) cells a 66-kDa cruciform-binding protein, CBP, with binding specificity for cruciform DNA regardless of its sequence. DNA cruciforms have been implicated in the regulation of initiation of DNA replication. CBP is a member of the 14-3-3 family of proteins, which are conserved regulatory molecules expressed in all eukaryotes. Here, the in vivo association of CBP/14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey replication origins ors8 and ors12, as assayed by a chromatin immunoprecipitation assay and quantitative PCR analysis. The association of the 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with these origins was found to be approximately 9-fold higher, compared with other portions of the genome, in logarithmically growing cells. In addition, the association of these isoforms with ors8 and ors12 was also analyzed as a function of the cell cycle. Higher binding of 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with ors8 and ors12 was found at the G(1)/S border, by comparison with other stages of the cell cycle. The CBP/14-3-3 cruciform binding activity was also found to be maximal at the G(1)/S boundary. The involvement of 14-3-3 in mammalian DNA replication was analyzed by studying the effect of anti-14-3-3beta, -epsilon, -gamma, and -zeta antibodies in the in vitro replication of p186, a plasmid containing the minimal replication origin of ors8. Anti-14-3-3epsilon, -gamma, and -zeta antibodies alone or in combination inhibited p186 replication by approximately 50-80%, while anti-14-3-3beta antibodies had a lesser effect ( approximately 25-50%). All of the antibodies tested were also able to interfere with CBP binding to cruciform DNA. The results indicate that CBP/14-3-3 is an origin-binding protein, acting at the initiation step of DNA replication by binding to cruciform-containing molecules, and dissociates after origin firing.

  7. Costunolide and dehydrocostuslactone combination treatment inhibit breast cancer by inducing cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 pathway.

    PubMed

    Peng, Zhangxiao; Wang, Yan; Fan, Jianhui; Lin, Xuejing; Liu, Chunying; Xu, Yang; Ji, Weidan; Yan, Chao; Su, Changqing

    2017-01-24

    Our previous studies demonstrated that volatile oil from saussurea lappa root (VOSL), rich in two natural sesquiterpene lactones, costunolide (Cos) and dehydrocostuslactone (Dehy), exerts better anti-breast cancer efficacy and lower side effects than Cos or Dehy alone in vivo, however, their anti-cancer molecular mechanisms were still unknown. In this study, we investigated the underlying mechanisms of Cos and Dehy combination treatment (CD) on breast cancer cells through proteomics technology coupled with Western blot validation. Ingenuity Pathways Analysis (IPA) results based on the differentially expressed proteins revealed that both VOSL and CD affect the 14-3-3-mediated signaling, c-Myc mediated apoptosis signaling and protein kinase A (PKA) signaling. Western blot coupled with cell cycle and apoptosis analysis validated the results of proteomics analysis. Cell cycle arrest and apoptosis were induced in a dose-dependent manner, and the expressions of p53 and p-14-3-3 were significantly up-regulated, whereas the expressions of c-Myc, p-AKT, p-BID were significantly down-regulated, furthermore, the ratio of BAX/BCL-2 were significantly increased in breast cancer cells after CD and VOSL treatment. The findings indicated that VOSL and CD could induce breast cancer cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 signaling pathways and may be novel effective candidates for breast cancer treatment.

  8. Accumulation and tolerance to cadmium heavy metal ions and induction of 14-3-3 gene expression in response to cadmium exposure in Coprinus atramentarius.

    PubMed

    Xie, Chengjian; Hu, Liujie; Yang, Yongzhu; Liao, Dunxiu; Yang, Xingyong

    2017-03-01

    Cadmium (Cd), one of the most toxic heavy-metal pollutants, has a strong and irreversible tendency to accumulate. Bioremediation is a promising technology to remedy and control heavy metal pollutants because of its low cost and ability to recycle heavy metals. Coprinus atramentarius is recognized as being able to accumulate heavy metal ions. In this work, C. atramentarius is cultivated on a solid medium containing Cd(2+) ions to analyze its ability to tolerate different concentrations of the heavy metal ion. It is found that the growth of C. atramentarius is not significantly inhibited when the concentration of Cd(2+) is less than 0.6mgL(-1). The accumulation capacity of C. atramentarius at different Cd(2+) concentrations also was determined. The results show that 76% of the Cd(2+) present can be accumulated even when the concentration of the Cd(2+) is 1mgL(-1). The different proteins of C. atramentarius exposed to Cd(2+) were further analyzed using gel electrophoresis. A 14-3-3 protein was identified and shown to be significantly up-regulated. In a further study, a full-length 14-3-3 gene was cloned containing a 759bp open reading frame encoding a polypeptide consisting of 252 amino acids and 3 introns. The gene expression work also showed that the 14-3-3 was significantly induced, and showed coordinated patterns of expression, with Cd(2+) exposure.

  9. Costunolide and dehydrocostuslactone combination treatment inhibit breast cancer by inducing cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 pathway

    PubMed Central

    Peng, Zhangxiao; Wang, Yan; Fan, Jianhui; Lin, Xuejing; Liu, Chunying; Xu, Yang; Ji, Weidan; Yan, Chao; Su, Changqing

    2017-01-01

    Our previous studies demonstrated that volatile oil from saussurea lappa root (VOSL), rich in two natural sesquiterpene lactones, costunolide (Cos) and dehydrocostuslactone (Dehy), exerts better anti-breast cancer efficacy and lower side effects than Cos or Dehy alone in vivo, however, their anti-cancer molecular mechanisms were still unknown. In this study, we investigated the underlying mechanisms of Cos and Dehy combination treatment (CD) on breast cancer cells through proteomics technology coupled with Western blot validation. Ingenuity Pathways Analysis (IPA) results based on the differentially expressed proteins revealed that both VOSL and CD affect the 14-3-3-mediated signaling, c-Myc mediated apoptosis signaling and protein kinase A (PKA) signaling. Western blot coupled with cell cycle and apoptosis analysis validated the results of proteomics analysis. Cell cycle arrest and apoptosis were induced in a dose-dependent manner, and the expressions of p53 and p-14-3-3 were significantly up-regulated, whereas the expressions of c-Myc, p-AKT, p-BID were significantly down-regulated, furthermore, the ratio of BAX/BCL-2 were significantly increased in breast cancer cells after CD and VOSL treatment. The findings indicated that VOSL and CD could induce breast cancer cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 signaling pathways and may be novel effective candidates for breast cancer treatment. PMID:28117370

  10. Search for Physiological Substrates of Protein Tyrosine Phosphatase Epsilon in Mammary Tumor Cell Lines

    DTIC Science & Technology

    2001-12-01

    BamHI/SmaI sites of pGEX2TK; the resulting plasmids were expressed in the BL-21 strain of E . coli bacteria . GST-PTPe fusion proteins were purified...attempted to overcome these difficulties by optimizing bacterial growth conditions. Several strains of E . coli bacteria were used to try and find one

  11. A Member of the 14-3-3 Gene Family in Brachypodium distachyon, BdGF14d, Confers Salt Tolerance in Transgenic Tobacco Plants

    PubMed Central

    He, Yuan; Zhang, Yang; Chen, Lihong; Wu, Chunlai; Luo, Qingchen; Zhang, Fan; Wei, Qiuhui; Li, Kexiu; Chang, Junli; Yang, Guangxiao; He, Guangyuan

    2017-01-01

    Plant 14-3-3 proteins are involved in diverse biological processes, but for the model monocotyledonous species, Brachypodium distachyon, their roles in abiotic stress tolerance are not well understood. In this study, a total of eight Bd14-3-3 genes were identified from B. distachyon and these were designated respectively as BdGF14a–BdGF14g. The qRT-PCR analyses of 3-month-old plants of B. distachyon showed that these genes were all expressed in the stems, leaves, and spikelets. By contrast, most of the plants had relatively lower transcriptional levels in their roots, except for the BdGF14g gene. The different expression profiles of the Bd14-3-3s under various stress treatments, and the diverse interaction patterns between Bd14-3-3s and BdAREB/ABFs, suggested that these gene products probably had a range of functions in the stress responses. The NaCl-induced Bd14-3-3 gene, BdGF14d, was selected for overexpression in tobacco. BdGF14d was found to be localized throughout the cell and it conferred enhanced tolerance to salt in the transgenic plants. Lowered contents of malondialdehyde, H2O2, and Na+, and lower relative electronic conductance (Rec%), yet greater activities of catalase and peroxidase, were observed in the overexpressing plants. Higher photosynthetic rate, transpiration rate, stomatal conductance, and water use efficiency were measured in the transgenic lines. Following abscisic acid (ABA) or NaCl treatment, stomatal aperture in leaves of the BdGF14d-overexpression plants was significantly lower than in leaves of the wild type (WT) controls. The stress-related marker genes involved in the ABA signaling pathway, the reactive oxygen species (ROS)-scavenging system, and the ion transporters were all up-regulated in the BdGF14d-overexpressing plants as compared with WT. Taken together, these results demonstrate that the Bd14-3-3 genes play important roles in abiotic stress tolerance. The ABA signaling pathway, the ROS-scavenging system, and ion

  12. Increased response to morphine in mice lacking protein kinase C epsilon

    PubMed Central

    Newton, P. M.; Kim, J. A.; McGeehan, A. J.; Paredes, J. P.; Chu, K.; Wallace, M. J.; Roberts, A. J.; Hodge, C. W.; Messing, R. O.

    2014-01-01

    The protein kinase C (PKC) family of serine–threonine kinases has been implicated in behavioral responses to opiates, but little is known about the individual PKC isozymes involved. Here, we show that mice lacking PKCε have increased sensitivity to the rewarding effects of morphine, revealed as the expression of place preference and intravenous self-administration at very low doses of morphine that do not evoke place preference or self-administration in wild-type mice. The PKCε null mice also show prolonged maintenance of morphine place preference in response to repeated testing when compared with wild-type mice. The supraspinal analgesic effects of morphine are enhanced in PKCε null mice, and the development of tolerance to the spinal analgesic effects of morphine is delayed. The density of μ-opioid receptors and their coupling to G-proteins are normal. These studies identify PKCε as a key regulator of opiate sensitivity in mice. PMID:16899053

  13. In-vivo administration of clozapine affects behaviour but does not reverse dendritic spine deficits in the 14-3-3ζ KO mouse model of schizophrenia-like disorders.

    PubMed

    Jaehne, Emily J; Ramshaw, Hayley; Xu, Xiangjun; Saleh, Eiman; Clark, Scott R; Schubert, Klaus Oliver; Lopez, Angel; Schwarz, Quenten; Baune, Bernhard T

    2015-11-01

    Clozapine is an atypical antipsychotic drug used in the treatment of schizophrenia, which has been shown to reverse behavioural and dendritic spine deficits in mice. It has recently been shown that deficiency of 14-3-3ζ has an association with schizophrenia, and that a mouse model lacking this protein displays several schizophrenia-like behavioural deficits. To test the effect of clozapine in this mouse model, 14-3-3ζ KO mice were administered clozapine (5mg/kg) for two weeks prior to being analysed in a test battery of cognition, anxiety, and despair (depression-like) behaviours. Following behavioural testing brain samples were collected for analysis of specific anatomical defects and dendritic spine formation. We found that clozapine reduced despair behaviour of 14-3-3ζ KO mice in the forced swim test (FST) and altered the behaviour of wild types and 14-3-3ζ KO mice in the Y-maze task. In contrast, clozapine had no effects on hippocampal laminar defects or decreased dendritic spine density observed in 14-3-3ζ KO mice. Our results suggest that clozapine may have beneficial effects on clinical behaviours associated with deficiencies in the 14-3-3ζ molecular pathway, despite having no effects on morphological defects. These findings may provide mechanistic insight to the action of this drug.

  14. Protein kinase C epsilon activates lens mitochondrial cytochrome c oxidase subunit IV during hypoxia.

    PubMed

    Barnett, Michael; Lin, Dingbo; Akoyev, Vladimir; Willard, Lloyd; Takemoto, Dolores

    2008-02-01

    Protein kinase C (PKC) isoforms have been identified as major cellular signaling proteins that act directly in response to oxidation conditions. In retina and lens two isoforms of PKC respond to changes in oxidative stress, PKCgamma and PKCepsilon, while only PKCepsilon is found in heart. In heart the PKCepsilon acts on connexin 43 to protect from hypoxia. The presence of both isoforms in the lens led to this study to determine if lens PKCepsilon had unique targets. Both lens epithelial cells in culture and whole mouse lens were examined using PKC isoform-specific enzyme activity assays, co-immunoprecipitation, confocal microscopy, immunoblots, and light and electron microscopy. PKCepsilon was found in lens epithelium and cortex but not in the nucleus of mouse lens. The PKCepsilon isoform was activated in both epithelium and whole lens by 5% oxygen when compared to activity at 21% oxygen. In hypoxic conditions (5% oxygen) the PKCepsilon co-immunoprecipitated with the mitochondrial cytochrome c oxidase IV subunit (CytCOx). Concomitant with this the CytCOx enzyme activity was elevated and increased co-localization of CytCOx with PCKvarepsilon was observed using immunolabeling and confocal microscopy. In contrast, no hypoxia-induced activation of CytCOx was observed in lenses from the PKCepsilon knockout mice. Lens from 6-week-old PKCepsilon knockout mice had a disorganized bow region which was filled with vacuoles indicating a possible loss of mitochondria but the size of the lens was not altered. Electron microscopy demonstrated that the nuclei of the PCKepsilon knockout mice were abnormal in shape. Thus, PKCepsilon is found to be activated by hypoxia and this results in the activation of the mitochondrial protein CytCOx. This could protect the lens from mitochondrial damage under the naturally hypoxic conditions observed in this tissue. Lens oxygen levels must remain low. Elevation of oxygen which occurs during vitreal detachment or liquification is associated with

  15. 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

  16. BIS targeting induces cellular senescence through the regulation of 14-3-3 zeta/STAT3/SKP2/p27 in glioblastoma cells.

    PubMed

    Lee, J-J; Lee, J-S; Cui, M N; Yun, H H; Kim, H Y; Lee, S H; Lee, J-H

    2014-11-20

    Cellular senescence is an important mechanism for preventing tumor progression. The elevated expression of Bcl-2-interacting cell death suppressor (BIS), an anti-apoptotic and anti-stress protein, often correlates with poor prognosis in several cancers including glioblastoma; however, the role of BIS in the regulation of senescence has not been well defined. Here, we describe for the first time that the depletion of BIS induces G1 arrest and cellular senescence through the accumulation of p27 that is independent of p53, p21 or p16. The increase in p27 expression in BIS-depleted cells was attributable to an impairment of the ubiquitin-mediated degradation of p27, which was caused by a decrease in S-phase kinase-associated protein 2 (SKP2) at the transcriptional level. As an underlying molecular mechanism, we demonstrate that the loss of activity of signal transducer and activator of transcription 3 (STAT3) was specifically linked to the suppression of SKP2 expression. Despite a reduction in phospho-STAT3 levels, total STAT3 levels were unexpectedly increased by BIS depletion, specifically in the insoluble fraction. Our results show that 14-3-3ζ expression is decreased by BIS knockdown and that 14-3-3ζ depletion per se significantly induced senescence phenotypes. In addition, the ectopic expression of 14-3-3ζ blocked senescence caused by BIS depletion, which was paralleled with a decrease in insoluble STAT3 in A172 glioblastoma cells. These findings indicate that the impairment of the protein quality control conferred by BIS and/or 14-3-3ζ is critical for BIS depletion-induced senescence. Moreover, BIS knockdown also induced senescence along with an accumulation of total STAT3 and p27 in several different cell types as well as embryonic fibroblasts derived from Bis-knock out mice with/without variations in 14-3-3ζ levels. Therefore, our findings suggest that a downregulation of BIS expression could serve as a potential strategy for restricting tumor progression

  17. 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

  18. Defective erythroid differentiation in miR-451 mutant mice mediated by 14-3-3ζ

    PubMed Central

    Patrick, David M.; Zhang, Cheng C.; Tao, Ye; Yao, Huiyu; Qi, Xiaoxia; Schwartz, Robert J.; Jun-Shen Huang, Lily; Olson, Eric N.

    2010-01-01

    Erythrocyte formation occurs throughout life in response to cytokine signaling. We show that microRNA-451 (miR-451) regulates erythropoiesis in vivo. Mice lacking miR-451 display a reduction in hematrocrit, an erythroid differentiation defect, and ineffective erythropoiesis in response to oxidative stress. 14-3-3ζ, an intracellular regulator of cytokine signaling that is repressed by miR-451, is up-regulated in miR-451−/− erythroblasts, and inhibition of 14-3-3ζ rescues their differentiation defect. These findings reveal an essential role of 14-3-3ζ as a mediator of the proerythroid differentiation actions of miR-451, and highlight the therapeutic potential of miR-451 inhibitors. PMID:20679397

  19. Metabolic-Stress-Induced Rearrangement of the 14-3-3ζ Interactome Promotes Autophagy via a ULK1- and AMPK-Regulated 14-3-3ζ Interaction with Phosphorylated Atg9

    PubMed Central

    Weerasekara, Vajira K.; Panek, David J.; Broadbent, David G.; Mortenson, Jeffrey B.; Mathis, Andrew D.; Logan, Gideon N.; Prince, John T.; Thomson, David M.; Thompson, J. Will

    2014-01-01

    14-3-3ζ promotes cell survival via dynamic interactions with a vast network of binding partners, many of which are involved in stress regulation. We show here that hypoxia (low glucose and oxygen) triggers a rearrangement of the 14-3-3ζ interactome to favor an interaction with the core autophagy regulator Atg9A. Our data suggest that the localization of mammalian Atg9A to autophagosomes requires phosphorylation on the C terminus of Atg9A at S761, which creates a 14-3-3ζ docking site. Under basal conditions, this phosphorylation is maintained at a low level and is dependent on both ULK1 and AMPK. However, upon induction of hypoxic stress, activated AMPK bypasses the requirement for ULK1 and mediates S761 phosphorylation directly, resulting in an increase in 14-3-3ζ interactions, recruitment of Atg9A to LC3-positive autophagosomes, and enhanced autophagosome production. These data suggest a novel mechanism whereby the level of autophagy induction can be modulated by AMPK/ULK1-mediated phosphorylation of mammalian Atg9A. PMID:25266655

  20. 14-3-3theta Protects against Neurotoxicity in a Cellular Parkinson's Disease Model through Inhibition of the Apoptotic Factor Bax

    PubMed Central

    Slone, Sunny R.; Lesort, Mathieu; Yacoubian, Talene A.

    2011-01-01

    Disruption of 14-3-3 function by alpha-synuclein has been implicated in Parkinson's disease. As 14-3-3s are important regulators of cell death pathways, disruption of 14-3-3s could result in the release of pro-apoptotic factors, such as Bax. We have previously shown that overexpression of 14-3-3θ reduces cell loss in response to rotenone and MPP+ in dopaminergic cell culture and reduces cell loss in transgenic C. elegans that overexpress alpha-synuclein. In this study, we investigate the mechanism for 14-3-3θ's neuroprotection against rotenone toxicity. While 14-3-3s can inhibit many pro-apoptotic factors, we demonstrate that inhibition of one factor in particular, Bax, is important to 14-3-3s' protection against rotenone toxicity in dopaminergic cells. We found that 14-3-3θ overexpression reduced Bax activation and downstream signaling events, including cytochrome C release and caspase 3 activation. Pharmacological inhibition or shRNA knockdown of Bax provided protection against rotenone, comparable to 14-3-3θ's neuroprotective effects. A 14-3-3θ mutant incapable of binding Bax failed to protect against rotenone. These data suggest that 14-3-3θ's neuroprotective effects against rotenone are at least partially mediated by Bax inhibition and point to a potential therapeutic role of 14-3-3s in Parkinson's disease. PMID:21799745

  1. Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization ▿ †

    PubMed Central

    Robens , Jeffrey M.; Yeow-Fong, Lee; Ng, Elsa; Hall, Christine; Manser, Ed

    2010-01-01

    Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia. PMID:19933840

  2. Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon.

    PubMed Central

    Schaap, D; van der Wal, J; van Blitterswijk, W J; van der Bend, R L; Ploegh, H L

    1993-01-01

    In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor. Images Figure 2 Figure 3 Figure 4 PMID:7679574

  3. Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14-3-3 isoforms to suppress effector-triggered immunity.

    PubMed

    Teper, Doron; Salomon, Dor; Sunitha, Sukumaran; Kim, Jung-Gun; Mudgett, Mary Beth; Sessa, Guido

    2014-01-01

    Effector-triggered immunity (ETI) to host-adapted pathogens is associated with rapid cell death at the infection site. The plant-pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI-associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI-associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein-protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14-3-3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14-3-3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI-associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity-associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.

  4. Selective 14-3-3γ induction quenches p-β-catenin Ser37/Bax-enhanced cell death in cerebral cortical neurons during ischemia

    PubMed Central

    Lai, X J; Ye, S Q; Zheng, L; Li, L; Liu, Q R; Yu, S B; Pang, Y; Jin, S; Li, Q; Yu, A C H; Chen, X Q

    2014-01-01

    Ischemia-induced cell death is a major cause of disability or death after stroke. Identifying the key intrinsic protective mechanisms induced by ischemia is critical for the development of effective stroke treatment. Here, we reported that 14-3-3γ was a selective ischemia-inducible survival factor in cerebral cortical neurons reducing cell death by downregulating Bax depend direct 14-3-3γ/p-β-catenin Ser37 interactions in the nucleus. 14-3-3γ, but not other 14-3-3 isoforms, was upregulated in primary cerebral cortical neurons upon oxygen–glucose deprivation (OGD) as measured by quantitative PCR, western blot and fluorescent immunostaining. The selective induction of 14-3-3γ in cortical neurons by OGD was verified by the in vivo ischemic stroke model. Knocking down 14-3-3γ alone or inhibiting 14-3-3/client interactions was sufficient to induce cell death in normal cultured neurons and exacerbate OGD-induced neuronal death. Ectopic overexpression of 14-3-3γ significantly reduced OGD-induced cell death in cultured neurons. Co-immunoprecipitation and fluorescence resonance energy transfer demonstrated that endogenous 14-3-3γ bound directly to more p-β-catenin Ser37 but not p-Bad, p-Ask-1, p-p53 and Bax. During OGD, p-β-catenin Ser37 but not p-β-catenin Ser45 was increased prominently, which correlated with Bax elevation in cortical neurons. OGD promoted the entry of 14-3-3γ into the nuclei, in correlation with the increase of nuclear p-β-catenin Ser37 in neurons. Overexpression of 14-3-3γ significantly reduced Bax expression, whereas knockdown of 14-3-3γ increased Bax in cortical neurons. Abolishing β-catenin phosphorylation at Ser37 (S37A) significantly reduced Bax and cell death in neurons upon OGD. Finally, 14-3-3γ overexpression completely suppressed β-catenin-enhanced Bax and cell death in neurons upon OGD. Based on these data, we propose that the 14-3-3γ/p-β-catenin Ser37/Bax axis determines cell survival or death of neurons during ischemia

  5. Differential 14-3-3 sigma DNA methylation and expression in c-myc- and activated H-ras-transformed cells under r- and K-selection.

    PubMed

    Sato, Hiroyuki; Nakamura, Yukari; Motokura, Toru

    2006-05-08

    We cloned rat 14-3-3 sigma, a mediator of p53 tumor suppressor, as a target of K-selection. 14-3-3 sigma expression is suppressed with DNA methylation in breast cancers while its overexpression with hypomethylation is frequent in pancreatic cancers. These opposite findings were recapitulated through r- and K-selection of transformed rat embryo fibroblasts. 14-3-3 sigma expression was suppressed with DNA methylation after r-selection and the gene was overexpressed and demethylated in K-selected cells. 5-aza-2'-deoxycytidine recovered 14-3-3 sigma expression in r-selected cells. The presence of heterogeneous methylation patterns and expression levels before selection suggests that different 14-3-3 sigma expression levels play a role as a prerequisite for selection and clonal evolution.

  6. CD3-epsilon overexpressed in prothymocytes acts as an oncogene.

    PubMed Central

    Wang, B.; She, J.; Salio, M.; Allen, D.; Lacy, E.; Lonberg, N.; Terhorst, C.

    1997-01-01

    BACKGROUND: Upon engagement of the T cell receptor for antigen, its associated CD3 proteins recruit signal transduction molecules, which in turn regulate T lymphocyte proliferation, apoptosis, and thymocyte development. Because some signal transducing molecules recruited by CD3-epsilon, i.e., p56lck and p59fyn, are oncogenic and since we previously found that overexpression of CD3-epsilon transgenes causes a block in T lymphocyte and NK cell development, we tested the hypothesis that aberrant CD3-epsilon signaling leads both to abnormal T lymphocyte death and lymphomagenesis. MATERIALS AND METHODS: Ten independently derived transgenic mouse lines were generated with four different genomic CD3-epsilon constructs. Mice either homozygous or hemizygous for each transgene were analyzed for an arrest in T lymphocyte development and for the occurrence of T cell lymphomas. RESULTS: Aggressive clonal T cell lymphomas developed at very high frequencies in seven mouse lines with intermediate levels of copies of CD3-epsilon derived transgenes. However, these lymphomas were not found when high copy numbers of CD3-epsilon transgenes caused a complete block in early thymic development or when a transgene was used in which the exons coding for the CD3-epsilon protein were deleted. Analyses of a series of double mutant mice, tgCD3-epsilon x RAG-2null, indicated that lymphomagenesis was initiated in lineage-committed prothymocytes, i.e., before rearrangement of the T cell receptor genes. In addition, the transgene coding for the CD3-epsilon cytoplasmic domain and its transmembrane region induced a T cell differentiation signal in premalignant tgCD3-epsilon x RAG-2null mice. CONCLUSION: The nonenzymatic CD3-epsilon protein acted as a potent oncogene when overexpressed early in T lymphocyte development. Lymphomagenesis was dependent on signal transduction events initiated by the cytoplasmic domain of CD3-epsilon. Images FIG. 2 FIG. 4 FIG. 5 PMID:9132282

  7. 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.

  8. 14-3-3ζ deficient mice in the BALB/c background display behavioural and anatomical defects associated with neurodevelopmental disorders.

    PubMed

    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; van den Buuse, Maarten; Lopez, Angel F; Ramshaw, Hayley S; Schwarz, Quenten

    2015-07-24

    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.

  9. 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

  10. Quantitative assessment of the impact of the gut microbiota on lysine epsilon-acetylation of host proteins using gnotobiotic mice.

    PubMed

    Simon, Gabriel M; Cheng, Jiye; Gordon, Jeffrey I

    2012-07-10

    The gut microbiota influences numerous aspects of human biology. One facet that has not been thoroughly explored is its impact on the host proteome. We hypothesized that the microbiota may produce certain of its effects through covalent modification of host proteins. We focused on protein lysine ε-acetylation because of its recently discovered roles in regulation of cell metabolism, and the potential for products of microbial fermentation to interact with the lysine acetylation machinery of host cells. Germ-free mice, fed a (15)N-labeled diet for two generations, were colonized as adults with a microbiota harvested from conventionally raised mouse donors. Using high-resolution mass spectrometry, we quantified 3,891 liver and proximal colonic proteins, 558 of which contained 1,602 sites of lysine acetylation, 43% not previously described. Multiple proteins from multiple subcellular compartments underwent microbiota-associated increases in their levels of lysine acetylation at one or more residues, in one or both tissues. Acetylated proteins were enriched in functions related to energy production, respiration, and primary metabolism. A number of the acetylation events affect lysine residues at or near the active sites of enzymes, whereas others occur at locations that may affect other facets of protein function. One of these modifications, affecting Lys292 in mouse α-1-antitrypsin, was detected in the corresponding lysine of the human serum protein. Methods described in this report can be applied to other co- or posttranslational modifications, and add quantitation of protein expression and covalent modification to the arsenal of techniques for characterizing the dynamic, important interactions between gut symbionts and their hosts.

  11. Inhibition of Specific NF-κB Activity Contributes to the Tumor Suppressor Function of 14-3-3σ in Breast Cancer

    PubMed Central

    Inglés-Esteve, Julia; Morales, Mònica; Dalmases, Alba; Garcia-Carbonell, Ricard; Jené-Sanz, Alba; López-Bigas, Núria; Iglesias, Mar; Ruiz-Herguido, Cristina; Rovira, Ana; Rojo, Federico; Albanell, Joan; Gomis, Roger R.

    2012-01-01

    14-3-3σ is frequently lost in human breast cancers by genetic deletion or promoter methylation. We have now investigated the involvement of 14-3-3σ in the termination of NF-κB signal in mammary cells and its putative role in cancer relapse and metastasis. Our results show that 14-3-3σ regulates nuclear export of p65-NF-κB following chronic TNFα stimulation. Restoration of 14-3-3σ in breast cancer cells reduces migration capacity and metastatic abilities in vivo. By microarray analysis, we have identified a genetic signature that responds to TNFα in a 14-3-3σ-dependent manner and significantly associates with different breast and other types of cancer. By interrogating public databases, we have found that over-expression of this signature correlates with poor relapse-free survival in breast cancer patients. Finally, screening of 96 human breast tumors showed that NF-κB activation strictly correlates with the absence of 14-3-3σ and it is significantly associated with worse prognosis in the multivariate analysis. Our findings identify a genetic signature that is important for breast cancer prognosis and for future personalized treatments based on NF-κB targeting. PMID:22675457

  12. High frequency of hypermethylation at the 14-3-3 σ locus leads to gene silencing in breast cancer

    PubMed Central

    Ferguson, Anne T.; Evron, Ella; Umbricht, Christopher B.; Pandita, Tej K.; Chan, Timothy A.; Hermeking, Heiko; Marks, Jeffrey R.; Lambers, Anouk R.; Futreal, P. Andrew; Stampfer, Martha R.; Sukumar, Saraswati

    2000-01-01

    Expression of 14-3-3 σ (σ) is induced in response to DNA damage, and causes cells to arrest in G2. By SAGE (serial analysis of gene expression) analysis, we identified σ as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, σ mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at σ such as loss of heterozygosity were rare (1/20 informative cases), and no mutations were detected (0/34). On the other hand, hypermethylation of CpG islands in the σ gene was detected in 91% (75/82) of breast tumors and was associated with lack of gene expression. Hypermethylation of σ is functionally important, because treatment of σ-non-expressing breast cancer cell lines with the drug 5-aza-2′-deoxycytidine resulted in demethylation of the gene and synthesis of σ mRNA. Breast cancer cells lacking σ expression showed increased number of chromosomal breaks and gaps when exposed to γ-irradiation. Therefore, it is possible that loss of σ expression contributes to malignant transformation by impairing the G2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of σ expression are the most consistent molecular alterations in breast cancer identified so far. PMID:10811911

  13. Deficiency of CCAAT/Enhancer Binding Protein-Epsilon Reduces Atherosclerotic Lesions in LDLR−/− Mice

    PubMed Central

    Okamoto, Ryoko; Gery, Sigal; Gombart, Adrian F.; Wang, Xuping; Castellani, Lawrence W.; Akagi, Tadayuki; Chen, Shuang; Arditi, Moshe; Ho, Quoc; Lusis, Aldons J.; Li, Quanlin; Koeffler, H. Phillip

    2014-01-01

    The CCAAT/enhancer binding proteins (C/EBPs) are transcription factors involved in hematopoietic cell development and induction of several inflammatory mediators. C/EBPε is expressed only in myeloid cells including monocytes/macrophages. Atherosclerosis is an inflammatory disorder of the vascular wall and circulating immune cells such as monocytes/macrophages. Mice deficient in the low density lipoprotein (LDL) receptor (Ldlr−/−) fed on a high cholesterol diet (HCD) show elevated blood cholesterol levels and are widely used as models to study human atherosclerosis. In this study, we generated Ldlr and Cebpe double-knockout (llee) mice and compared their atherogenic phenotypes to Ldlr single deficient (llEE) mice after HCD. Macrophages from llee mice have reduced lipid uptake by foam cells and impaired phagokinetic motility in vitro compared to macrophages from llEE mice. Also, compared to llEE mice, llee mice have alterations of lipid metabolism, and reduced atheroma and obesity, particularly the males. Peritoneal macrophages of llee male mice have reduced mRNA expression of FABP4, a fatty acid binding protein implicated in atherosclerosis. Overall, our study suggests that the myeloid specific factor C/EBPε is involved in systemic lipid metabolism and that silencing of C/EBPε could decrease the development of atherosclerosis. PMID:24489659

  14. Protein Kinase C isoform epsilon (ε) negatively regulates ADP-induced calcium mobilization and thromboxane generation in platelets

    PubMed Central

    Bynagari-Settipalli, Yamini S; Lakhani, Parth; Jin, Jianguo; Bhavaraju, Kamala; Rico, Mario C.; Kim, Soochong; Woulfe, Donna; Kunapuli, Satya P

    2012-01-01

    Objective Members of Protein Kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. In this study we investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. Methods and Results A pan-PKC inhibitor GF109203X potentiated ADP-induced cPLA2 phosphorylation and thromboxane generation, as well as ERK activation and intracellular calcium (Ca2+i) mobilization, two signaling molecules, upstream of cPLA2 activation. Thus, PKCs inhibit cPLA2 activation by inhibiting ERK and Ca2+i mobilization. Since, the inhibitor of Classical PKC isoforms, GO-6976 did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP- induced thromboxane generation, calcium mobilization and ERK phosphorylation were potentiated in PKCε null murine platelets compared to platelets from wild type (WT) littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKCε KO and WT was similar, suggesting that PKCε does not affect ADP-induced aggregation directly. PKCε knockout mice exhibited shorter times to occlusion in FeCl3-induced arterial injury model and shorter bleeding times in tail bleeding experiments. Conclusion We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis. PMID:22362759

  15. 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.

  16. 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

  17. Differential protein levels and post-translational modifications in spinal cord injury of the rat.

    PubMed

    Afjehi-Sadat, Leila; Brejnikow, Mika; Kang, Sung Ung; Vishwanath, Vinay; Walder, Nadja; Herkner, Kurt; Redl, Heinz; Lubec, Gert

    2010-03-05

    Although changes in protein expression in spinal cord injury (SCI) would be of pivotal interest, information so far is limited. It was therefore the aim of the study to determine protein levels and post-translational modifications in the early phase following SCI in the rat. SCI was induced in Sprague-Dawley rats and sham operated rats served as controls. A gel-based proteomic approach using two-dimensional gel electrophoresis followed by quantification with specific software and subsequent identification of differentially expressed proteins by nano-ESI-LC-MS/MS was applied. Proteins of several pathways and cascades were dysregulated in SCI: 14-3-3 epsilon protein, dynein light chain 1, and tubulin beta-5 chain showed higher levels in SCI, whereas adenylyl cyclase associated protein 1, dihydropyrimidinase-related protein 2, F-actin capping protein subunit beta, glyceraldehyde-3-phosphate dehydrogenase, stress-induced phosphoprotein 1 and transthyretin showed lower levels in the injured tissue. Post-translational modifications indicated free oxygen radical attack on proteins in SCI. The occurrence of stress is indicated by deranged stress-induced phosphoprotein 1 and signaling abnormalities are reflected by adenylyl cyclase-associated protein 1 and 14-3-3 epsilon protein. The findings propose the involvement of the corresponding cascades and challenge further work into aberrant signaling and oxidative stress in SCI, which may form the basis for experimental intervention for spinal cord trauma.

  18. Evidence against a Role for the JIL-1 Kinase in H3S28 Phosphorylation and 14-3-3 Recruitment to Active Genes in Drosophila

    PubMed Central

    Li, Yeran; Cai, Weili; Bao, Xiaomin; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

    2013-01-01

    JIL-1 is the major kinase controlling phosphorylation of histone H3S10 and has been demonstrated to function to counteract heterochromatization and gene silencing. However, an alternative model has been proposed in which JIL-1 is required for transcription to occur, additionally phosphorylates H3S28, and recruits 14-3-3 to active genes. Since these findings are incompatible with our previous demonstration that there are robust levels of transcription in the complete absence of JIL-1 and that JIL-1 is not present at developmental or heat shock-induced polytene chromosome puffs, we have reexamined JIL-1’s possible role in H3S28 phosphorylation and 14-3-3 recruitment. Using two different H3S28ph antibodies we show by immunocytochemistry and immunoblotting that in Drosophila the H3S28ph mark is not present at detectable levels above background on polytene chromosomes at interphase but only on chromosomes at pro-, meta-, and anaphase during cell division in S2 cells and third instar larval neuroblasts. Moreover, this mitotic H3S28ph signal is also present in a JIL-1 null mutant background at undiminished levels suggesting that JIL-1 is not the mitotic H3S28ph kinase. We also demonstrate that H3S28ph is not enriched at heat shock puffs. Using two different pan-specific 14-3-3 antibodies as well as an enhancer trap 14-3-3ε-GFP line we show that 14-3-3, while present in salivary gland nuclei, does not localize to chromosomes but only to the nuclear matrix surrounding the chromosomes. In our hands 14-3-3 is not recruited to developmental or heat shock puffs. Furthermore, using a lacO repeat tethering system to target LacI-JIL-1 to ectopic sites on polytene chromosomes we show that only H3S10ph is present and upregulated at such sites, not H3S28ph or 14-3-3. Thus, our results argue strongly against a model where JIL-1 is required for H3S28 phosphorylation and 14-3-3 recruitment at active genes. PMID:23638096

  19. A 14-3-3γ dimer-based scaffold bridges CtBP1-S/BARS to PI(4)KIIIβ to regulate post-Golgi carrier formation.

    PubMed

    Valente, Carmen; Turacchio, Gabriele; Mariggiò, Stefania; Pagliuso, Alessandro; Gaibisso, Renato; Di Tullio, Giuseppe; Santoro, Michele; Formiggini, Fabio; Spanò, Stefania; Piccini, Daniele; Polishchuk, Roman S; Colanzi, Antonino; Luini, Alberto; Corda, Daniela

    2012-02-26

    Large pleiomorphic carriers leave the Golgi complex for the plasma membrane by en bloc extrusion of specialized tubular domains, which then undergo fission. Several components of the underlying molecular machinery have been identified, including those involved in the budding/initiation of tubular carrier precursors (for example, the phosphoinositide kinase PI(4)KIIIβ, the GTPase ARF, and FAPP2), and in the fission of these precursors (for example, PKD, CtBP1-S/BARS). However, how these proteins interact to bring about carrier formation is poorly understood. Here, we describe a protein complex that mediates carrier formation and contains budding and fission molecules, as well as other molecules, such as the adaptor protein 14-3-3γ. Specifically, we show that 14-3-3γ dimers bridge CtBP1-S/BARS with PI(4)KIIIβ, and that the resulting complex is stabilized by phosphorylation by PKD and PAK. Disrupting the association of these proteins inhibits the fission of elongating carrier precursors, indicating that this complex couples the carrier budding and fission processes.

  20. Gene expressions levels of 14-3-3a, NKCCla, APO-14, and Na(+)-K(+)-ATPaseβ in gill tissue of Mugil cephalus acclimated to low salinity.

    PubMed

    Li, L; Jiang, M; Shen, X Q

    2017-02-16

    Fishes adapt to salinity changes primarily through osmotic pressure regulation, a process often associated with several genes, including 14-3-3a, NKCCla, APO-14, and Na(+)-K(+)-ATPaseβ. The present study investigated the differential expression of genes 14-3-3a, NKCCla, APO-14, and Na(+)-K(+)-ATPaseβ in the gill tissue of Mugil cephalus acclimated to low salinity. Susceptibility relationships between the four gene expressions levels and salinity were detected and analyzed using polymerase chain reaction-restriction fragment length polymorphism. Homology analysis results indicated significant differences in the correlation between gene expression and salinity. Under low-salt conditions, expression levels for genes Na(+)-K(+)-ATPaseβ and NKCC1a were significantly elevated (P < 0.05), whereas those of genes 14-3-3a and APO-14 were significantly reduced (P < 0.05). Thus, when compared to 14-3-3a and APO-14, Na(+)-K(+)-ATPaseβ, and NKCC1a may be better suited to promoting the development of osmotic-regulation mechanisms and increased resistance to environmental stress under low-salt conditions. Furthermore, Na(+)-K(+)-ATPaseβ and NKCC1a were identified as suitable potential molecular biomarkers for regulating and controlling genes in low-salinity aquatic environments.

  1. 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.

  2. 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

  3. 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

  4. Measurement of /var epsilon/'//var epsilon/ at Fermilab

    SciTech Connect

    Hsiung, Yee B.

    1988-10-01

    The current status of the measurement of ''direct'' CP violation parameters /var epsilon/'//var epsilon/ in the Fermilab experiment E731 is reviewed. Preliminary results on upper limit for the decays K/sub L/ ..-->.. ..pi../sup 0/e/sup +/e/sup /minus// and ..pi../sup 0/ ..-->.. e/sup +/e/sup /minus// (from 20% of the data taken in 1987-88) are also reported. 9 refs., 9 figs., 1 tab.

  5. 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

  6. 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

  7. Quantitative proteomic analysis of protein complexes: concurrent identification of interactors and their state of phosphorylation.

    PubMed

    Pflieger, Delphine; Jünger, Martin A; Müller, Markus; Rinner, Oliver; Lee, Hookeun; Gehrig, Peter M; Gstaiger, Matthias; Aebersold, Ruedi

    2008-02-01

    Protein complexes have largely been studied by immunoaffinity purification and (mass spectrometric) analysis. Although this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications, and detecting quantitative changes in complex composition or state of modification of complex components. We have developed a protocol that enables us to determine, in a single LC-MALDI-TOF/TOF analysis, the true protein constituents of a complex, to detect changes in the complex composition, and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of fourplex iTRAQ (isobaric tags for relative and absolute quantification) isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate homologue Chico. These experiments allowed us to identify 14-3-3epsilon, 14-3-3zeta, and the insulin receptor as specific Chico interactors. In a further experiment, we compared the immunopurified materials obtained from tagged Chico-expressing cells that were either treated with insulin or left unstimulated. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins.

  8. Proteomic pathway analysis of the hippocampus in schizophrenia and bipolar affective disorder implicates 14-3-3 signaling, aryl hydrocarbon receptor signaling, and glucose metabolism: potential roles in GABAergic interneuron pathology.

    PubMed

    Schubert, Klaus Oliver; Föcking, Melanie; Cotter, David R

    2015-09-01

    Neuropathological changes of the hippocampus have been associated with psychotic disorders such as schizophrenia and bipolar disorder. Recent work has particularly implicated hippocampal GABAergic interneurons in the pathophysiology of these diseases. However, the molecular mechanisms underlying structural and cellular hippocampal pathology remain poorly understood. We used data from comprehensive difference-in-gel electrophoresis (2-D DIGE) investigations of postmortem human hippocampus of people with schizophrenia and bipolar disorder, covering the acidic (isoelectric point (pI) between pH4 and 7) and, separately, the basic (pI between pH6 and 11) sub-proteome, for Ingenuity Pathway Analysis (IPA) of implicated protein networks and pathways. Comparing disease and control cases, we identified 58 unique differentially expressed proteins in schizophrenia, and 70 differentially expressed proteins in bipolar disorder, using mass spectrometry. IPA implicated, most prominently, 14-3-3 and aryl hydrocarbon receptor signaling in schizophrenia, and gluconeogenesis/glycolysis in bipolar disorder. Both disorders were characterized by alterations of proteins involved in the oxidative stress response, mitochondrial function, and protein-endocytosis, -trafficking, -degradation, and -ubiquitination. These findings are interpreted with a focus on GABAergic interneuron pathology in the hippocampus.

  9. 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

  10. 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

    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.

  11. 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.

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

    PubMed

    Kambach, D M; Sodi, V L; Lelkes, P I; Azizkhan-Clifford, J; Reginato, M J

    2014-01-30

    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.

  13. Pharmacological properties of GABAA receptors in rat hypothalamic neurons expressing the epsilon-subunit.

    PubMed

    Sergeeva, Olga A; Andreeva, Nadja; Garret, Maurice; Scherer, Annette; Haas, Helmut L

    2005-01-05

    The pharmacological properties and functional role of native GABA(A) receptors (GABA(A)Rs) were investigated in rat hypothalamic neurons expressing the epsilon-subunit with the help of whole-cell patch-clamp recording and single-cell reverse transcription-PCR. Two cell groups were identified: histaminergic tuberomamillary and orexinergic/hypocretinergic neurons. Approximately 25% of histaminergic and 70% of orexinergic neurons contained mRNA encoding for the epsilon-subunit. Double-immunofluorescence staining revealed a somatic localization of this protein in these two neuronal groups. Constitutive activity, diazepam modulation, fast desensitization of maximal currents, and activation by propofol (6-98 microm) of GABA(A)Rs did not correlate with epsilon-subunit expression. Propofol at 3-12 microm potentiated GABA-mediated currents similarly in all neurons. However, noise variance analysis of GABA-mediated currents enhanced by propofol revealed a significant difference between epsilon-positive and epsilon-negative neurons. The former displayed no difference between control and potentiated responses, and, in the latter, noise was decreased in the presence of propofol. Spontaneous IPSCs recorded in cultured hypothalamic neurons were prolonged in the presence of propofol in all epsilon-negative neurons, whereas propofol-resistant IPSCs were recorded in epsilon-positive cells. The infrequent expression of the epsilon-subunit may be a key factor in the recently discovered central role of the tuberomamillary nucleus in anesthesia.

  14. Coincident inactivation of 14-3-3sigma and p16INK4a is an early event in vulval squamous neoplasia.

    PubMed

    Gasco, Milena; Sullivan, Alex; Repellin, Claire; Brooks, Louise; Farrell, Paul J; Tidy, John A; Dunne, Barbara; Gusterson, Barry; Evans, David J; Crook, Tim

    2002-03-14

    The structure and expression of 14-3-3 sigma(sigma) was analysed in squamous carcinomas (SCC) of the vulva and in the vulval pre-malignant lesion vulval intraepithelial neoplasia (VIN). Sequence analysis of the sigma coding region did not detect mutations in any case of SCC or VIN III and loss of heterozygosity (LOH) occurred in only 2 out of 27 informative cases. In contrast to the absence of genetic change, methylation-specific PCR (MSP) analysis revealed dense CpG methylation within the sigma gene in approximately 60% of cases of vulval SCC, but methylation was not detected in matched, normal epithelial tissue. Methylation was associated in all cases with reduced or absent expression of sigma mRNA. There was no correlation between sigma methylation and HPV or p53 status. Analysis of pre-malignant vulval intraepithelial neoplasia (VIN) revealed that sigma methylation was detectable early in neoplastic development. Co-incident methylation, accompanied by loss of expression, of sigma and p16INK4a was commonly detected in both SCC and VIN III, suggesting that epigenetic silencing of these two genes is an early and important event in vulval neoplasia.

  15. 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.

  16. 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.

  17. 14-3-3ζ turns TGF-β’s function from tumor suppressor to metastasis promoter in breast cancer by contextual changes of Smad partners from p53 to Gli2

    PubMed Central

    Xu, Jia; Acharya, Sunil; Sahin, Ozgur; Zhang, Qingling; Saito, Yohei; Yao, Jun; Wang, Hai; Li, Ping; Zhang, Lin; Lowery, Frank J; Kuo, Wen-Ling; Xiao, Yi; Ensor, Joe; Sahin, Aysegul A; Zhang, Xiang H.-F.; Hung, Mien-Chie; Zhang, Jitao David; Yu, Dihua

    2015-01-01

    Summary Transforming growth factor-β (TGF-β) functions as a tumor suppressor in pre-malignant cells but as a metastasis promoter in cancer cells. The dichotomous functions of TGF-β are proposed to be dictated by different partners of its downstream effectors Smads. However, the mechanism for the contextual changes of Smad partners remained undefined. Here, we demonstrate that 14-3-3ζ destabilizes p53, a Smad partner in pre-malignant mammary epithelial cells, by downregulating 14-3-3σ, thus turning off TGF-β’s tumor suppression function. Conversely, 14-3-3ζ stabilizes Gli2 in breast cancer cells, and Gli2 partners with Smads to activate PTHrP and promote TGF-β-induced bone metastasis. The 14-3-3ζ-driven contextual changes of Smad partners from p53 to Gli2 may serve as biomarkers and therapeutic targets of TGF-β-mediated cancer progression. PMID:25670079

  18. IκB kinase-induced interaction of TPL-2 kinase with 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP kinases

    PubMed Central

    Ben-Addi, Abduelhakem; Mambole-Dema, Agnes; Brender, Christine; Martin, Stephen R.; Janzen, Julia; Kjaer, Sven; Smerdon, Stephen J.; Ley, Steven C.

    2014-01-01

    The MEK-1/2 kinase TPL-2 is critical for Toll-like receptor activation of the ERK-1/2 MAP kinase pathway during inflammatory responses, but it can transform cells following C-terminal truncation. IκB kinase (IKK) complex phosphorylation of the TPL-2 C terminus regulates full-length TPL-2 activation of ERK-1/2 by a mechanism that has remained obscure. Here, we show that TPL-2 Ser-400 phosphorylation by IKK and TPL-2 Ser-443 autophosphorylation cooperated to trigger TPL-2 association with 14-3-3. Recruitment of 14-3-3 to the phosphorylated C terminus stimulated TPL-2 MEK-1 kinase activity, which was essential for TPL-2 activation of ERK-1/2. The binding of 14-3-3 to TPL-2 was also indispensible for lipopolysaccharide-induced production of tumor necrosis factor by macrophages, which is regulated by TPL-2 independently of ERK-1/2 activation. Our data identify a key step in the activation of TPL-2 signaling and provide a mechanistic insight into how C-terminal deletion triggers the oncogenic potential of TPL-2 by rendering its kinase activity independent of 14-3-3 binding. PMID:24912162

  19. 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.

  20. 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.

  1. {epsilon}{prime}/{epsilon} and rare KL decays from KTeV Experiment

    SciTech Connect

    Yee Bob Hsiung

    2001-01-16

    The authors update the current status for the measurement of the direct-CP violation parameters {epsilon}{prime}/{epsilon} in the KTeV experiment at Fermilab. Substantial statistics have been accumulated during the 1996-7 run and 1999 run for both {epsilon}{prime}/{epsilon} and rare K{sub L} decay searches. The first KTeV result on {epsilon}{prime}/{epsilon} published last year was Re({epsilon}{prime}/{epsilon}) = (28.0 {+-} 4.1) x 10{sup {minus}4} based on the 23% data from 1996-7 run. Combining with the previous E731, NA31 and the recent preliminary NA48 results, the grand average is Re({epsilon}{prime}/{epsilon}) = (19.3 {+-} 3.6) x 10{sup {minus}4} (with S = 1:49), more than 5{sigma} above zero. More data from both KTeV and NA48 after completing the analysis will further improve the precision of this measurement in the near future and hopefully further improve the agreement. New results on the branching ratio and form factor measurements of K{sub L} {r_arrow} {mu}{sup +} {mu}{sup {minus}} {gamma} using 1997 data are also presented. We find that BR(K{sub L} {r_arrow} {mu}{sup +} {mu}{sup {minus}} {gamma}) = (3.66 {+-} 0.04{sub stat} {+-} 0.07{sub syst}) x 10{sup {minus}7}. The form factor parameter {alpha}{sub K}* is measured to be {alpha}{sub K}* = {minus}0.157{sub {minus}0.027}{sup +0.025}. We make the first measurement of the parameter {alpha} from the D'Ambrosio, Isidori, and Portoles form factor, finding {alpha} = {minus}1.53 {+-} 0.09. This measurement of {alpha} limits the CKM parameter {rho} > {minus}0.2.

  2. YAP and 14-3-3γ are involved in HS-OA-induced growth inhibition of hepatocellular carcinoma cells: A novel mechanism for hydrogen sulfide releasing oleanolic acid

    PubMed Central

    Xu, Guanglin; Wang, Jing; Wu, Fangfang; Wang, Na; Zhou, Wenli; Wang, Qian; Pan, Wang; Ao, Guizhen; Yang, Jiquan

    2016-01-01

    Hydrogen sulfide-releasing oleanolic acid (HS-OA) is an emerging novel class of compounds and consists of an oleanolic acid (OA) and a H2S-releasing moiety. Although it exhibits improved anti-inflammatory activity, its potency in human cancers has not been understood yet. In this study, we examined the effects of HS-OA on the growth of liver cancer cell lines and the underlying mechanisms. HS-OA inhibited the growth of all four cancer cell lines studied, with potencies of 10- to 30-fold greater than that of its counterpart (OA). HS-OA induced significant apoptosis and decreased viability, clonogenic activity and migration of Hep G2 cells. Further studies showed that HS-OA resulted in the reduction of YAP expression and its downstream targets, CTGF and CYR 61, thus promoting cell apoptosis. In addition, HS-OA caused a decrease of 14-3-3γ expression, which led to Bad translocation to the mitochondria, ΔΨm loss, cytochrome c release, caspase activation and a recovery of 14-3-3γ reversed these effects induced by HS-OA. These findings indicate that YAP and 14-3-3γ are involved in HS-OA's effects on liver cancer cells and identifying HS-OA as a potential new drug candidate for cancer therapy. PMID:27437776

  3. The biology of the circadian Ck1epsilon tau mutation in mice and Syrian hamsters: a tale of two species.

    PubMed

    Loudon, A S I; Meng, Q J; Maywood, E S; Bechtold, D A; Boot-Handford, R P; Hastings, M H

    2007-01-01

    The tau mutation in the Syrian hamster resides in the enzyme casein kinase 1 epsilon (CK1epsilon), resulting in a dramatic acceleration of wheel-running activity cycles to about 20 hours. tau also impacts growth, energy, metabolism, feeding behavior, and circadian mechanisms underpinning seasonal timing, causing accelerated reproductive and neuroendocrine responses to photoperiodic changes. Modeling and experimental studies suggest that tau acts as a gain of function on specific residues of PER, consistent with hamster studies showing accelerated degradation of PER in the suprachiasmatic nucleus in the early circadian night. We have created null and tau mutants of Ck1epsilon in mice. Circadian period lengthens in CK1epsilon(/), whereas CK1epsilon(tau/tau) shortens circadian period of behavior in vivo in a manner nearly identical to that of the Syrian hamster. CK1epsilon(tau/tau) also accelerates molecular oscillations in peripheral tissues, demonstrating its global circadian role. CK1epsilon(tau) acts by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Our studies reveal that tau acts as a gain-of-function mutation, to accelerate degradation of PERIOD proteins. tau has consistent effects in both hamsters and mice on the circadian organization of behavior and metabolism, highlighting the global impact of this mutation on mammalian clockwork in brain and periphery.

  4. Apparatus and method to measure dielectric properties (epsilon(') and epsilon(")) of ionic liquids.

    PubMed

    Göllei, Attila; Vass, András; Pallai, Elisabeth; Gerzson, Miklós; Ludányi, Lajos; Mink, János

    2009-04-01

    Conventional techniques for measurement of dielectric properties of ionic liquids or electrolyte solutions fail because the samples are largely short circuited by the high electrical conductance. The object of the author's research activity was to elaborate an apparatus (microwave dielectrometer) and method suitable to measure the dielectric constant (epsilon(')) and loss factor (epsilon(")) of well conducting ionic liquids and other solvents. This process is based on a revised waveguide method completed with an automatic calibration possibility. Contrary to conventional measuring methods this technique uses about 20 W/g power density. The measurements were carried out at 2.45 GHz frequency in the temperature range from 10 up to 100 degrees C. The obtained (epsilon(')) and (epsilon(")) values of different solvents were compared with several published (calculated and measured) data. Statistical analysis was used to determine the error of measurements and distilled water was chosen as a standard for study of data dispersion. To accomplish statistical analysis, namely, the dielectric characteristics have to be determined at the same temperature. The values of variances were less or equal 1 in case of epsilon(') and decrease with increasing temperature. In case of epsilon(") the variance data were much smaller.

  5. 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.

  6. cDNA cloning and expression of a gamma-aminobutyric acid A receptor epsilon-subunit in rat brain.

    PubMed

    Moragues, N; Ciofi, P; Lafon, P; Odessa, M F; Tramu, G; Garret, M

    2000-12-01

    A cDNA encoding a GABA(A) receptor subunit was isolated from rat brain. The predicted protein is 70% identical to the human epsilon-subunit. It was recently reported [Sinkkonen et al. (2000), J. Neurosci., 20, 3588-3595] that the rodent epsilon-subunit mRNA encoded an additional sequence ( approximately 400 residues). We provide evidence that human and rat epsilon-subunit are similar in size. The distribution of cells expressing the GABA(A) epsilon-subunit was examined in the rat brain. In situ hybridization histochemistry revealed that epsilon-subunit mRNA is expressed by neurons located in septal and preoptic areas, as well as in various hypothalamic nuclei, including paraventricular, arcuate, dorsomedial and medial tuberal nuclei. The mRNA was also detected in major neuronal groups with broad-range influence, such as the cholinergic (basal nucleus), dopaminergic (substantia nigra compacta), serotonergic (raphe nuclei), and noradrenergic (locus coeruleus) systems. Immunohistochemistry using an affinity-purified antiserum directed towards the N-terminal sequence unique to the rat epsilon-subunit revealed the presence of epsilon-subunit immunoreactivity over the somatodendritic domain of neurons with a distribution closely matching that of mRNA-expressing cells. Moreover, using in situ hybridization, alpha3, theta and epsilon GABA(A) subunit mRNAs were all detected with an overlapping distribution in neurons of the dorsal raphe and the locus coeruleus. Our results suggest that novel GABA(A) receptors may regulate, neuroendocrine and modulatory systems in the brain.

  7. 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.

  8. 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

  9. 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.

  10. epsilon-COP is a structural component of coatomer that functions to stabilize alpha-COP.

    PubMed Central

    Duden, R; Kajikawa, L; Wuestehube, L; Schekman, R

    1998-01-01

    We isolated a novel yeast alpha-COP mutant, ret1-3, in which alpha-COP is degraded after cells are shifted to a restrictive temperature. ret1-3 cells cease growth at 28 degrees C and accumulate the ER precursor of carboxypeptidase Y (p1 CPY). In a screen for high copy suppressors of these defects, we isolated the previously unidentified yeast epsilon-COP gene. epsilon-COP (Sec28p) overproduction suppresses the defects of ret1-3 cells up to 34 degrees C, through stabilizing levels of alpha-COP. Surprisingly, cells lacking epsilon-COP (sec28 Delta) grow well up to 34 degrees C and display normal trafficking of carboxypeptidase Y and KKXX-tagged proteins at a permissive temperature. epsilon-COP is thus non-essential for yeast cell growth, but sec28 Delta cells are thermosensitive. In sec28 Delta cells shifted to 37 degrees C, wild-type alpha-COP (Ret1p) levels diminish rapidly and cells accumulate p1 CPY; these defects can be suppressed by alpha-COP overproduction. Mutant coatomer from sec28 Delta cells behaves as an unusually large protein complex in gel filtration experiments. The sec28 Delta mutation displays allele-specific synthetic-lethal interactions with alpha-COP mutations: sec28 Delta ret1-3 double mutants are unviable at all temperatures, whereas sec28 Delta ret1-1 double mutants grow well up to 30 degrees C. Our results suggest that a function of epsilon-COP is to stabilize alpha-COP and the coatomer complex. PMID:9463377

  11. 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

  12. Phospholipase C-epsilon augments epidermal growth factor-dependent cell growth by inhibiting epidermal growth factor receptor down-regulation.

    PubMed

    Yun, Sanguk; Hong, Won-Pyo; Choi, Jang Hyun; Yi, Kye Sook; Chae, Suhn-Kee; Ryu, Sung Ho; Suh, Pann-Ghill

    2008-01-04

    The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-epsilon. The overexpression of PLC-epsilon led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-epsilon RA2 domain independently of Ras. The interaction of PLC-epsilon with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-epsilon led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-epsilon in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-epsilon could promote EGF-dependent cell growth by suppressing receptor down-regulation.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  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. The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

    PubMed

    Wieczorek, Anna; McHenry, Charles S

    2006-05-05

    The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

  2. Reverse pharmacognosy: application of selnergy, a new tool for lead discovery. The example of epsilon-viniferin.

    PubMed

    Do, Quoc-Tuan; Renimel, Isabelle; Andre, Patrice; Lugnier, Claire; Muller, Christian D; Bernard, Philippe

    2005-09-01

    The aim of reverse pharmacognosy is to find new biological targets for natural compounds by virtual or real screening and identify natural resources that contain the active molecules. To demonstrate the applicability of this concept, we report here a study on epsilon-viniferin, an active ingredient for cosmetic development. Nevertheless, this natural substance is weakly defined in terms of biological properties. SELNERGY, an inverse docking computer software, was used to identify putative binding biological targets for epsilon-viniferin. Among the 400 screened proteins two targets were retained. For cosmetic application, cyclic nucleotide phosphodiesterase 4 (PDE4) was the most interesting candidate. Moreover, other PDE subtypes (1, 2, 3, 5 and 6) were not retained, indicating a selectivity for PDE4. The experimental binding tests on the 6 subtypes of PDE revealed a significant selectivity of epsilon-viniferin for the PDE4 subtype. This selectivity was confirmed by evaluation of epsilon-viniferin on the secretion of TNF-alpha and Interleukin-8. Our data demonstrated that epsilon-viniferin possesses anti-inflammatory properties by inhibiting PDE4 subtype. In conclusion, reverse pharmacognosy and its inverse docking component cannot only be integrated into a program for new lead discovery but is also a useful approach to find new applications for identified compounds.

  3. A tyrosine-containing motif mediates ER retention of CD3-epsilon and adopts a helix-turn structure.

    PubMed Central

    Mallabiabarrena, A; Jiménez, M A; Rico, M; Alarcón, B

    1995-01-01

    The CD3-epsilon endoplasmic reticulum (ER) retention motif has been characterized by mutagenesis and NMR spectroscopy. Tyr177, Leu180 and Arg183 are involved in ER retention. The motif forms an elongated alpha-helix in which the tyrosine and leucine residues are closely apposed, followed by a beta I' turn that places Arg183 in the vicinity of Leu180. The structure formed by Tyr177 and the leucine in position +3 is reminiscent of the beta-turn structure adopted by tyrosine-containing endocytosis signals. Moreover, substitution of the transferrin receptor (TfR) internalization sequence by the CD3-epsilon motif still allowed the rapid internalization of the TfR and, conversely, the chimeric protein resulting from the substitution of the CD3-epsilon motif by the endocytosis signal of the low density lipoprotein receptor was ER located. These data support the idea of a functional homology between the two types of signal. Images PMID:7774584

  4. Rotational dynamics of type I Fc epsilon receptors on individually-selected rat mast cells studied by polarized fluorescence depletion.

    PubMed Central

    Rahman, N A; Pecht, I; Roess, D A; Barisas, B G

    1992-01-01

    We report the first application of polarized fluorescence depletion (PFD), a technique which combines the sensitivity of fluorescence detection with the long lifetimes of triplet probes, to the measurement of membrane protein rotational diffusion on individually selected, intact mammalian cells. We have examined the rotation of type I Fc epsilon receptors (Fc epsilon RI) on rat mucosal mast cells of the RBL-2H3 line in their resting monomeric and differently oligomerized states using as probes IgE and three monoclonal antibodies (mAbs; H10, J17, and F4) specific for the Fc epsilon RI. PFD experiments using eosin (EITC)-IgE show that individual Fc epsilon RI on cells have a rotational correlation time (RCT) at 4 degrees C of 79 +/- 4 microseconds. Similarly, Fc epsilon RI-bound EITC-Fab fragments of the J17 Fc epsilon RI-specific mAb exhibit an RCT of 76 +/- 6 microseconds. These values agree with previous measurements of Fc epsilon RI-bound IgE rotation by time-resolved phosphorescence anisotropy methods. Receptor-bound EITC-conjugated divalent J17 antibody exhibits an increased RCT of 140 +/- 6 microseconds. This is consistent with the ability of this mAb to form substantial amounts of Fc epsilon RI dimers on these cell surfaces. The ratio of limiting to initial anisotropy in these experiments remains constant at about 0.5 from 5 degrees C through 25 degrees C for IgE, Fab, and intact mAb receptor ligands. Extensive cross-linking by second antibody of cell-bound IgE, of intact Fc epsilon RI-specific mAbs or of their Fab fragments, however, produced large fixed anisotropies demonstrating, under these conditions, receptor immobilization in large aggregates. PFD using the mAbs H10 and F4 as receptor probes yielded values for triplet lifetimes, RCT values, and anisotropy parameters essentially indistinguishable from those obtained with the mAb J17 clone. Possible explanations for these observations are discussed. PMID:1547323

  5. Calf thymus RF-C as an essential component for DNA polymerase delta and epsilon holoenzymes function.

    PubMed Central

    Podust, V N; Georgaki, A; Strack, B; Hübscher, U

    1992-01-01

    By using a complementation assay that enabled DNA polymerase delta and DNA polymerase epsilon to replicate a singly-DNA primed M13 DNA in the presence of proliferating cell nuclear antigen (PCNA) and Escherichia coli single-stranded DNA binding protein (SSB), we have purified from calf thymus in a five step procedure a multipolypeptide complex with molecular masses of polypeptides of 155, 70, 60, 58, 39 (doublet), 38 (doublet) and 36 kDa. The protein is very likely replication factor C (Tsurimoto, T. and Stillman, B. (1989) Mol. Cell. Biol. 9, 609-619). This conclusion is based on biochemical and physicochemical data and the finding that it contains a DNA stimulated ATPase which is under certain conditions stimulated by PCNA. Together RF-C, PCNA and ATP convert DNA polymerases delta and epsilon to holoenzyme forms, which were able to replicate efficiently SSB-covered singly-DNA primed M13 DNA. Calf thymus RF-C could form a primer recognition complex on a 3'-OH primer terminus in the presence of calf thymus PCNA and ATP. Holoenzyme complexes of DNA polymerase delta and epsilon could be isolated suggesting that these enzymes directly interact with the auxiliary proteins in a similar way. Under optimal replication conditions on singly-DNA primed M13 DNA the DNA synthesis rate of DNA polymerase delta was higher than of DNA polymerase epsilon. Based on these functional date possible roles of these two DNA polymerases in eukaryotic DNA replication are discussed. Images PMID:1354854

  6. 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.

  7. 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.

  8. Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors.

    PubMed

    Jones, Kristen L G; Holloway, M Katharine; Su, Hua-Poo; Carroll, Steven S; Burlein, Christine; Touch, Sinoeun; DiStefano, Daniel J; Sanchez, Rosa I; Williams, Theresa M; Vacca, Joseph P; Coburn, Craig A

    2010-07-15

    A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-L-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.

  9. 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.

  10. Determination of N epsilon-(carboxymethyl)lysine in foods and related systems.

    PubMed

    Ames, Jennifer M

    2008-04-01

    The sensitive and specific determination of advanced glycation end products (AGEs) is of considerable interest because these compounds have been associated with pro-oxidative and proinflammatory effects in vivo. AGEs form when carbonyl compounds, such as glucose and its oxidation products, glyoxal and methylglyoxal, react with the epsilon-amino group of lysine and the guanidino group of arginine to give structures including N epsilon-(carboxymethyl)lysine (CML), N epsilon-(carboxyethyl)lysine, and hydroimidazolones. CML is frequently used as a marker for AGEs in general. It exists in both the free or peptide-bound forms. Analysis of CML involves its extraction from the food (including protein hydrolysis to release any peptide-bound adduct) and determination by immunochemical or instrumental means. Various factors must be considered at each step of the analysis. Extraction, hydrolysis, and sample clean-up are all less straight forward for food samples, compared to plasma and tissue. The immunochemical and instrumental methods all have their advantages and disadvantages, and no perfect method exists. Currently, different procedures are being used in different laboratories, and there is an urgent need to compare, improve, and validate methods.

  11. Formation of N epsilon-(gamma-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells.

    PubMed Central

    Fesus, L; Tarcsa, E

    1989-01-01

    N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present. PMID:2574570

  12. [Bleeding after extracorporeal circulation and epsilon-aminocaproic acid].

    PubMed

    Saussine, M; Delpech, S; Allien, M; Grolleau, D; Daures, M F; Coulon, P; Chaptal, P A

    1985-01-01

    In order to assess the efficacy of epsilon aminocaproic acid in reducing bleeding after extracorporeal circulation for aorto-coronary bypass grafting, a double blind study was carried out in 57 patients. The efficiency of epsilon aminocaproic acid was assessed by the fibrinolytic activity as measured by a Von Kaulla test one hour after injection of protamine, by the amount of blood transfusions required and by the measurement of blood losses between the end of the injection of protamine and transfer of the patient to the intensive care unit, and then during the first 24 h following operation. No significant difference (p less than 0.05) between the group of treated patients and the group with placebo could be found concerning the postoperative bleeding, the amount of blood transfusions necessary and the occurrence of fibrinolysis. It was therefore concluded that there was no reason to routinely use epsilon aminocaproic acid after aorto-coronary bypass grafting.

  13. Efficient Vortex Generation in Subwavelength Epsilon-Near-Zero Slabs

    NASA Astrophysics Data System (ADS)

    Ciattoni, Alessandro; Marini, Andrea; Rizza, Carlo

    2017-03-01

    We show that a homogeneous and isotropic slab, illuminated by a circularly polarized beam with no topological charge, produces vortices of order 2 in the opposite circularly polarized components of the reflected and transmitted fields, as a consequence of the transverse magnetic and transverse electric asymmetric response of the rotationally invariant system. In addition, in the epsilon-near-zero regime, we find that vortex generation is remarkably efficient in subwavelength thick slabs up to the paraxial regime. This physically stems from the fact that a vacuum paraxial field can excite a nonparaxial field inside an epsilon-near-zero slab since it hosts slowly varying fields over physically large portions of the bulk. Our theoretical predictions indicate that epsilon-near-zero media hold great potential as nanophotonic elements for manipulating the angular momentum of the radiation, since they are available without resorting to complicated micro- or nanofabrication processes and can operate even at very small (ultraviolet) wavelengths.

  14. Framework for improved lattice calculations of epsilion'/epsilon

    NASA Astrophysics Data System (ADS)

    Laiho, Jack

    In this thesis we show that it is possible to construct epsilon '/epsilon to NLO using both full and partially quenched chiral perturbation theory (PQChPT) from amplitudes that are computable using numerical lattice gauge theory. We find that the electro-weak penguin (Delta I = 3/2 and 1/2) contributions to epsilon'/epsilon in PQChPT can be determined to NLO using only degenerate (mK = mpi) K → pi computations without momentum insertion. All one-loop formulas needed to extract the necessary NLO constants from the lattice are presented in this work. Issues pertaining to power divergent contributions, originating from mixing with lower dimensional operators in a lattice regularization, are addressed. In embedding the QCD penguin left-right operator onto PQChPT an ambiguity arises when the number of light sea quarks is not the physical value of three, as first emphasized by Golterman and Pallante. In the quenched theory they have pointed out that there are additional effective operators that appear in the quenched chiral perturbation theory needed to make contact with K → pipi amplitudes at physical kinematics. They have also proposed a method for determining the leading order low-energy constant, aNSq , associated with the new operators. We show that their method has difficulties due to power divergent contributions and propose a new method to obtain this constant from the lattice which does not suffer from this problem. Using this alternative method, we obtain aNSq , and show that our value implies a large ambiguity in the quenched contribution of Q6 to epsilon'/epsilon.

  15. Cloning and expression of Clostridium perfringens type D vaccine strain epsilon toxin gene in E. coli as a recombinant vaccine candidate

    PubMed Central

    Aziminia, Parastoo; Pilehchian-Langroudi, Reza; Esmaeilnia, Kasra

    2016-01-01

    Background and Objectives: Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene. Materials and Methods: Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase. The PCR product was cloned into pJET1.2/blunt cloning vector. The recombinant vector (pJETε) was sequenced using universal primers. At the next step epsilon toxin gene was subcloned into pET22b(+) expression vector and transformed into E. coli Rosetta (DE3) host strain. Results: The recombinant protein has been expressed in E. coli Rosetta (DE3) cells after subcloning of C. perfringens etx gene (1008 bp) into the expression vector. Conclusion: We concluded that E. coli Rosetta strain was suitable for the expression of recombinant C. perfringens epsilon toxin protein from pET22ε expression vector. This recombinant cell can be used for further research on recombinant vaccine development. PMID:28210460

  16. Localisation of GABA(A) receptor epsilon-subunit in cholinergic and aminergic neurones and evidence for co-distribution with the theta-subunit in rat brain.

    PubMed

    Moragues, N; Ciofi, P; Tramu, G; Garret, M

    2002-01-01

    In situ hybridisation and immunohistochemical methodologies suggest the existence of a large diversity of GABA(A) receptor subtypes in the brain. These are hetero-oligomeric proteins modulated by a number of clinically important drugs, depending on their subunit composition. We recently cloned and localised the rat GABA(A) receptor epsilon-subunit by in situ hybridisation and immunohistochemical procedures. Here, in a dual-labelling immunohistochemical study in the rat brain, we used our affinity-purified antiserum to epsilon with antisera to markers of cholinergic, catecholaminergic, and serotonergic neurones. As far as cholinergic systems were concerned, epsilon-immunoreactivity was expressed in all forebrain cell-groups, as well as in the caudal lateral pontine tegmentum and dorsal motor nucleus of the vagus nerve. As far as dopaminergic systems were concerned, epsilon-immunoreactivity was found to be expressed in a great number of hypothalamic cell-groups (A15, A14 and A12) and in the substantia nigra pars compacta. The noradrenergic, and to a lesser extent, adrenergic cell-groups were all epsilon-immunoreactive. Also, epsilon-immunoreactivity was detected in all serotonergic cell-groups. We also revealed by in situ hybridisation in a monkey brain that epsilon mRNA was expressed in the locus coeruleus, as previously observed in rats. Finally, by using in situ hybridisation in rat brains, we compared the distribution of the mRNA of epsilon with that of the recently cloned theta-subunit of the GABA(A) receptor. Both subunits showed strikingly overlapping expression patterns throughout the brain, especially in the septum, preoptic areas, various hypothalamic nuclei, amygdala, and thalamus, as well as the aforementioned monoaminergic cell-groups. No theta-mRNA signals were detected in cholinergic cell-groups. Taken together with previously published evidence of the presence of the alpha3-subunit in monoamine- or acetylcholine-containing systems, our data suggest

  17. Does Planet Exist in Epsilon Eridani? A Comparison Between Observations and Numerical Simulations

    NASA Technical Reports Server (NTRS)

    Liou, J.-C.; Zook, H. A.; Greaves, J. S.; Holland, W. S.

    2000-01-01

    We report results from our numerical simulations of the Epsilon Eridani dust disk. We also compare our results with actual observations of the disk and address the question of whether or not planet(s) exist in Epsilon Eridani.

  18. Characterization of thymus-derived lymphocytes expressing Ti alpha-beta CD3 gamma delta epsilon zeta-zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta- eta antigen receptor isoforms: analysis by gene transfection

    PubMed Central

    1990-01-01

    To characterize the function of the CD3 eta subunit of the T cell receptor (TCR), we have used cDNAs encoding CD3 zeta, CD3 eta, or both to reconstitute a variant of a cytochrome c-specific, I-Ek-restricted murine T cell hybridoma, termed MA5.8, which lacks CD3 zeta and CD3 eta proteins. We provide direct evidence that assembly and surface expression of TCRs can be mediated by either of these subunits separately or together. However, the level of TCR expression on zeta transfectants is up to one order of magnitude greater than that on eta transfectants, implying that CD3 eta is weakly associated with the pentameric Ti alpha-beta CD3 gamma delta epsilon complex and/or inefficient at salvaging the incomplete TCR from lysosomal degradation. As a component of the TCR, the CD3 eta subunit preferentially forms a heterodimer with CD3 zeta, but is also able to form a CD3 eta-eta homodimer. Crosslinking of Ti alpha-beta CD3 gamma delta epsilon zeta- zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta, or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta-eta TCR isotypes with anti-CD3 epsilon monoclonal antibody or a cytochrome c peptide epitope on I-Ek antigen-presenting cells mediates signal transduction resulting in reversible cell-cycle arrest of transfected clones. Given the potential for diversity of signals generated by these functional TCR isotypes and the expression of the CD3 eta gene product in the thymus, CD3 eta is likely to play a role in selection and/or activation of thymocytes during development. PMID:2145389

  19. 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.

  20. Synergistic activation of the germline epsilon promoter mediated by Stat6 and C/EBP beta.

    PubMed

    Mikita, T; Kurama, M; Schindler, U

    1998-08-15

    Transcription of the Ig H chain germline transcripts is a prerequisite for class switching. Expression of the epsilon germline transcript is induced by IL-4 and requires the integrity of a composite IL-4 response element. The element is bound by the IL-4-inducible transcription factor Stat6 and one or more members of the CAAT/enhancer-binding protein (C/EBP) family, a constitutively expressed class of transcription factors. Here, we show that Stat6 and C/EBP beta cooperate to synergistically activate transcription from the epsilon element. The effect was most pronounced in lymphoid cells, and the activation domains of both proteins were required to achieve this synergy. Although other members of the C/EBP family are able to bind the element, very little cooperativity was seen with C/EBP alpha and none with C/EBP gamma. In fact, C/EBP gamma was able to inhibit IL-4-induced reporter activity. Stat6 and C/EBP beta bind the IL-4 response element simultaneously. The fast dissociation rate apparent when Stat6 binds this DNA element alone is slowed when C/EBP beta binds at the neighboring site. These data suggest a mechanism whereby C/EBP beta stabilizes Stat6 binding at this element, thereby increasing the likelihood that both of their activation domains will interact, possibly with other factors, to activate transcription in an IL-4-dependent manner.

  1. Characterization of the interface between gamma and epsilon subunits of Escherichia coli F1-ATPase.

    PubMed

    Tang, C; Capaldi, R A

    1996-02-09

    The interaction faces of the gamma and epsilon subunits in the Escherichia coli F1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant epsilon subunits as follows: epsilonS10C, epsilonH38C, epsilonT43C, epsilonS65C, epsilonS108C, and epsilonM138C, along with a mutant of the gamma subunit, gammaT106C. The replacement of Ser-10 by a Cys or Met-138 by a Cys reduced the inhibition of ECF1 by the epsilon subunit, while the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimide further reduced the binding affinity of, and the maximal inhibition by, the epsilon subunit. Similar chemical modification of the Cys at position 43 of the epsilon subunit (in the mutant epsilonT43C) and a Cys at position 106 of the gamma subunit (gammaT106C) also affected the inhibition of ECF1 by the epsilon subunit. The various epsilon subunit mutants were reacted with TFPAM3, and the site(s) of cross-linking within the ECF1 complex was determined. Previous studies have shown cross-linking from the Cys at positions 10 and 38 with the gamma subunit and from a Cys at position 108 to an alpha subunit (Aggeler, R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 2956-2961; Aggeler, R., Weinreich, F., and Capaldi, R. A. (1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross-linking was found from a Cys at position 43 to the gamma subunit and from the Cys at position 138 to a beta subunit. The site of cross-linking from Cys-10 of epsilon to the gamma subunit was localized by peptide mapping to a region of the gamma subunit between residues 222 and 242. Cross-linking from a Cys at position 38 and at position 43 was with the C-terminal part of the gamma subunit, between residues 202 and 286. ECF1 treated with trypsin at pH 7.0 still binds purified epsilon subunit, while enzyme treated with the protease at pH 8.0 does

  2. 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.

  3. 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.

  4. The embedded objects in epsilon Cha I cloud

    NASA Technical Reports Server (NTRS)

    Prusti, Timo; Assendorp, R.; Wesselius, P. R.

    1989-01-01

    A study was made of the embedded objects in the epsilon Cha I cloud. General shapes of the spectra were constructed for the members in the cloud. The near infrared data were compiled from the literature and combined with the IRAS Point Source Catalog information. Pointed observations by the IRAS were used in the regions of high source density where the Point Source Catalog is confused. Member objects near the late B star HD 97300 were measured recently in the 3 to 10 micron bands using the ESO 2.2 m telescope in order to study the effects of disks seen in other young stellar objects. A picture is presented of the complete initial luminosity function in the epsilon Cha I cloud. The observations were compared with the theoretical views on low mass star formation.

  5. PKC Epsilon: A Novel Oncogenic Player in Prostate Cancer

    DTIC Science & Technology

    2013-09-01

    levels. (A) RWPE-1 cells were infected with ViraPower lentiviruses containing a human PKCε construct cloned into pLenti6/V-5 DESTRWPE-1. Expression...kinase C epsilon (PKCε), a member of the PKC family of phorbol ester/diacylglycerol receptors, is up-regulated in many human cancers, including prostate...In prostate cancer cells PKCε mediates survival through Bad -dependent and Bad -independent mechanisms and is involved in the transition to androgen

  6. 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.

  7. 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.

  8. An Attempt to Derive the epsilon Equation from a Two-Point Closure

    NASA Technical Reports Server (NTRS)

    Canuto, V. M.; Cheng, Y.; Howard, A. M.

    2010-01-01

    The goal of this paper is to derive the equation for the turbulence dissipation rate epsilon for a shear-driven flow. In 1961, Davydov used a one-point closure model to derive the epsilon equation from first principles but the final result contained undetermined terms and thus lacked predictive power. Both in 1987 and in 2001, attempts were made to derive the epsilon equation from first principles using a two-point closure, but their methods relied on a phenomenological assumption. The standard practice has thus been to employ a heuristic form of the equation that contains three empirical ingredients: two constants, c(sub 1 epsilon), and c(sub 2 epsilon), and a diffusion term D(sub epsilon) In this work, a two-point closure is employed, yielding the following results: 1) the empirical constants get replaced by c(sub 1), c(sub 2), which are now functions of Kappa and epsilon; 2) c(sub 1) and c(sub 2) are not independent because a general relation between the two that are valid for any Kappa and epsilon are derived; 3) c(sub 1), c(sub 2) become constant with values close to the empirical values c(sub 1 epsilon), c(sub epsilon 2), (i.e., homogenous flows); and 4) the empirical form of the diffusion term D(sub epsilon) is no longer needed because it gets substituted by the Kappa-epsilon dependence of c(sub 1), c(sub 2), which plays the role of the diffusion, together with the diffusion of the turbulent kinetic energy D(sub Kappa), which now enters the new equation (i.e., inhomogeneous flows). Thus, the three empirical ingredients c(sub 1 epsilon), c(sub epsilon 2), D (sub epsilon)are replaced by a single function c(sub 1)(Kappa, epsilon ) or c(sub 2)(Kappa, epsilon ), plus a D(sub Kappa)term. Three tests of the new equation for epsilon are presented: one concerning channel flow and two concerning the shear-driven planetary boundary layer (PBL).

  9. Tissue angiotensin II during progression or ventricular hypertrophy to heart failure in hypertensive rats; differential effects on PKC epsilon and PKC beta.

    PubMed

    Inagaki, Koichi; Iwanaga, Yoshitaka; Sarai, Nobuaki; Onozawa, Yoko; Takenaka, Hiroyuki; Mochly-Rosen, Daria; Kihara, Yasuki

    2002-10-01

    The protein kinase C (PKC) family has been implicated as second messengers in mechanosensitive modulation of cardiac hypertrophy. However, little information is available on the role of expression and activation of specific cardiac PKC isozymes during development of left ventricular hypertrophy (LVH) and failure (LVF). Dahl salt-sensitive rats fed an 8% salt diet developed systemic hypertension and concentric LVH at 11 weeks of age that is followed by left ventricle (LV) dilatation and global hypokinesis at 17 weeks. Among several PKC isozymes expressed in the LV myocardium, only PKC epsilon showed a 94% increase at the LVH stage. At the LVF stage, however, PKC epsilon returned to the control level, whereas PKC beta I and beta II increased by 158% and 155%, respectively. Hearts were studied at each stage using the Langendorff set-up, and a LV balloon was inflated to achieve an equivalent diastolic wall stress. Following mechanical stretch, PKC epsilon was significantly activated in LVH myocardium in which tissue angiotensin II levels were increased by 59%. Pre-treatment with valsartan, an AT(1)-receptor blocker, abolished the stretch-mediated PKC epsilon activation. Mechanical stretch no longer induced PKC epsilon activation in LVF. Chronic administration of valsartan blunted the progression of LVF and inhibited the increase in PKC beta. Mechanosensitive PKC epsilon activation is augmented and therefore may contribute to the development of compensatory hypertrophy. This effect was dependent on activation of tissue angiotensin II. However, this compensatory mechanism becomes inactive in LVF, where PKC beta may participate in the progression to cardiac dysfunction and LV remodeling.

  10. Specific BACE1 genotypes provide additional risk for late-onset Alzheimer disease in APOE epsilon 4 carriers.

    PubMed

    Gold, Gabriel; Blouin, Jean-Louis; Herrmann, François R; Michon, Agnès; Mulligan, Reinhild; Duriaux Saïl, Geneviève; Bouras, Constantin; Giannakopoulos, Panteleimon; Antonarakis, Stylianos E

    2003-05-15

    Alzheimer disease (AD) is characterized neuropathologically by neurofibrillary tangles and senile plaques. A key component of plaques is A beta, a polypeptide derived from A beta-precursor protein (APP) through proteolytic cleavage catalyzed by beta and gamma-secretase. We hypothesized that sequence variation in genes BACE1 (on chromosome 11q23.3) and BACE2 (on chromosome 21q22.3), which encode two closely related proteases that seem to act as the APP beta-secretase, may represent a genetic risk factor for AD. We analyzed the frequencies of single nucleotide polymorphisms (SNPs) in BACE1 and BACE2 genes in a community-based sample of 96 individuals with late-onset AD and 170 controls selected randomly among residents of the same community. The genotype data in both study groups did not demonstrate any association between AD and BACE1 or BACE2. After stratification for APOE status, however, an association between a BACE1 polymorphism located within codon V262 and AD in APOE epsilon 4 carriers was observed (P = 0.03). We conclude that sequence variation in the BACE1 or BACE 2 gene is not a significant risk factor for AD; however, a combination of a specific BACE1 allele and APOE epsilon 4 may increase the risk for Alzheimer disease over and above that attributed to APOE epsilon 4 alone.

  11. Interaction of p72syk with the gamma and beta subunits of the high-affinity receptor for immunoglobulin E, Fc epsilon RI.

    PubMed Central

    Shiue, L; Green, J; Green, O M; Karas, J L; Morgenstern, J P; Ram, M K; Taylor, M K; Zoller, M J; Zydowsky, L D; Bolen, J B

    1995-01-01

    Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gamma subunit motif. These data suggest that Syk associates with the gamma subunit of the high-affinity receptor for immunoglobulin E through an interaction between the tandem SH2 domains of SH2 domains of Syk and the phosphorylated tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Fc epsilon RI tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Dc epsilon

  12. Determination of poly(epsilon-caprolactone) solubility parameters: application to solvent substitution in a microencapsulation process.

    PubMed

    Bordes, C; Fréville, V; Ruffin, E; Marote, P; Gauvrit, J Y; Briançon, S; Lantéri, P

    2010-01-04

    The evolution of regulation on chemical substances (i.e. REACH regulation) calls for the progressive substitution of toxic chemicals in formulations when suitable alternatives have been identified. In this context, the method of Hansen solubility parameters was applied to identify an alternative solvent less toxic than methylene chloride used in a microencapsulation process. During the process based on a multiple emulsion (W/O/W) with solvent evaporation/extraction method, the solvent has to dissolve a polymer, poly(epsilon-caprolactone) (PCL), which forms a polymeric matrix encapsulating or entrapping a therapeutic protein as the solvent is extracted. Therefore the three partial solubility parameters of PCL have been determined by a group contribution method, swelling experiments and turbidimetric titration. The results obtained allowed us to find a solvent, anisole, able to solubilize PCL and to form a multiple emulsion with aqueous solutions. A feasibility test was conducted under standard operating conditions and allowed the production of PCL microspheres.

  13. Identification of epsilon martensite in a Fe-based shape memory alloy by means of EBSD.

    PubMed

    Verbeken, K; Van Caenegem, N; Raabe, D

    2009-01-01

    Ferrous shape memory alloys (SMAs) are often thought to become a new, important group of SMAs. The shape memory effect in these alloys is based on the reversible, stress-induced martensitic transformation of austenite to epsilon martensite. The identification and quantification of epsilon martensite is crucial when evaluating the shape memory behaviour of this material. Previous work displayed that promising results were obtained when studying the evolution of the amount of epsilon martensite after different processing steps with Electron BackScatter Diffraction (EBSD). The present work will discuss in detail, on the one hand, the challenges and opportunities arising during the identification of epsilon martensite by means of EBSD and, on the other hand, the possible interpretations that might be given to these findings. It will be illustrated that although the specific nature of the austenite to epsilon martensite transformation can still cause some points of discussion, EBSD has a high potential for identifying epsilon martensite.

  14. 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.

  15. New epsilon-Bi2O3 metastable polymorph.

    PubMed

    Cornei, Nicoleta; Tancret, Nathalie; Abraham, Francis; Mentré, Olivier

    2006-06-26

    The new metastable epsilon-Bi2O3 polymorph has been prepared by hydrothermal treatment and structurally characterized. It shows strong relationships with the room temperature alpha form and the metastable beta form through rearrangements of [Bi2O3] columns formed by edge-sharing OBi4 tetrahedra. Its fully ordered crystal structure yields an ionic insulating character. It irreversibly transforms at 400 degrees C to the alpha form. The chemical analysis indicates its undoped bismuth oxide nature, then leading to the fifth characterized Bi2O3 polymorph to date.

  16. Epsilon Canis Majoris and the ionization of the local cloud

    NASA Astrophysics Data System (ADS)

    Vallerga, J. V.; Welsh, B. Y.

    1995-05-01

    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 10-15/s. This figure 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 ne 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, chiH 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 ne 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 toward Sirius A. We show

  17. 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

  18. Mutation screening in the human epsilon-globin gene using single-strand conformation polymorphism analysis.

    PubMed

    Papachatzopoulou, Adamantia; Menounos, Panagiotis G; Kolonelou, Christina; Patrinos, George P

    2006-02-01

    The human epsilon-globin gene is necessary for primitive human erythropoiesis in the yolk sac. Herein we report a non-radioactive single-strand conformation polymorphism (SSCP) approach to screen the human epsilon-globin gene and its regulatory regions for possible mutations and single-nucleotide polymorphisms in normal adult subjects, in order to determine those genomic regions, which are not necessary for its proper regulation and function. We identified no sequence variations apart from the expected 5'epsilon /HincII polymorphism in the fragments analyzed, suggesting that genomic alterations in the epsilon-globin gene are most likely incompatible with normal erythropoiesis and proper embryonic development.

  19. 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.

  20. 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.

  1. Applications of Epsilon Radial Networks in Neuroimage Analyses

    PubMed Central

    Adluru, Nagesh; Chung, Moo K.; Lange, Nicholas T.; Lainhart, Janet E.; Alexander, Andrew L.

    2016-01-01

    “Is the brain ’wiring’ different between groups of populations?” is an increasingly important question with advances in diffusion MRI and abundance of network analytic tools. Recently, automatic, data-driven and computationally efficient framework for extracting brain networks using tractography and epsilon neighborhoods were proposed in the diffusion tensor imaging (DTI) literature [1]. In this paper we propose new extensions to that framework and show potential applications of such epsilon radial networks (ERN) in performing various types of neuroimage analyses. These extensions allow us to use ERNs not only to mine for topo-physical properties of the structural brain networks but also to perform classical region-of-interest (ROI) analyses in a very efficient way. Thus we demonstrate the use of ERNs as a novel image processing lens for statistical and machine learning based analyses. We demonstrate its application in an autism study for identifying topological and quantitative group differences, as well as performing classification. Finally, these views are not restricted to ERNs but can be effective for population studies using any computationally efficient network-extraction procedures. PMID:28251191

  2. {epsilon}-optimality conditions for weakly convex problems

    SciTech Connect

    Pappalardo, M.

    1994-12-31

    There are several generalizations concerning the concept of convexity both for sets and for functions. Weak convexity, among these, has showed many possibilities of applications and many theoretical properties. It has, in fact, been applied in several fields of mathematics: see for example geometry and optimization. We want to analyze this generalization of the concept of convexity via the image-space approach. This kind of approach has showed its utility in many fields of optimization. In particular, we introduce a new concept of {open_quotes}image{close_quotes} based on a suitable relaxation or reduction (lower and upper) of the image itself. Moreover we analyze the main properties of this concept and we show how to utilize it in the study of weakly convex constrained extremum problems in order to obtain {epsilon}-optimality conditions. The paper is divided in three parts: in the first we introduce the concept of perturbed image and we investigate the main theoretical properties. In the second we state {epsilon}-optimality conditions for weakly convex constrained extremum problems. In the third one we study relationships between this type of image and the augmented lagrangian.

  3. 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

  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. Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling

    PubMed Central

    Baker, Matthew L.; Hryc, Corey F.; Zhang, Qinfen; Wu, Weimin; Jakana, Joanita; Haase-Pettingell, Cameron; Afonine, Pavel V.; Adams, Paul D.; King, Jonathan A.; Jiang, Wen; Chiu, Wah

    2013-01-01

    High-resolution structures of viruses have made important contributions to modern structural biology. Bacteriophages, the most diverse and abundant organisms on earth, replicate and infect all bacteria and archaea, making them excellent potential alternatives to antibiotics and therapies for multidrug-resistant bacteria. Here, we improved upon our previous electron cryomicroscopy structure of Salmonella bacteriophage epsilon15, achieving a resolution sufficient to determine the tertiary structures of both gp7 and gp10 protein subunits that form the T = 7 icosahedral lattice. This study utilizes recently established best practice for near-atomic to high-resolution (3–5 Å) electron cryomicroscopy data evaluation. The resolution and reliability of the density map were cross-validated by multiple reconstructions from truly independent data sets, whereas the models of the individual protein subunits were validated adopting the best practices from X-ray crystallography. Some sidechain densities are clearly resolved and show the subunit–subunit interactions within and across the capsomeres that are required to stabilize the virus. The presence of the canonical phage and jellyroll viral protein folds, gp7 and gp10, respectively, in the same virus suggests that epsilon15 may have emerged more recently relative to other bacteriophages. PMID:23840063

  6. Further studies on T*{sub {epsilon}} integral for curved crack growth

    SciTech Connect

    Lam, P.W.; Kobayashi, A.S.; Atluri, S.N.; Tan, P.W.

    1999-07-01

    T*{sub {epsilon}} integral values associated with stable, curved crack growth in biaxially loaded, fatigued precracked, 2024-T3 single edge notched (SEN) specimens were determined. The SEN specimens were loaded under combined Modes 1 and 2 and mimicked the flapping of a failed lap splice joint of a pressurized airplane fuselage. Most specimens were provided with a tear strap, which was either bonded, bonded and riveted, or integrally machined (machined pad-up) in the specimen. The stably growing crack curved and either penetrated or curved again upon hitting the tear strap. The displacement field, which was determined by Moire interferometry as well as with finite element analysis, was used to directly determine the T*{sub 2{epsilon}} and T*{sub 2{epsilon}} integral values. These T*{sub {epsilon}} values agreed reasonably well with those determined by an elastic-plastic finite element modeling of the experiments. T*{sub 1{epsilon}} was identical to that obtained previously for pure Mode I crack extension while the T*{sub 2{epsilon}} integral oscillated about its null value. The results of this study suggest that T*{sub 1{epsilon}} could represent the resistance for locally self-similar crack growth and that a crack will curve in the direction of vanishing T*{sub 2{epsilon}}.

  7. The application of an emerging technique for protein-protein interaction interface mapping: the combination of photo-initiated cross-linking protein nanoprobes with mass spectrometry.

    PubMed

    Ptáčková, Renata; Ječmen, Tomáš; Novák, Petr; Hudeček, Jiří; Stiborová, Marie; Šulc, Miroslav

    2014-05-26

    Protein-protein interaction was investigated using a protein nanoprobe capable of photo-initiated cross-linking in combination with high-resolution and tandem mass spectrometry. This emerging experimental approach introduces photo-analogs of amino acids within a protein sequence during its recombinant expression, preserves native protein structure and is suitable for mapping the contact between two proteins. The contact surface regions involved in the well-characterized interaction between two molecules of human 14-3-3ζ regulatory protein were used as a model. The employed photo-initiated cross-linking techniques extend the number of residues shown to be within interaction distance in the contact surface of the 14-3-3ζ dimer (Gln8-Met78). The results of this study are in agreement with our previously published data from molecular dynamic calculations based on high-resolution chemical cross-linking data and Hydrogen/Deuterium exchange mass spectrometry. The observed contact is also in accord with the 14-3-3ζ X-ray crystal structure (PDB 3dhr). The results of the present work are relevant to the structural biology of transient interaction in the 14-3-3ζ protein, and demonstrate the ability of the chosen methodology (the combination of photo-initiated cross-linking protein nanoprobes and mass spectrometry analysis) to map the protein-protein interface or regions with a flexible structure.

  8. Neural regulation of muscle acetylcholine receptor epsilon- and alpha- subunit gene promoters in transgenic mice

    PubMed Central

    1993-01-01

    The effects of denervation were investigated in mice with transgenes containing promoter elements from the muscle acetylcholine receptor epsilon- and alpha-subunit genes. The promoter sequences were coupled to a nuclear localization signal-beta-galactosidase fusion gene (nlacZ) as a reporter. While many postsynaptic specializations form in the embryo, expression of the epsilon subunit is induced during the first two postnatal weeks. When muscles were denervated at birth, before the onset of epsilon expression, epsilon nlacZ still appeared at the former synaptic sites on schedule. This result suggests that the nerve leaves a localized "trace" in the muscle that can continue to regulate transcription. An additional finding was that epsilon nlacZ expression was much stronger in denervated than in intact muscles. This suggests that the epsilon promoter is similar to the other subunits in containing elements that are activated on cessation of neural activity. However, even after denervation, epsilon nlacZ expression was always confined to the synaptic region whereas alpha nlacZ expression increased in nuclei along the entire length of the fiber. This suggests that while the epsilon gene is similar in its activity dependence to other subunit genes, it is unique in that local nerve-derived signals are essential for its expression. Consequently, inactivity enhances epsilon expression only in synaptic nuclei where such signals are present, but enhances expression throughout the muscle fiber. Truncations and an internal deletion of the epsilon promoter indicate that cis-elements essential for the response to synaptic signals are contained within 280 bp of the transcription start site. In contrast to these results in young animals, denervation in older animals leads to an unexpected reduction in nlacZ activity. However, mRNA measurements indicated that transgene expression was increased in these animals. This discordance between nlacZ mRNA and enzyme activity, demonstrates a

  9. 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.

  10. Improved solution for system identification equations by Epsilon-Decomposition

    NASA Technical Reports Server (NTRS)

    Ojalvo, Irving U.

    1990-01-01

    Matrix eigenvalue theory is used to examine the source of ill-conditioning in linear algebraic equations. This approach highlights the crucial role played by the zero and near-zero eigenvalues and corresponding eigenvectors of poorly conditioned systems. Insight gained from this approach is used to significantly improve a recently developed solution procedure called Epsilon-Decomposition (E-D). E-D is an efficient alternative to Singular Value Decomposition (SVD) for ill-conditioned systems arising in parameter estimation and system identification studies. The efficiency of the improved E-D over SVD resides in the need to only obtain the zero and near-zero eigenvalues of the coefficient matrix as opposed to all of its eigenvalues and vectors (as required by SVD). Thus, the efficiency of E-D is significant for large matrices with small rank deficiency.

  11. 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

    2013-04-08

    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.

  12. 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.

  13. 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.

  14. Noise reduction combining time-frequency epsilon-filter and M-transform.

    PubMed

    Abe, Tomomi; Matsumoto, Mitsuharu; Hashimoto, Shuji

    2008-08-01

    This paper introduces noise reduction combining time-frequency epsilon-filter (TF epsilon-filter) and time-frequency M-transform (TF M-transform). Musical noise is an offensive noise generated due to noise reduction in the time-frequency domain such as spectral subtraction and TF epsilon-filter. It has a deleterious effect on speech recognition. To solve the problem, M-transform is introduced. M-transform is a linear transform based on M-sequence. The method combining the time-domain epsilon-filter (TD epsilon-filter) and time-domain M-transform (TD M-transform) can reduce not only white noise but also impulse noise. Musical noise is isolated in the time-frequency domain, which is similar to impulse noise in the time domain. On these prospects, this paper aims to reduce musical noise by improving M-transform for the time-frequency domain. Noise reduction by using TD M-transform and the TD epsilon-filter is first explained to clarify its features. Then, an improved method applying M-transform to the time-frequency domain, namely TF M-transform, is described. Noise reduction combining the TF epsilon-filter and TF M-transform is also proposed. The proposed method can reduce not only high-level nonstationary noise but also musical noise. Experimental results are also given to demonstrate the performance of the proposed method.

  15. Hydroxyl radical mediates N epsilon-(carboxymethyl)lysine formation from Amadori product.

    PubMed

    Nagai, R; Ikeda, K; Higashi, T; Sano, H; Jinnouchi, Y; Araki, T; Horiuchi, S

    1997-05-08

    Recent studies demonstrated N epsilon-(carboxymethyl)lysine (CML) in several tissue proteins. Incubation of proteins with glucose leads through a Schiff base to Amadori products. Oxidative cleavage of Amadori products is considered as a major route to CML formation in vivo, whereas it is not known which reactive oxygen species (ROS) is involved. The present study is undertaken to identify such a ROS. We prepared heavily glycated human serum albumin (HSA) which contained a high level of Amadori products, but an undetectable level of CML. Incubation of glycated HSA with FeCl2, but not with H2O2, led to CML formation which was enhanced by H2O2, but inhibited by catalase or mannitol, whereas superoxide dismutase had no effect. Similar data were obtained by experiments using Boc-fructose-lysine as a model Amadori compound. These data indicate that hydroxyl radical generated by the reaction of Fe2+ with H2O2 mediates CML formation from Amadori compounds.

  16. Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

    NASA Astrophysics Data System (ADS)

    Jiang, Wen; Chang, Juan; Jakana, Joanita; Weigele, Peter; King, Jonathan; Chiu, Wah

    2006-02-01

    The critical viral components for packaging DNA, recognizing and binding to host cells, and injecting the condensed DNA into the host are organized at a single vertex of many icosahedral viruses. These component structures do not share icosahedral symmetry and cannot be resolved using a conventional icosahedral averaging method. Here we report the structure of the entire infectious Salmonella bacteriophage epsilon15 (ref. 1) determined from single-particle cryo-electron microscopy, without icosahedral averaging. This structure displays not only the icosahedral shell of 60 hexamers and 11 pentamers, but also the non-icosahedral components at one pentameric vertex. The densities at this vertex can be identified as the 12-subunit portal complex sandwiched between an internal cylindrical core and an external tail hub connecting to six projecting trimeric tailspikes. The viral genome is packed as coaxial coils in at least three outer layers with ~90 terminal nucleotides extending through the protein core and the portal complex and poised for injection. The shell protein from icosahedral reconstruction at higher resolution exhibits a similar fold to that of other double-stranded DNA viruses including herpesvirus, suggesting a common ancestor among these diverse viruses. The image reconstruction approach should be applicable to studying other biological nanomachines with components of mixed symmetries.

  17. Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class.

    PubMed Central

    Sawicki, Rafał; Singh, Sharda P; Mondal, Ashis K; Benes, Helen; Zimniak, Piotr

    2003-01-01

    From the fruitfly, Drosophila melanogaster, ten members of the cluster of Delta-class glutathione S-transferases (GSTs; formerly denoted as Class I GSTs) and one member of the Epsilon-class cluster (formerly GST-3) have been cloned, expressed in Escherichia coli, and their catalytic properties have been determined. In addition, nine more members of the Epsilon cluster have been identified through bioinformatic analysis but not further characterized. Of the 11 expressed enzymes, seven accepted the lipid peroxidation product 4-hydroxynonenal as substrate, and nine were active in glutathione conjugation of 1-chloro-2,4-dinitrobenzene. Since the enzymically active proteins included the gene products of DmGSTD3 and DmGSTD7 which were previously deemed to be pseudogenes, we investigated them further and determined that both genes are transcribed in Drosophila. Thus our present results indicate that DmGSTD3 and DmGSTD7 are probably functional genes. The existence and multiplicity of insect GSTs capable of conjugating 4-hydroxynonenal, in some cases with catalytic efficiencies approaching those of mammalian GSTs highly specialized for this function, indicates that metabolism of products of lipid peroxidation is a highly conserved biochemical pathway with probable detoxification as well as regulatory functions. PMID:12443531

  18. Cell-bound IgE and increased expression of Fc epsilon-receptors on dendritic cells in cutaneous infiltrates of mycosis fungoides.

    PubMed Central

    Preesman, A H; Van de Winkel, J G; Magnusson, C G; Toonstra, J; van der Putte, S C; van Vloten, W A

    1991-01-01

    Skin biopsies of 31 non-atopic patients, 20 with mycosis fungoides, six with psoriasis and five with contact dermatitis, and of five non-atopic healthy controls were compared for the presence of cell-bound IgE and vacant IgE binding sites. IgE+ cells were demonstrated in the cutaneous infiltrate of nine (45%) patients with mycosis fungoides, two (33%) with psoriasis and one (20%) with contact dermatitis. Following pre-incubation of skin sections with IgE myeloma protein to saturate vacant IgE-binding sites, 14 out of 16 patients (88%) with stage I mycosis fungoides, five (83%) patients with psoriasis and one (20%) with contact dermatitis showed an increase in the number of IgE+ cells. While cell-bound IgE was positively related to serum IgE levels the expression of IgE-binding sites was not. All IgE+ cells were HLA-DR+ dendritic cells identified as either macrophages (CD68+, CD14+) or Langerhans cells (CD1+). Skin biopsies of non-atopic healthy controls or clinically uninvolved skin in mycosis fungoides had neither any IgE+ cells nor any vacant binding sites. Inhibition studies with IgG1, IgG4 and IgE myeloma proteins as well as with several enzymatic fragments of IgE demonstrated that IgE interacted with Fc epsilon-receptors through isotype-specific structures on the Fc epsilon-fragment. Four anti-CD23 monoclonal antibodies, however, were unable to stain vacant Fc epsilon-receptors nor could they block IgE-binding. We hypothesize that locally-secreted lymphokines, like IL-4 or interferon-gamma, induce Fc epsilon-receptors on dendritic cells in the cutaneous infiltrate and that these receptors become occupied in parallel with elevated serum IgE levels. Images Fig. 1 Fig. 2 PMID:1834378

  19. Preparation of magnetic microspheres based on poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone) copolymers by modified solvent diffusion method.

    PubMed

    Men, Ke; Zeng, Shi; Gou, MaLing; Guo, Gang; Gu, Ying Chun; Luo, Feng; Zhao, Xia; Wei, YuQuan; Qian, ZhiYong

    2010-06-01

    Magnetic microspheres have promising application in biomedical field. In this paper, biodegradable poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone) (PCEC) triblock copolymers were synthesized by ring-opening polymerization method. Through adjusting the epsilon-CL/PEG weight ratio in feed, PCEC copolymers with different block ratio were obtained. A novel modified solvent diffusion method was described to prepare magnetic PCEC composite microspheres containing magnetite nanoparticles. The particle size of microsphere decreased with increase in the PEG/PCL block ratio. The obtained microspheres could response to external magnetic field. This study described a novel method to prepare magnetic microspheres. The obtained magnetic polymeric microspheres might have potential application in drug delivery system or disease diagnosis field.

  20. 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.

  1. 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.

  2. 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

  3. Preferential emission into epsilon-near-zero metamaterial [Invited

    DOE PAGES

    Galfsky, Tal; Sun, Zheng; Jacob, Zubin; ...

    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

  4. September epsilon Perseid cluster as a result of orbital fragmentation

    NASA Astrophysics Data System (ADS)

    Koten, P.; Čapek, D.; Spurný, P.; Vaubaillon, J.; Popek, M.; Shrbený, L.

    2017-04-01

    Context. A bright fireball was observed above the Czech Republic on September 9, 2016, at 23:06:59 UT. Moreover, the video cameras at two different stations recorded eight fainter meteors flying on parallel atmospheric trajectories within less than 2 s. All the meteors belong to the September epsilon Perseid meteor shower. The measured proximity of all meteors during a very low activity meteor shower suggests that a cluster of meteors was observed. Aims: The goal of the paper is first to determine whether this event was a random occurrence or a real meteor cluster and second, if it was a cluster, to determine the epoch and at what distance from the Earth the separation of the particles occurred. Methods: The atmospheric trajectories of the observed meteors, masses, and relative distances of individual particles were determined using a double-station observation. According to the distances and masses of the particles, the most probable distance and time of fragmentation is determined. Results: The observed group of meteors is interpreted as the result of the orbital fragmentation of a bigger meteoroid. The fragmentation happened no earlier than 2 or 3 days before the encounter with the Earth at a distance smaller than 0.08 AU from the Earth.

  5. 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.

  6. Automatic parameter optimization in epsilon-filter for acoustical signal processing utilizing correlation coefficient.

    PubMed

    Abe, Tomomi; Hashimoto, Shuji; Matsumoto, Mitsuharu

    2010-02-01

    epsilon-filter can reduce most kinds of noise from a single-channel noisy signal while preserving signals that vary drastically such as speech signals. It can reduce not only stationary noise but also nonstationary noise. However, it has some parameters whose values are set empirically. So far, there have been few studies to evaluate the appropriateness of the parameter settings for epsilon-filter. This paper employs the correlation coefficient of the filter output and the difference between the filter input and output as the evaluation function of the parameter setting. This paper also describes the algorithm to set the optimal parameter value of epsilon-filter automatically. To evaluate the adequateness of the obtained parameter, the mean absolute error is calculated. The experimental results show that the adequate parameter in epsilon-filter can be obtained automatically by using the proposed method.

  7. SYNTHESIZING OPTIMAL STRATEGIES IN PURSUIT-EVASION GAMES BY THE EPSILON TECHNIQUE,

    DTIC Science & Technology

    A constructive method for synthesizing optimal strategies in pursuit-evasion games is described using the epsilon technique as described by Balakrishnan. An illustrative example is worked out. (Author)

  8. DNA replication: polymerase epsilon as a non-catalytic converter of the helicase.

    PubMed

    Zegerman, Philip

    2013-04-08

    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.

  9. 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.

  10. Navier-Stokes cascade analysis with a stiff k-epsilon turbulence solver

    NASA Technical Reports Server (NTRS)

    Liu, Jong-Shang; Sockol, Peter M.; Prahl, Joseph M.

    1988-01-01

    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.

  11. 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.

  12. 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.

  13. Inactivating I kappa B epsilon mutations in Hodgkin/Reed-Sternberg cells.

    PubMed

    Emmerich, Florian; Theurich, Sebastian; Hummel, Michael; Haeffker, Antje; Vry, Magnus S; Döhner, Konstanze; Bommert, Kurt; Stein, Harald; Dörken, Bernd

    2003-11-01

    The pathogenesis of Hodgkin lymphoma (HL) is still unclear. Previous investigations have demonstrated constitutive nuclear activity of the transcription factor NF kappa B (NF-kappaB) in Hodgkin/Reed-Sternberg (HRS) cells as an important prerequisite in protecting these cells from apoptosis. As a molecular mechanism leading to constitutive NF-kappaB activity in HRS cells, mutations of the NF-kappaB inhibitor I kappa B alpha (IkappaBalpha) have recently been identified in classical (c) HL-derived cell lines in a patient with cHL. In the present study, the NF-kappaB inhibitor I kappa B epsilon (IkappaBepsilon) has been analysed for somatic mutations in the same group of six patients already studied for IkappaBalpha mutations, as well as in cHL-derived cell lines. In one cHL-derived cell line (L428), a hemizygous frame-shift mutation generating a pre-terminal stop codon resulting in a severely truncated protein was found. Moreover, in the HRS cells of one patient, a hemizygous mutation affecting the 5'-splicing site of intron 1 of the IkappaBepsilon gene was found. These results, in combination with recently described IkappaBalpha mutations, indicate that defective NF-kappaB inhibitors appear more frequent than previously thought and might explain the constitutive nuclear activity of NF-kappaB in a significant proportion of cHL cases.

  14. Computational evidence for the detoxifying mechanism of epsilon class glutathione transferase toward the insecticide DDT.

    PubMed

    Li, Yanwei; Shi, Xiangli; Zhang, Qingzhu; Hu, Jingtian; Chen, Jianmin; Wang, Wenxing

    2014-05-06

    A combined quantum mechanics/molecular mechanics (QM/MM) computation of the detoxifying mechanism of an epsilon class glutathione transferases (GSTs) toward organochlorine insecticide DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, has been carried out. The exponential average barrier of the proton transfer mechanism is 15.2 kcal/mol, which is 27.6 kcal/mol lower than that of the GS-DDT conjugant mechanism. It suggests that the detoxifying reaction proceeds via a proton transfer mechanism where GSH acts as a cofactor rather than a conjugate. The study reveals that the protein environment has a strong effect on the reaction barrier. The experimentally proposed residues Arg112, Glu116 and Phe120 were found to have a strong influence on the detoxifying reaction. The influence of residues Pro13, Cys15, His53, Ile55, Glu67, Ser68, Phe115, and Leu119 was detected as well. It is worth noticing that Ile55 facilitates the detoxifying reaction most. On the basis of the structure of DDT, structure 2, (BrC6H4)2CHCCl3, is the best candidate among all the tested structures in resisting the detoxification of enzyme agGSTe2.

  15. Nasal mast cells in perennial allergic rhinitics exhibit increased expression of the Fc epsilonRI, CD40L, IL-4, and IL-13, and can induce IgE synthesis in B cells.

    PubMed Central

    Pawankar, R; Okuda, M; Yssel, H; Okumura, K; Ra, C

    1997-01-01

    Cross-linking of allergen specific IgE bound to the high affinity IgE receptor (FC epsilonRI) on the surface of mast cells with multivalent allergens results in the release of both pre-formed and newly generated mediators, and in the manifestation of allergic symptoms. The expression of Fc epsilonRI, and the synthesis of IgE are therefore critical for the development of allergic diseases. In this study, we report that nasal mast cells (NMC) from patients with perennial allergic rhinitis (PAR) expressed significantly greater levels of the Fc epsilonRI, CD40L, IL-4, and IL-13 as compared to NMC from patients with chronic infective rhinitis (CIR). The level of Fc epsilonRI expression in NMC of PAR patients strongly correlated with the levels of serum total (r = 0.8, P < 0.003) and specific IgE (r = 0.89, P < 0.0004) antibodies. In addition, stimulation of NMC with IL-4, upregulated the Fc epsilonRIalpha chain expression both at the protein and mRNA levels, as detected by flow cytometry and reverse transcriptase-polymerase chain reaction. Furthermore, NMC from PAR, but not CIR, patients induced IgE synthesis by purified B cells in the presence of Der fII (mite antigen). These results suggest novel and critical roles for mast cells in promoting the allergic reaction through the increased expression of Fc epsilonRI and by enhancing and amplifying the IgE production, within the local microenvironment. PMID:9119992

  16. Long-term pioglitazone treatment augments insulin sensitivity and PKC-epsilon and PKC-theta activation in skeletal muscles in sucrose fed rats.

    PubMed

    Marková, I; Zídek, V; Musilová, A; Simáková, M; Mlejnek, P; Kazdová, L; Pravenec, M

    2010-01-01

    It has been suggested that thiazolidinediones (TZDs) ameliorate insulin resistance in muscle tissue by suppressing muscle lipid storage and the activity of novel protein kinase C (nPKC) isoforms. To test this hypothesis, we analyzed long-term metabolic effects of pioglitazone and the activation of nPKC-epsilon and -theta isoforms in an animal model of the metabolic syndrome, the spontaneously hypertensive rat (a congenic SHR strain with wild type Cd36 gene) fed a diet with 60 % sucrose from the age of 4 to 8 months. Compared to untreated controls, pioglitazone treatment was associated with significantly increased basal (809+/-36 vs 527+/-47 nmol glucose/g/2h, P<0.005) and insulin-stimulated glycogenesis (1321+/-62 vs 749+/-60 nmol glucose/g/2h, P<0.0001) in isolated gastrocnemius muscles despite increased concentrations of muscle triglycerides (3.83+/-0.33 vs 2.25+/-0.12 micromol/g, P<0.005). Pioglitazone-treated rats exhibited significantly increased membrane/total (cytosolic plus membrane) ratio of both PKC-epsilon and PKC-theta isoforms compared to untreated controls. These results suggest that amelioration of insulin resistance after long-term pioglitazone treatment is associated with increased activation of PKC-epsilon and -theta isoforms in spite of increased lipid concentration in skeletal muscles.

  17. The epsilon subunit of DNA polymerase III Is involved in the nalidixic acid-induced SOS response in Escherichia coli.

    PubMed

    Pohlhaus, Jennifer Reineke; Long, David T; O'Reilly, Erin; Kreuzer, Kenneth N

    2008-08-01

    Quinolone antibacterial drugs such as nalidixic acid target DNA gyrase in Escherichia coli. These inhibitors bind to and stabilize a normally transient covalent protein-DNA intermediate in the gyrase reaction cycle, referred to as the cleavage complex. Stabilization of the cleavage complex is necessary but not sufficient for cell killing--cytotoxicity apparently results from the conversion of cleavage complexes into overt DNA breaks by an as-yet-unknown mechanism(s). Quinolone treatment induces the bacterial SOS response in a RecBC-dependent manner, arguing that cleavage complexes are somehow converted into double-stranded breaks. However, the only proteins known to be required for SOS induction by nalidixic acid are RecA and RecBC. In hopes of identifying additional proteins involved in the cytotoxic response to nalidixic acid, we screened for E. coli mutants specifically deficient in SOS induction upon nalidixic acid treatment by using a dinD::lacZ reporter construct. From a collection of SOS partially constitutive mutants with disruptions of 47 different genes, we found that dnaQ insertion mutants are specifically deficient in the SOS response to nalidixic acid. dnaQ encodes DNA polymerase III epsilon subunit, the proofreading subunit of the replicative polymerase. The deficient response to nalidixic acid was rescued by the presence of the wild-type dnaQ gene, confirming involvement of the epsilon subunit. To further characterize the SOS deficiency of dnaQ mutants, we analyzed the expression of several additional SOS genes in response to nalidixic acid using real-time PCR. A subset of SOS genes lost their response to nalidixic acid in the dnaQ mutant strain, while two tested SOS genes (recA and recN) continued to exhibit induction. These results argue that the replication complex plays a role in modulating the SOS response to nalidixic acid and that the response is more complex than a simple on/off switch.

  18. Functional study of a monoclonal antibody to IgE Fc receptor (Fc epsilon R2) of eosinophils, platelets, and macrophages

    PubMed Central

    1986-01-01

    An IgM mAb (BB10) was produced by immunization of mice with human eosinophils purified according to their abnormal low density ("hypodense" cells), and previously shown to exhibit increased IgE- dependent antiparasite cytotoxicity. This BB10 antibody, selected for positive fluorescence staining of hypodense blood or lung eosinophils and low or negative staining of normodense eosinophils or neutrophils, could strongly inhibit IgE-dependent cytotoxicity of human eosinophils and platelets. The specificity for the IgE Fc receptor was suggested by the high levels of inhibition of IgE rosettes formed by eosinophils after incubation with the purified IgM fraction of BB10, whereas other receptors (Fc gamma R, CR1) were not affected. On the other hand, BB10, able to inhibit rat eosinophil Fc epsilon R, did not react with the IgE Fc receptor on mast cells or basophils. A technique using radioiodinated BB10 allowed us to quantify the specific binding of BB10 to human eosinophils and platelets. Competition experiments revealed a crossinhibition between the binding of BB10 and IgE, suggesting the specificity of BB10 for the IgE binding site of eosinophil, platelet, and monocyte Fc epsilon R. Three proteins having extrapolated Mr of 32,000, 43,000-45,000, and 97,000 were found in the platelet extract eluted from a BB10 or from an IgE immunosorbent column. These findings confirm the similarities between IgE Fc receptors on human eosinophils, platelets, and macrophages, already observed with polyclonal antibodies directed against the B lymphocyte Fc epsilon receptor. They suggest, moreover, that the mAb BB10 can represent a good reagent for further investigations on the structure and the functions of this IgE Fc receptor (Fc epsilon R2). PMID:2425032

  19. 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.

  20. New insights into Clostridium perfringens epsilon toxin activation and action on the brain during enterotoxemia.

    PubMed

    Freedman, John C; McClane, Bruce A; Uzal, Francisco A

    2016-10-01

    Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is responsible for diseases that occur mostly in ruminants. ETX is produced in the form of an inactive prototoxin that becomes proteolytically-activated by several proteases. A recent ex vivo study using caprine intestinal contents demonstrated that ETX prototoxin is processed in a step-wise fashion into a stable, active ∼27 kDa band on SDS-PAGE. When characterized further by mass spectrometry, the stable ∼27 kDa band was shown to contain three ETX species with varying C-terminal residues; each of these ETX species is cytotoxic. This study also demonstrated that, in addition to trypsin and chymotrypsin, proteases such as carboxypeptidases are involved in processing ETX prototoxin. Once absorbed, activated ETX species travel to several internal organs, including the brain, where this toxin acts on the vasculature to cross the blood-brain barrier, produces perivascular edema and affects several types of brain cells including neurons, astrocytes, and oligodendrocytes. In addition to perivascular edema, affected animals show edema within the vascular walls. This edema separates the astrocytic end-feet from affected blood vessels, causing hypoxia of nervous system tissue. Astrocytes of rats and sheep affected by ETX show overexpression of aquaporin-4, a membrane channel protein that is believed to help remove water from affected perivascular spaces in an attempt to resolve the perivascular edema. Amyloid precursor protein, an early astrocyte damage indicator, is also observed in the brains of affected sheep. These results show that ETX activation in vivo seems to be more complex than previously thought and this toxin acts on the brain, affecting vascular permeability, but also damaging neurons and other cells.

  1. Human IgE-binding protein: A soluble lectin exhibiting a highly conserved interspecies sequence and differential recognition of IgE glycoforms

    SciTech Connect

    Robertson, M.W.; Albrandt, K.; Keller, D.; Liu, Fu-Tong )

    1990-09-04

    IgE-binding protein ({epsilon}BP) refers to a protein originally identified in rat basophilic leukemia cells by virtue of its affinity for IgE. It is now known to be a {beta}-galactoside-binding lectin equivalent to carbohydrate-binding protein 35 (CBP 35). More recently, its identity to Mac-2, a macrophage cell-surface protein, has been established. cDNA coding for human {epsilon}BP has been cloned from a human HeLa cell cDNA library and contains an open reading frame of 750 base pairs encoding a 250 amino acid protein. Like the rat and murine counterparts, the human {epsilon}BP amino acid sequence can be divided into two domains with the amino-terminal domain consisting of a highly conserved repetitive sequence (YPGXXXPGA) and the carboxyl-terminal domain containing sequences shared by other S-type lectins. The human {epsilon}BP sequence exhibits extensive homology to murine and rat {epsilon}BP with 84% and 82% identity, respectively. The homology is particularly striking in the carboxyl-terminal domain where 95% identity is found between human and murine sequences in a stretch of over 70 amino acids. A survey of {epsilon}BP mRNA expression from several lymphocyte cell lines revealed that the level of {epsilon}BP transcription may reflect a relationship between cell differentiation and {epsilon}BP expression. Finally, human {epsilon}BP was purified from several human cell lines and shown to possess lactose-binding characteristics and cross-species reactivity to murine IgE. Surprisingly, three different human myeloma IgE proteins did not show reactivity to human {epsilon}BP. However, after neuraminidase treatment of each human IgE, pronounced binding to {epsilon}BP was observed, thereby indicating that only specific IgE glycoforms can be recognized by {epsilon}BP.

  2. 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.

  3. Altered Prion Protein Expression Pattern in CSF as a Biomarker for Creutzfeldt-Jakob Disease

    PubMed Central

    Torres, Mauricio; Cartier, Luis; Matamala, José Manuel; Hernández, Nancy; Woehlbier, Ute; Hetz, Claudio

    2012-01-01

    Creutzfeldt-Jakob disease (CJD) is the most frequent human Prion-related disorder (PrD). The detection of 14-3-3 protein in the cerebrospinal fluid (CSF) is used as a molecular diagnostic criterion for patients clinically compatible with CJD. However, there is a pressing need for the identification of new reliable disease biomarkers. The pathological mechanisms leading to accumulation of 14-3-3 protein in CSF are not fully understood, however neuronal loss followed by cell lysis is assumed to cause the increase in 14-3-3 levels, which also occurs in conditions such as brain ischemia. Here we investigated the relation between the levels of 14-3-3 protein, Lactate dehydrogenase (LDH) activity and expression of the prion protein (PrP) in CSF of sporadic and familial CJD cases. Unexpectedly, we found normal levels of LDH activity in CJD cases with moderate levels of 14-3-3 protein. Increased LDH activity was only observed in a percentage of the CSF samples that also exhibited high 14-3-3 levels. Analysis of the PrP expression pattern in CSF revealed a reduction in PrP levels in all CJD cases, as well as marked changes in its glycosylation pattern. PrP present in CSF of CJD cases was sensitive to proteases. The alterations in PrP expression observed in CJD cases were not detected in other pathologies affecting the nervous system, including cases of dementia and tropical spastic paraparesis/HTLV-1 associated myelopathy (HAM/TSP). Time course analysis in several CJD patients revealed that 14-3-3 levels in CSF are dynamic and show a high degree of variability during the end stage of the disease. Post-mortem analysis of brain tissue also indicated that 14-3-3 protein is upregulated in neuronal cells, suggesting that its expression is modulated during the course of the disease. These results suggest that a combined analysis of 14-3-3 and PrP expression pattern in CSF is a reliable biomarker to confirm the clinical diagnosis of CJD patients and follow disease progression

  4. 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.

  5. Enhancing the carotenoid content of Brassica napus seeds by downregulating lycopene epsilon cyclase.

    PubMed

    Yu, Bianyun; Lydiate, Derek J; Young, Lester W; Schäfer, Ulrike A; Hannoufa, Abdelali

    2008-08-01

    The accumulation of carotenoids in higher plants is regulated by the environment, tissue type and developmental stage. In Brassica napus leaves, beta-carotene and lutein were the main carotenoids present while petals primarily accumulated lutein and violaxanthin. Carotenoid accumulation in seeds was developmentally regulated with the highest levels detected at 35-40 days post anthesis. The carotenoid biosynthesis pathway branches after the formation of lycopene. One branch forms carotenoids with two beta rings such as beta-carotene, zeaxanthin and violaxanthin, while the other introduces both beta- and epsilon-rings in lycopene to form alpha-carotene and lutein. By reducing the expression of lycopene epsilon-cyclase (epsilon-CYC) using RNAi, we investigated altering carotenoid accumulation in seeds of B. napus. Transgenic seeds expressing this construct had increased levels of beta-carotene, zeaxanthin, violaxanthin and, unexpectedly, lutein. The higher total carotenoid content resulting from reduction of epsilon-CYC expression in seeds suggests that this gene is a rate-limiting step in the carotenoid biosynthesis pathway. epsilon-CYC activity and carotenoid production may also be related to fatty acid biosynthesis in seeds as transgenic seeds showed an overall decrease in total fatty acid content and minor changes in the proportions of various fatty acids.

  6. Phase Stability of Epsilon and Gamma Hniw (CL-20) at High-Pressure and Temperature

    NASA Astrophysics Data System (ADS)

    Gump, Jared C.; Stoltz, Chad A.; Peiris, Suhithi M.

    2007-12-01

    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 (α, β, γ, ɛ, ζ). 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 °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 °C then underwent a transition to the gamma phase above 125 °C and then thermal decomposition occurred above 150 °C. Upon compression, the gamma phase underwent a phase transition at both ambient temperature and 140 °C. Pressure—volume data for the epsilon and gamma phase at ambient temperature and the epsilon phase at 75 °C were fit to the Birch-Murnaghan formalism to obtain isothermal equations of state.

  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. 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

  9. Comparative Genomics of the Anopheline Glutathione S-Transferase Epsilon Cluster

    PubMed Central

    Ayres, Constância; Müller, Pie; Dyer, Naomi; Wilding, Craig; Rigden, Daniel; Donnelly, Martin

    2011-01-01

    Enzymes of the glutathione S-transferase (GST) family play critical roles in detoxification of xenobiotics across many taxa. While GSTs are ubiquitous both in animals and plants, the GST epsilon class (GSTE) is insect-specific and has been associated with resistance to chemical insecticides. While both Aedes aegypti and Anopheles gambiae GSTE clusters consist of eight members, only four putative orthologs are identifiable between the species, suggesting independent expansions of the class in each lineage. We used a primer walking approach, sequencing almost the entire cluster from three Anopheles species (An. stephensi, An. funestus (both Cellia subgenus) and An. plumbeus (Anopheles subgenus)) and compared the sequences to putative orthologs in An. gambiae (Cellia) in an attempt to trace the evolution of the cluster within the subfamily Anophelinae. Furthermore, we measured transcript levels from the identified GSTE loci by real time reverse transcription PCR to determine if all genes were similarly transcribed at different life stages. Among the species investigated, gene order and orientation were similar with three exceptions: (i) GSTE1 was absent in An. plumbeus; (ii) GSTE2 is duplicated in An. plumbeus and (iii) an additional transcriptionally active pseudogene (ψAsGSTE2) was found in An. stephensi. Further statistical analysis and protein modelling gave evidence for positive selection on codons of the catalytic site in GSTE5 albeit its origin seems to predate the introduction of chemical insecticides. Gene expression profiles revealed differences in expression pattern among genes at different life stages. With the exception of GSTE1, ψAsGSTE2 and GSTE2b, all Anopheles species studied share orthologs and hence we assume that GSTE expansion generally predates radiation into subgenera, though the presence of GSTE1 may also suggest a recent duplication event in the Old World Cellia subgenus, instead of a secondary loss. The modifications of the catalytic site

  10. Enhanced gene delivery using biodegradable poly(ester amine)s (PEAs) based on low-molecular-weight polyethylenimine and poly(epsilon-caprolactone)-pluronic-poly(epsilon-caprolactone).

    PubMed

    Liu, Tingting; Yu, Xiujun; Kan, Bing; Guo, Qingfa; Wang, Xiuhong; Shi, Shuai; Guo, Gang; Luo, Feng; Zhao, Xia; Wei, Yuquan; Qian, Zhiyong

    2010-08-01

    In this paper, the poly(ester amine)s (PEAs) were successfully prepared from low-molecular-weight PEI (Mn = 2000) and Poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol)-poly(epsilon-caprolactone) (PCFC) copolymers using isophorone diisocyanate (IPDI) as cross-linker. The obtained PEAs copolymers are biodegradable and water-soluble. The PEAs/DNA complexes showed effective and stable DNA condensation with the particle size < or = 200 nm and zeta potential > or =10 mV, indicating its potential for intracellular delivery. Compared to the unmodified low-molecular-weight PEI, PEAs displayed similarly low cytotoxicity in all two cell lines (293T: Human kidney carcinoma, HUVEC: Human umbilical vein Endothelial cell) and revealed much higher transfection efficiency in 293T cell lines. Therefore these PEAs might be a novel safe and efficient polymeric gene delivery vectors.

  11. Directional and monochromatic thermal emitter from epsilon-near-zero conditions in semiconductor hyperbolic metamaterials.

    PubMed

    Campione, Salvatore; Marquier, Francois; Hugonin, Jean-Paul; Ellis, A Robert; Klem, John F; Sinclair, Michael B; Luk, Ting S

    2016-10-05

    The development of novel thermal sources that control the emission spectrum and the angular emission pattern is of fundamental importance. In this paper, we investigate the thermal emission properties of semiconductor hyperbolic metamaterials (SHMs). Our structure does not require the use of any periodic corrugation to provide monochromatic and directional emission properties. We show that these properties arise because of epsilon-near-zero conditions in SHMs. The thermal emission is dominated by the epsilon-near-zero effect in the doped quantum wells composing the SHM. Furthermore, different properties are observed for s and p polarizations, following the characteristics of the strong anisotropy of hyperbolic metamaterials.

  12. GABAA receptor epsilon subunit expression in identified peptidergic neurons of the rat hypothalamus.

    PubMed

    Moragues, Nathalie; Ciofi, Philippe; Lafon, Pierrette; Tramu, Gérard; Garret, Maurice

    2003-03-28

    Dual-labeling immunohistochemical or in situ hybridization studies for the recently cloned epsilon-subunit and several neuropeptides were performed in the rat hypothalamus. We revealed an extensive co-expression (>90%) with hypocretin (Hcrt), oxytocin (OT), the gonadotropin-releasing hormone (GnRH), and the melanin-concentrating hormone (MCH) peptides, whereas occasional co-expression (<10%) with cocaine-amphetamine-regulated transcript (CART) was found. Our results suggest that novel GABA(A) receptor subtypes comprising epsilon-subunit are important for metabolic and neuroendocrine functions.

  13. Directional and monochromatic thermal emitter from epsilon-near-zero conditions in semiconductor hyperbolic metamaterials

    PubMed Central

    Campione, Salvatore; Marquier, Francois; Hugonin, Jean-Paul; Ellis, A. Robert; Klem, John F.; Sinclair, Michael B.; Luk, Ting S.

    2016-01-01

    The development of novel thermal sources that control the emission spectrum and the angular emission pattern is of fundamental importance. In this paper, we investigate the thermal emission properties of semiconductor hyperbolic metamaterials (SHMs). Our structure does not require the use of any periodic corrugation to provide monochromatic and directional emission properties. We show that these properties arise because of epsilon-near-zero conditions in SHMs. The thermal emission is dominated by the epsilon-near-zero effect in the doped quantum wells composing the SHM. Furthermore, different properties are observed for s and p polarizations, following the characteristics of the strong anisotropy of hyperbolic metamaterials. PMID:27703223

  14. Broadband epsilon-near-zero metamaterials with steplike metal-dielectric multilayer structures

    NASA Astrophysics Data System (ADS)

    Sun, Lei; Gao, Jie; Yang, Xiaodong

    2013-04-01

    The concept of the broadband epsilon-near-zero meta-atom consisting of layered stacks with specified metallic filling ratio and thickness is proposed based on the Bergman spectral representation of the effective permittivity. The steplike metal-dielectric multilayer structures are designed to achieve realistic broadband epsilon-near-zero meta-atoms in optical frequency range. These meta-atoms can be integrated as building blocks for unconventional optical components with exotic electromagnetic properties over a wide frequency range, such as the demonstrated broadband directional emission and phase front shaping.

  15. A new formulation for the epsilon method applied to the minimum-time-to-climb problem

    NASA Technical Reports Server (NTRS)

    Taylor, L. W., Jr.; Smith, H. J.

    1978-01-01

    Balakrishnan's epsilon technique is used to compute minimum-time profiles for the F-104 airplane. This technique differs from the classical gradient method in that a quadratic penalty on the error in satisfying the equation of motion is included in the cost function to be minimized as a means of eliminating the requirement of satisfying the equations of motion. Although the number of unknown independent functions is increased to include the state variables, the evaluation of the gradient of the cost function is simplified, resulting in considerable computational savings, thereby making it appear feasible to use the epsilon method for real-time application.

  16. Directional and monochromatic thermal emitter from epsilon-near-zero conditions in semiconductor hyperbolic metamaterials

    SciTech Connect

    Campione, Salvatore; Marquier, Francois; Hugonin, Jean -Paul; Ellis, A. Robert; Klem, John F.; Sinclair, Michael B.; Luk, Ting S.

    2016-10-05

    The development of novel thermal sources that control the emission spectrum and the angular emission pattern is of fundamental importance. In this paper, we investigate the thermal emission properties of semiconductor hyperbolic metamaterials (SHMs). Our structure does not require the use of any periodic corrugation to provide monochromatic and directional emission properties. We show that these properties arise because of epsilon-near-zero conditions in SHMs. The thermal emission is dominated by the epsilon-near-zero effect in the doped quantum wells composing the SHM. In conclusion, different properties are observed for s and p polarizations, following the characteristics of the strong anisotropy of hyperbolic metamaterials.

  17. Experimental Investigation of the Kinetics of the Shock-Induced Alpha to Epsilon Phase Transformation in Armco Iron

    DTIC Science & Technology

    1977-12-15

    163 kbar while Mao, et al. 3 3 report no signs of alpha iron above 145 kbar. Giles, et al. 32 found that the epsilon phase persisted down to 45 kbar...Bowden, et al.31 concluded that markings from recovered shocked alpha iron samples resulted from shear transformation to the epsilon phase, body

  18. 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.

  19. 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

  20. Mapping of the high affinity Fc epsilon receptor binding site to the third constant region domain of IgE.

    PubMed Central

    Nissim, A; Jouvin, M H; Eshhar, Z

    1991-01-01

    Identification of the precise region(s) on the IgE molecule that take part in the binding of IgE to its high affinity receptor (Fc epsilon RI) may lead to the design of IgE analogues able to block the allergic response. To localize the Fc epsilon RI-binding domain of mouse IgE, we attempted to confer on human IgE, which normally does not bind to the rodent receptor, the ability to bind to the rat Fc epsilon RI. Employing exon shuffling, we have expressed chimeric epsilon-heavy chain genes composed of a mouse (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH domain, and human C epsilon in which various domains were replaced by their murine counterparts. This has enabled us to test the Fc epsilon RI-binding of each mouse IgE domain while maintaining the overall conformation of the molecule. All of the chimeric IgE molecules which contain the murine C epsilon 3, bound equally to both the rodent and human receptor, as well as to monoclonal antibodies recognizing a site on IgE which is identical or very close to the Fc epsilon RI binding site. Deletion of the second constant region domain did not impair either the binding capacity of the mutated IgE or its ability to mediate mast cell degradation. These results assign the third epsilon domain of IgE as the principal region involved in the interaction with the Fc epsilon RI. Images PMID:1824934

  1. 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.

  2. 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).

  3. 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…

  4. 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.

  5. Epsilon metal waste form for immobilization of noble metals from used nuclear fuel

    NASA Astrophysics Data System (ADS)

    Crum, Jarrod V.; Strachan, Denis; Rohatgi, Aashish; Zumhoff, Mac

    2013-10-01

    Epsilon metal (ɛ-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, thus the processing problems related to their 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 alloying temperatures, 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).

  6. Molecular cloning and nucleotide sequencing of human immunoglobulin epsilon chain cDNA.

    PubMed Central

    Seno, M; Kurokawa, T; Ono, Y; Onda, H; Sasada, R; Igarashi, K; Kikuchi, M; Sugino, Y; Nishida, Y; Honjo, T

    1983-01-01

    DNA complementary to mRNA of human immunoglobulin E heavy chain (epsilon chain) isolated and purified from U266 cells has been synthesized and inserted into the PstI site of pBR322 by G-C tailing. This recombinant plasmid was used to transform E. coli chi 1776 to screen 1445 tetracycline resistant colonies. Nine clones (pGETI - 9) containing cDNA coding for the human epsilon chain were recognized by colony hybridization and Southern blotting analysis with a nick-translated human IgE genome fragment. The nucleotide sequence of the longest cDNA contained in pGET2 was determined. The results indicate that the sequence of 1657 nucleotides codes for 494 amino acids covering a part of the variable region and all of the constant region of the human epsilon chain. Most of the amino acid sequence deduced from the nucleotide sequence is in substantial agreement with that reported. Furthermore a termination codon after the -COOH terminal amino acid marks the beginning of a 3' untranslated region of 125 nucleotides with a poly A tail. Taking this into account, the structure of the human epsilon chain mRNA, except a part of the 5' end, is conserved fairly well in the cDNA insert in pGET2. Images PMID:6300763

  7. 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…

  8. Analysis of Epsilon Aurigae light curve from the Solar Mass Ejection Imager

    NASA Astrophysics Data System (ADS)

    Clover, John; Jackson, B. V.; Buffington, A.; Hick, P. P.; Kloppenborg, B.; Stencel, R.

    2011-01-01

    The Solar Mass Ejection Imager (SMEI) was launched aboard the Coriolis spacecraft in 2003. It is equipped with 3 CCD cameras to measure the brightness of Thomson-scattered electrons in the heliosphere. Each CCD images a strip of the sky that is 3°x60°. The three cameras are mounted on the satellite with their fields of view aligned end-to-end so that SMEI sweeps nearly the entire sky each 102 minute orbit. SMEI has now accumulated stellar time series for about 5700 bright stars, including epsilon Aurigae, for each orbit where data is available. SMEI data provide nearly year-round coverage of epsilon Aurigae. The baffled SMEI optics provide more accurate photometric data than ground-based observations, particularly at mid-eclipse when epsilon Aurigae is close to the Sun. We present an analysis of the brightness variations of the epsilon Aurigae system, before and during the eclipse. The University of Denver participants are grateful for support under NSFgrant 10-16678 and the bequest of William Hershel Womble in support of astronomy at the University of Denver.

  9. 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.

  10. 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.

  11. Mapping of the continuous epitopes displayed on the Clostridium perfringens type D epsilon-toxin.

    PubMed

    Alves, Guilherme Guerra; Machado-de-Ávila, Ricardo Andrez; Chávez-Olórtegui, Carlos Delfin; Silva, Rodrigo Otávio Silveira; Lobato, Francisco Carlos Faria

    2017-02-03

    The epsilon toxin, produced by Clostridium perfringens, is responsible for enterotoxemia in ruminants and is a potential bioterrorism agent. In the present study, 15 regions of the toxin were recognized by antibodies present in the serum, with different immunodominance scales, and may be antigen determinants that can be used to formulate subunit vaccines.

  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.

  13. FcR epsilon+ lymphocytes and regulation of the IgE antibody system. IV. Delineation of target cells and mechanisms of action of SFA and EFA in inhibiting in vitro induction of FcR epsilon expression.

    PubMed

    Marcelletti, J F; Katz, D H

    1984-12-01

    SFA and EFA are derived from distinct mouse T cell hybridomas secreting one or the other (but not both) factor, and although both are capable of inhibiting FcR epsilon expression by unfractionated spleen cells induced by monomeric IgE, neither was inhibitory for EIRT-induced FcR epsilon expression by T cells in the same cell population. This suggests that the final target cell for the inhibitory effects of SFA and EFA is the FcR epsilon+ B lymphocyte. T cells are required for both SFA- and EFA-mediated FcR epsilon inhibition, and more precisely, as shown in this study, SFA stimulates Lyt-1+ cells in the presence or absence of IgE to produce a suppressive effector molecule (SEM), and EFA together with IgE stimulates Lyt-2+ cells to produce an enhancing effector molecule (EEM), both of which can directly inhibit FcR epsilon expression by B cells. SFA and SEM can inhibit both IgE- and EIRB-induced FcR epsilon expression by B cells, indicating that SFA may act by blocking the EIRB-mediated expansion of the FcR epsilon+ B cell population. EFA and EEM, in contrast, can inhibit IgE-induced but not EIRB-induced FcR epsilon expression, indicating that EFA may act at some point before the release of EIR, perhaps involving those FcR epsilon+ B cells that respond to IgE and produce EIRB. Finally, although neither SFA and EFA display IgE binding properties, both SEM and EEM, in contrast, are IgE binding factors (IgE-BF) and may be homologous to the suppressive IgE binding factor and potentiating IgE binding factor described by other investigators. The possible interrelationships between these various cells and factors are discussed.

  14. PKC Epsilon: A Novel Oncogenic Player in Prostate Cancer

    DTIC Science & Technology

    2014-09-01

    Numerous evidences have implicated PKCε with multiple cellular processes including cell proliferation and survival in human cancers13, 21, 23, 27. We have...prostate cancer. These findings suggest a crucial role of PKCε in several important cellular processes relevant to prostate cancer progression...therapy. Cancer research 65: 8366-8371. 22 Caino, M. C., Lopez- Haber , C., Kim, J., Mochly-Rosen, D., Kazanietz, M. G. (2012). Proteins kinase

  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.

  16. Inhibitory transmission in locus coeruleus neurons expressing GABAA receptor epsilon subunit has a number of unique properties.

    PubMed

    Belujon, P; Baufreton, J; Grandoso, L; Boué-Grabot, E; Batten, T F C; Ugedo, L; Garret, M; Taupignon, A I

    2009-10-01

    Fast inhibitory synaptic transmission in the brain relies on ionotropic GABA(A) receptors (GABA(A)R). Eighteen genes code for GABA(A)R subunits, but little is known about the epsilon subunit. Our aim was to identify the synaptic transmission properties displayed by native receptors incorporating epsilon. Immunogold localization detected epsilon at synaptic sites on locus coeruleus (LC) neurons. In situ hybridization revealed prominent signals from epsilon, and mRNAs, some low beta1 and beta3 signals, and no gamma signal. Using in vivo extracellular and in vitro patch-clamp recordings in LC, we established that neuron firing rates, GABA-activated currents, and mIPSC charge were insensitive to the benzodiazepine flunitrazepam (FLU), in agreement with the characteristics of recombinant receptors including an epsilon subunit. Surprisingly, LC provided binding sites for benzodiazepines, and GABA-induced currents were potentiated by diazepam (DZP) in the micromolar range. A number of GABA(A)R ligands significantly potentiated GABA-induced currents, and zinc ions were only active at concentrations above 1 muM, further indicating that receptors were not composed of only alpha and beta subunits, but included an epsilon subunit. In contrast to recombinant receptors including an epsilon subunit, GABA(A)R in LC showed no agonist-independent opening. Finally, we determined that mIPSCs, as well as ensemble currents induced by ultra-fast GABA application, exhibited surprisingly slow rise times. Our work thus defines the signature of native GABA(A)R with a subunit composition including epsilon: differential sensitivity to FLU and DZP and slow rise time of currents. We further propose that alpha(3,) beta(1/3,) and epsilon subunits compose GABA(A)R in LC.

  17. T cell receptor complexes containing Fc epsilon RI gamma homodimers in lieu of CD3 zeta and CD3 eta components: a novel isoform expressed on large granular lymphocytes

    PubMed Central

    1992-01-01

    CD3 zeta and CD3 eta form disulfide-linked homo- or heterodimers important in targeting partially assembled Ti alpha-beta/CD3 gamma delta epsilon T cell receptor (TCR) complexes to the cell surface and transducing stimulatory signals after antigen recognition. Here we identify a new TCR isoform expressed on splenic CD2+, CD3/Ti alpha- beta+, CD4-, CD8-, CD16+, NK1.1+ mouse large granular lymphocytes (LGL), which are devoid of CD3 zeta and CD3 eta proteins. The TCRs of this subset contain homodimers of the gamma subunit of the high affinity receptor for IgE (Fc epsilon RI gamma) in lieu of CD3 zeta and/or CD3 eta proteins. The LGL display natural killer-like activity and are cytotoxic for B cell hybridomas producing anti-CD3 epsilon and anti-CD16 monoclonal antibodies, demonstrating the signaling capacity of both TCR and CD16 in this cell type. These findings provide evidence for an additional level of complexity of TCR signal transduction isoforms in naturally occurring T cell subsets. PMID:1530959

  18. 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.

  19. 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.

  20. 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.

  1. Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

    PubMed Central

    Cai, H; Smola, U; Wixler, V; Eisenmann-Tappe, I; Diaz-Meco, M T; Moscat, J; Rapp, U; Cooper, G M

    1997-01-01

    The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo. PMID:9001227

  2. Directional and monochromatic thermal emitter from epsilon-near-zero conditions in semiconductor hyperbolic metamaterials

    DOE PAGES

    Campione, Salvatore; Marquier, Francois; Hugonin, Jean -Paul; ...

    2016-10-05

    The development of novel thermal sources that control the emission spectrum and the angular emission pattern is of fundamental importance. In this paper, we investigate the thermal emission properties of semiconductor hyperbolic metamaterials (SHMs). Our structure does not require the use of any periodic corrugation to provide monochromatic and directional emission properties. We show that these properties arise because of epsilon-near-zero conditions in SHMs. The thermal emission is dominated by the epsilon-near-zero effect in the doped quantum wells composing the SHM. In conclusion, different properties are observed for s and p polarizations, following the characteristics of the strong anisotropy ofmore » hyperbolic metamaterials.« less

  3. The Invisible Monster Has Two Faces: Observations of epsilon Aurigae with the Herschel Space Observatory

    NASA Astrophysics Data System (ADS)

    Hoard, D. W.; Ladjal, D.; Stencel, R. E.; Howell, S. B.

    2012-04-01

    We present Herschel Space Observatory photometric observations of the unique, long-period eclipsing binary star epsilon Aurigae. Its extended spectral energy distribution is consistent with our previously published cool (550 K) dust disk model. We also present an archival infrared spectral energy distribution of the side of the disk facing the bright F-type star in the binary, which is consistent with a warmer (1150 K) disk model. The lack of strong molecular emission features in the Herschel bands suggests that the disk has a low gas-to-dust ratio. The spectral energy distribution and Herschel images imply that the 250 GHz radio detection reported by Altenhoff et al. is likely contaminated by infrared-bright, extended background emission associated with a nearby nebular region and should be considered an upper limit to the true flux density of epsilon Aur.

  4. Hyperbranched poly(epsilon-caprolactone) as a nonmigrating alternative plasticizer for phthalates in flexible PVC.

    PubMed

    Choi, Jeongsoo; Kwak, Seung-Yeop

    2007-05-15

    Hyperbranched (dendritic) poly(epsilon-caprolactone)s (HPCLs) were synthesized to have architectural variations, which are the different lengths of linear segments and different numbers of branches, and were used as plasticizers for flexible poly(vinyl chloride) (PVC). The plasticization efficiency estimated by the lowering of glass transition temperature and the enhancement in ultimate elongation indicated that the HPCLs with the shorter linear segments and the larger number of branches imparted as high flexibility as di(ethylhexyl) phthalate (DEHP) and much higher flexibility than their linear analogue, linear poly(epsilon-caprolactone), which is one of currently used polymer plasticizers. Volatility, extractability, and exudation tests for PVC/HPCL samples showed that there was no plasticizer migration even at very harsh condition, while ca. 7-78% of additives in PVC/DEHP was migrated out of samples, indicating that the HPCL can be used as an alternative plasticizer to remove the potential health risk from migrating phthalates during end use.

  5. 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.

  6. THE INVISIBLE MONSTER HAS TWO FACES: OBSERVATIONS OF {epsilon} AURIGAE WITH THE HERSCHEL SPACE OBSERVATORY

    SciTech Connect

    Hoard, D. W.; Ladjal, D.; Stencel, R. E.; Howell, S. B.

    2012-04-01

    We present Herschel Space Observatory photometric observations of the unique, long-period eclipsing binary star {epsilon} Aurigae. Its extended spectral energy distribution is consistent with our previously published cool (550 K) dust disk model. We also present an archival infrared spectral energy distribution of the side of the disk facing the bright F-type star in the binary, which is consistent with a warmer (1150 K) disk model. The lack of strong molecular emission features in the Herschel bands suggests that the disk has a low gas-to-dust ratio. The spectral energy distribution and Herschel images imply that the 250 GHz radio detection reported by Altenhoff et al. is likely contaminated by infrared-bright, extended background emission associated with a nearby nebular region and should be considered an upper limit to the true flux density of {epsilon} Aur.

  7. First detection of the astrometric orbit of the single-lined spectroscopic binary epsilon Librae

    NASA Technical Reports Server (NTRS)

    Castelaz, Michael W.

    1989-01-01

    The Multichannel Astrometric Photometer of the University of Pittsburgh's Allegheny Observatory was used to successfully measure for the first time the astrometric orbit of the single-lined spectroscopic binary epsilon Lib. Solution of the orbit indicates that the secondary component is a 0.5-solar-mass star, assuming that the F5 IV primary is 1.3 solar mass. If the secondary is on the main sequence, then it is later than a K5 star, but earlier than an M2 star. In addition to detection of the astrometric orbit of epsilon Lib, two references stars used in the observations, AO 754 and AO 755, are found to be a common-proper-motion pair.

  8. Turbulent small-scale neutral and ion density fluctuations as measured during MAC/Epsilon

    NASA Technical Reports Server (NTRS)

    Luebken, F.-J.; Hillert, W.; Vonzahn, U.; Blix, T. A.; Thrane, E. V.

    1989-01-01

    During the MAC/Epsilon campaign (Fall 1987, from Andoya, Northern Norway, 69 N, 16 E) a total of four altitude profiles of neutral gas number densities and six profiles of ion number densities were measured with high spatial resolution in the height range from 60 to 120 km. First results of these rocket-borne experiments are presented with emphasis on small scale turbulent density variations and related turbulent parameter as structure function constants and energy dissipation rates.

  9. Epsilon-near-zero strong coupling in metamaterial-semiconductor hybrid structures.

    PubMed

    Jun, Young Chul; Reno, John; Ribaudo, Troy; Shaner, Eric; Greffet, Jean-Jacques; Vassant, Simon; Marquier, Francois; Sinclair, Mike; Brener, Igal

    2013-01-01

    We present a new type of electrically tunable strong coupling between planar metamaterials and epsilon-near-zero modes that exist in a doped semiconductor nanolayer. The use of doped semiconductors makes this strong coupling tunable over a wide range of wavelengths through the use of different doping densities. We also modulate this coupling by depleting the doped semiconductor layer electrically. Our hybrid approach incorporates strong optical interactions into a highly tunable, integrated device platform.

  10. 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).

  11. 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.

  12. 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.

  13. 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].

  14. IgE isotype suppression in anti-epsilon-treated mice.

    PubMed Central

    Bozelka, B E; McCants, M L; Salvaggio, J E; Lehrer, S B

    1982-01-01

    Two groups of CBA/J mice received a total of eight intraperitoneal (i.p.) injections of heavy-chain-specific rabbit anti-IgE or rabbit gammaglobulin within 48 hr of birth through day 38. A third group of animals was untreated. All mice were subsequently immunized with four i.p. injections of castor allergen plus aluminum hydroxide. Results indicate that anti-treatments severely suppressed murine serum IgE levels as compared with control mice. In addition anti-epsilon-treated mice were initially unable to produce detectable reaginic antibody upon immunization with castor bean allergens (CA). Upon further CA immunization, these animals did produce an IgE antibody response, but this was still lower than that detected in control immunized mice. Other immunoglobulin levels in the anti-epsilon-treated mice were not suppressed as compared with those in the control mice. These results suggest that neonatally administered anti-epsilon antisera selectively diminished total IgE levels as well as antigen-induced IgE antibodies in mice. PMID:6807838

  15. Direct measurement of the alpha-epsilon transition stress and kinetics for shocked iron

    SciTech Connect

    Jensen, Brian J; Gray, Ill, George T; Hixson, Robert S

    2009-01-01

    Iron undergoes a polymorphic phase transformation from alpha phase (bcc) to the epsilon phase (hcp) when compressed to stresses exceeding 13 CPa. Bccause the epsilon phase is denser than the alpha phase, a single shock wave is unstable and breaks up into an elastic wave, a plastic wave, and a phase transition wave. Examination of this structured wave coupled with various phase transformation models has been used to indirectly examine the transition kinetics. Recently, multimillion atom simulations (molecular dynamics) have been used to examine the shock-induced transition in single crystal iron illustrating an orientation dependence of the transition stress, mechanisms, and kinetics. The objective of the current work was to perform plate impact experiments to examine the shock-response of polycrystalline and single crystal iron with nanosecond resolution for impact stresses spanning the {alpha} - {epsilon} transition. The current data reveal an orientation dependence of the transition stress coupled with a transition time that is nonlinearly dependent on the impact stress with a duration ranging from picoseconds to hundreds of nanoseconds. The higher transition stress for iron[100] is in agreement with the predictions from MD calculations that describe an orientation dependence of the transition stress. However, MD calculations do not capture the complexity of the continuum states achieved or the transition kinetics. Further results and implications are discussed in this paper.

  16. Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines

    PubMed Central

    Ferreira, Marcos Roberto A.; Moreira, Gustavo Marçal S. G.; da Cunha, Carlos Eduardo P.; Mendonça, Marcelo; Salvarani, Felipe M.; Moreira, Ângela N.; Conceição, Fabricio R.

    2016-01-01

    Clostridium perfringens is a spore-forming, commensal, ubiquitous bacterium that is present in the gastrointestinal tract of healthy humans and animals. This bacterium produces up to 18 toxins. The species is classified into five toxinotypes (A–E) according to the toxins that the bacterium produces: alpha, beta, epsilon, or iota. Each of these toxinotypes is associated with myriad different, frequently fatal, illnesses that affect a range of farm animals and humans. Alpha, beta, and epsilon toxins are the main causes of disease. Vaccinations that generate neutralizing antibodies are the most common prophylactic measures that are currently in use. These vaccines consist of toxoids that are obtained from C. perfringens cultures. Recombinant vaccines offer several advantages over conventional toxoids, especially in terms of the production process. As such, they are steadily gaining ground as a promising vaccination solution. This review discusses the main strategies that are currently used to produce recombinant vaccines containing alpha, beta, and epsilon toxins of C. perfringens, as well as the potential application of these molecules as vaccines for mammalian livestock animals. PMID:27879630

  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.

  18. Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines.

    PubMed

    Ferreira, Marcos Roberto A; Moreira, Gustavo Marçal S G; Cunha, Carlos Eduardo P da; Mendonça, Marcelo; Salvarani, Felipe M; Moreira, Ângela N; Conceição, Fabricio R

    2016-11-21

    Clostridium perfringens is a spore-forming, commensal, ubiquitous bacterium that is present in the gastrointestinal tract of healthy humans and animals. This bacterium produces up to 18 toxins. The species is classified into five toxinotypes (A-E) according to the toxins that the bacterium produces: alpha, beta, epsilon, or iota. Each of these toxinotypes is associated with myriad different, frequently fatal, illnesses that affect a range of farm animals and humans. Alpha, beta, and epsilon toxins are the main causes of disease. Vaccinations that generate neutralizing antibodies are the most common prophylactic measures that are currently in use. These vaccines consist of toxoids that are obtained from C. perfringens cultures. Recombinant vaccines offer several advantages over conventional toxoids, especially in terms of the production process. As such, they are steadily gaining ground as a promising vaccination solution. This review discusses the main strategies that are currently used to produce recombinant vaccines containing alpha, beta, and epsilon toxins of C. perfringens, as well as the potential application of these molecules as vaccines for mammalian livestock animals.

  19. SPITZER/INFRARED ARRAY CAMERA LIMITS TO PLANETARY COMPANIONS OF FOMALHAUT AND {epsilon} ERIDANI

    SciTech Connect

    Marengo, Massimo; Hora, Joseph L.; Fazio, Giovanni G.; Schuster, Michael T.; Stapelfeldt, Karl; Werner, Michael W.; Carson, Joseph C.; Megeath, S. Thomas

    2009-08-01

    Fomalhaut and {epsilon} Eridani are two young, nearby stars that possess extended debris disks whose structures suggest the presence of perturbing planetary objects. With its high sensitivity and stable point-spread function, Spitzer/Infrared Array Camera (IRAC) is uniquely capable of detecting cool, Jupiter-like planetary companions whose peak emission is predicted to occur near 4.5 {mu}m. We report on deep IRAC imaging of these two stars, taken at 3.6 and 4.5 {mu}m using subarray mode and in all four channels in wider-field full array mode. Observations acquired at two different telescope roll angles allowed faint surrounding objects to be separated from the stellar diffraction pattern. No companion candidates were detected at the reported position of Fomalhaut b with 3{sigma} model-dependent mass upper limits of 3M {sub J} (for an age of 200 Myr). Around {epsilon} Eridani, we instead set a limit of 4 and {approx}<1M {sub J} (1 Gyr model age) at the inner and outer edge of the submillimeter debris ring, respectively. These results are consistent with non-detections in recent near-infrared imaging searches, and set the strongest limits to date on the presence of planets outside {epsilon} Eridani submillimeter ring.

  20. Numerical Evaluation by Harmonic Analysis of the (Epsilon)-Function of the Theodorsen Arbitrary-Airfoil Potential Theory

    NASA Technical Reports Server (NTRS)

    Naiman, Irven

    1945-01-01

    Finite trigonometric series is fitted by harmonic analysis as an approximation function to the psi function of the Theodorsen arbitrary-airfoil potential theory. By harmonic synthesis, the corresponding conjugate trigonometric series is used as an approximation to the epsilon function. A set of coefficients of particularly simple form is obtained algebraically for direct calculation of the epsilon values from the corresponding set of psi values. Complete derivation of this process is presented.

  1. Search for Physiological Substrates of Protein Tyrosine Phosphatase Epsilon in Mammary Tumor Cell Lines

    DTIC Science & Technology

    1999-12-01

    WT -+80mV IP pTyr Kv2.1 -0mV WB Kv2.1 +80mV ......... IgG KO WB Kv 2.1 V O Kv2.1 Crude . ___ -60mV extracts WB Src - 0 - Src WB PTPe W-- Cyt-PTPe... Kv 2.1 .+ . . + + + IP FLAG (PTPe) -K 2.1 WB Kv2.1 IP FLAG (PTP), WB PTPE 10O -Cyt-PTPF Crude,I - v .WB Kv 2.1 -Kv2.1 Crude, jr- ON -Cyt-PTP

  2. Role of Protein Kinase C Epsilon in Prostate Cancer and Metastasis

    DTIC Science & Technology

    2012-06-01

    Overexpression of PKCε in non-transformed prostate cells: We overexpressed PKCε in RWPE -1 cells, a model used as “normal” non-transformed epithelial prostate...in the presence of blasticidin (0.3 μg/ml). High levels of PKCε were detected in these cells ( RWPE -PKCε) by Western blot (Fig. 6a). 6. PKCε...overexpression increases proliferation of RWPE -1 cells: RWPE -PKCε display high proliferative rates compared to control cells (Fig. 6b), as determined by

  3. Search for Physiological Substrates of Protein Tyrosine Phosphatase Epsilon in Mammary Tumor Cell Lines

    DTIC Science & Technology

    2000-12-01

    PTPe. 7. Reportable Outcomes: Manuscripts: 1. Peretz, A*., Gil-Henn, H*., Sobko, A., Shinder, V., Attali , B and Elson, A. (2000) - Hypomyelination and...contributing first authors). (listed as in preparation in previous report). 2. Tiran, Z., Peretz, A., Gil-Henn, H., Attali , B. and Elson, A. - Tyrosine 124 of...myelination of peripheral nerves in early post- natal mice. 21 Ari Elson, Bernard Attali , Asher Peretz, Hava Gil-Henn, Alex Sobko, and Vera Shinder. (Oral

  4. Role of Protein Kinase C Epsilon in Prostate Cancer and Metastasis

    DTIC Science & Technology

    2013-08-01

    agar over a 0.5% agar layer. After 10 days, the number of colonies was quantified by MTT assay (a colorimetric cell viability assay). (c) PC3-ML...at io n (A .U .) time (h) 17 PC3-ML (4 x 106 cells) se ct io n ar ea (m m 2 ) Figure 2 a se ct io n ar ea (m m 2 ) 0 30 60 90 120 150 180 0 7 14 21...28 35 42 49 NTC 844 848 days C ro ss se ct io n ar ea (m m 2 ) b c PC3-ML (1.2 x 106 cells) PC3 (4 x 106 cells) NTC

  5. 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

  6. 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

  7. A Soluble Form of the High Affinity IgE Receptor, Fc-Epsilon-RI, Circulates in Human Serum

    PubMed Central

    Dehlink, Eleonora; Platzer, Barbara; Baker, Alexandra H.; LaRosa, Jessica; Pardo, Michael; Dwyer, Peter; Yen, Elizabeth H.; Szépfalusi, Zsolt

    2011-01-01

    Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI), the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum. PMID:21544204

  8. Structural organization and splice variants of the POLE1 gene encoding the catalytic subunit of human DNA polymerase epsilon.

    PubMed Central

    Huang, D; Pospiech, H; Kesti, T; Syväoja, J E

    1999-01-01

    The catalytic subunit of human DNA polymerase epsilon, an enzyme involved in nuclear DNA replication and repair, is encoded by the POLE1 gene. This gene is composed of 51 exons spanning at least 97 kb of genomic DNA. It was found to encode three alternative mRNA splice variants that differ in their 5'-terminal sequences and in the N-termini of the predicted proteins. A CpG island covers the promoter region for the major transcript in HeLa cells. This promoter is TATA-less and contains several putative binding sites for transcription factors typical of S-phase-up-regulated and serum-responsive promoters. Potential promoter regions were also identified for the two other alternative transcripts. Interestingly, no nuclear polyadenylation signal sequence was detected in the 3'-untranslated region, although a poly(A) tail was present. These results suggest a complicated regulatory machinery for the expression of the human POLE1 gene, including three alternative transcripts expressed from three promoters. PMID:10215605

  9. Identification of genetic variations associated with epsilon-poly-lysine biosynthesis in Streptomyces albulus ZPM by genome sequencing

    PubMed Central

    Wang, Lin; Gao, Chunhui; Tang, Nan; Hu, Songnian; Wu, Qingfa

    2015-01-01

    The biosynthesis of the antibiotic epsilon-poly-lysine (ε-PL) in Streptomyces albulus is performed by polylysine synthase (pls); however, the regulatory mechanism of this process is still unknown. Here, we first obtained the complete genome sequence of S. albulus ZPM, which consists of 9,784,577 bp and has a GC content of 72.2%. The genome houses 44 gene clusters for secondary metabolite biosynthesis, in which 20 gene clusters are involved in the biosynthesis of polyketides and nonribosomally synthesized peptides. High-throughput sequencing was further performed, and genetic variants were identified from pooled libraries consisting of the 30 highest-yield mutants or 30 lowest-yield mutants. More than 350 genetic variants associated with ε-PL yield have been identified. One hundred sixty-two affected proteins, from important metabolic enzymes to novel transcriptional regulators, were identified as being related to ε-PL synthesis. HrdD, one of the affected genes, is a sigma factor that shows the most sensitive response to pH change and contains a non-synonymous mutation (A132V) in mutant strains with lower ε-PL yields. Electrophoretic mobility shift assays showed that the pls gene is likely regulated by transcriptional activator HrdD. The data obtained in this study will facilitate future studies on ε-PL yield improvement and industrial bioprocess optimization. PMID:25776564

  10. The RGK family: a regulatory tail of small GTP-binding proteins.

    PubMed

    Kelly, Kathleen

    2005-12-01

    RGK proteins are small Ras-related GTP-binding proteins that function as potent inhibitors of voltage-dependent calcium channels, and two members of the family, Gem and Rad, modulate Rho-dependent remodeling of the cytoskeleton. Within the Ras superfamily, RGK proteins have distinct structural and regulatory characteristics. It is an open question as to whether RGK proteins catalyze GTP hydrolysis in vivo. Binding of calmodulin and the 14-3-3 protein to RGK proteins controls downstream pathways. Here, we discuss the structural and functional properties of RGK proteins and highlight recent work by Beguin and colleagues addressing the mechanism of Gem regulation by calmodulin and 14-3-3.

  11. Coaxial electrospinning of (fluorescein isothiocyanate-conjugated bovine serum albumin)-encapsulated poly(epsilon-caprolactone) nanofibers for sustained release.

    PubMed

    Zhang, Y Z; Wang, X; Feng, Y; Li, J; Lim, C T; Ramakrishna, S

    2006-04-01

    As an aim toward developing biologically mimetic and functional nanofiber-based tissue engineering scaffolds, we demonstrated the encapsulation of a model protein, fluorescein isothiocyanate-conjugated bovine serum albumin (fitcBSA), along with a water-soluble polymer, poly(ethylene glycol) (PEG), within the biodegradable poly(epsilon-caprolactone) (PCL) nanofibers using a coaxial electrospinning technique. By variation of the inner flow rates from 0.2 to 0.6 mL/h with a constant outer flow rate of 1.8 mL/h, fitcBSA loadings of 0.85-2.17 mg/g of nanofibrous membranes were prepared. Variation of flow rates also resulted in increases of fiber sizes from ca. 270 nm to 380 nm. The encapsulation of fitcBSA/PEG within PCL was subsequently characterized by laser confocal scanning microscopy, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS) analysis. In vitro release studies were conducted to evaluate sustained release potential of the core-sheath-structured composite nanofiber PCL-r-fitcBSA/PEG. As a negative control, composite nanofiber PCL/fitcBSA/PEG blend was prepared from a normal electrospinning method. It was found that core-sheath nanofibers PCL-r-fitcBSA/PEG pronouncedly alleviated the initial burst release for higher protein loading and gave better sustainability compared to that of PCL/fitcBSA/PEG nanofibers. The present study would provide a basis for further design and optimization of processing conditions to control the nanostructure of core-sheath composite nanofibers and ultimately achieve desired release kinetics of bioactive proteins (e.g., growth factors) for practical tissue engineering applications.

  12. 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

  13. Expression of GABA(A) receptor alpha3-, theta-, and epsilon-subunit mRNAs during rat CNS development and immunolocalization of the epsilon subunit in developing postnatal spinal cord.

    PubMed

    Pape, J-R; Bertrand, S S; Lafon, P; Odessa, M-F; Chaigniau, M; Stiles, J K; Garret, M

    2009-04-21

    Ionotropic GABA(A) receptors are heteromeric structures composed of a combination of five from at least 16 different subunits. Subunit genes are expressed in distinct cell types at specific times during development. The most abundant native GABA(A) receptors consist of alpha1-, beta2-, and gamma2-subunits that are co-expressed in numerous brain areas. alpha3-, theta-, And epsilon-subunits are clustered on the X chromosome and show striking overlapping expression patterns throughout the adult rat brain. To establish whether these subunits are temporally and spatially co-expressed, we used in situ hybridization to analyze their expression throughout rat development from embryonic stage E14 to postnatal stage P12. Each transcript exhibited a unique or a shared regional and temporal developmental expression profile. The thalamic expression pattern evolved from a restricted expression of epsilon and theta transcripts before birth, to a theta and alpha3 expression at birth, and finally to a grouped epsilon, theta and alpha3 expression postpartum. However, strong similarities occurred, such as a grouped expression of the three subunits within the hypothalamus, tegmentum and pontine nuclei throughout the developmental process. At early stages of development (E17), epsilon and theta appeared to have a greater spatial distribution before the dominance of the alpha3 subunit transcript around birth. We also revealed expression of alpha3, theta, and epsilon in the developing spinal cord and identified neurons that express epsilon in the postnatal dorsal horn, intermediolateral column and motoneurons. Our findings suggest that various combinations of alpha3-, theta- and epsilon-subunits may be assembled at a regional and developmental level in the brain.

  14. 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 tempera