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Sample records for activated protein map

  1. Functional Protein Pathway Activation Mapping of the Progression of Normal Skin to Squamous Cell Carcinoma

    PubMed Central

    Einspahr, Janine G.; Calvert, Valerie; Alberts, David S.; Curiel-Lewandrowski, Clara; Warneke, James; Krouse, Robert; Stratton, Steven P; Liotta, Lance; Longo, Caterina; Pellicani, Giovanni; Prasad, Anil; Sagerman, Paul; Bermudez, Yira; Deng, Jianghong; Bowden, G. Timothy; Petricoin, Emanuel F.

    2012-01-01

    Reverse Phase Protein Microarray analysis was used to identify cell-signaling derangements in squamous cell carcinoma (SCC) compared to actinic keratosis (AK) and upper inner arm (UIA). We analyzed 2 independent tissue sets with isolation and enrichment of epithelial cells by Laser Capture Microdissection. Set 1 served as a pilot and a means to identify protein pathway activation alterations that could be further validated in a second independent set. Set 1 was comprised of 4 AK, 13 SCC, and 20 UIA. Set 2 included 15 AK, 9 SCC and 20 UIA. Activation of 51 signaling proteins known to be involved in tumorigenesis were assessed for set 1 and demonstrated the MEK-ERK pathway was activated in SCC compared to AK and UIA, that EGFR and mTOR pathways were aberrantly activated in SCC. Unsupervised two-way hierarchical clustering revealed that AK and UIA shared a common signaling network activation architecture while SCC was dramatically different. Statistical analysis found that pro-survival signaling through phosphorylation of ASK and 4EBP1 as well as increased Bax and Bak expression was higher in AK compared to UIA. We expanded pathway network activation mapping in Set 2 to 101 key signaling proteins, which corroborated activation of MEK-ERK, EGFR, and mTOR pathways through discovery of a number of upstream, and downstream signaling molecules within these pathways to conclude that SCC is indeed a pathway activation-driven disease. Pathway activation mapping of SCC compared to AK revealed several interconnected networks that could be targeted with drug therapy for potential chemoprevention and therapeutic applications. PMID:22389437

  2. Active Mapping.

    ERIC Educational Resources Information Center

    Day, Dennis

    1994-01-01

    Explains a social studies lesson for third graders that uses KidPix, a computer software graphics program to help students make maps and map keys. Advantages to using the computer versus hand drawing maps are discussed, and an example of map requirements for the lesson is included. (LRW)

  3. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    SciTech Connect

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J.

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  4. Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.

    PubMed Central

    Nebreda, A R; Gannon, J V; Hunt, T

    1995-01-01

    The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone. Images PMID:8521817

  5. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

    PubMed

    Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. PMID:27226575

  6. Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways*

    PubMed Central

    Dickinson, Robin J.; Delavaine, Laurent; Cejudo-Marín, Rocío; Stewart, Graeme; Staples, Christopher J.; Didmon, Mark P.; Trinidad, Antonio Garcia; Alonso, Andrés; Pulido, Rafael; Keyse, Stephen M.

    2011-01-01

    MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site 55RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo. PMID:21908610

  7. Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists.

    PubMed Central

    Nahas, N; Molski, T F; Fernandez, G A; Sha'afi, R I

    1996-01-01

    The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulation of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated. This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mitogen-activated protein (MAP) kinase. This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody immunoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP kinase antibody, and conversely, anti-p38 MAP kinase antibody immunoprecipitates a 38 kDa protein that can be recognized by anti-PY antibody. Moreover, this tyrosine phosphorylated protein is found associated entirely with the cytosol. It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum. This conclusion is based on three experimental findings. First, soluble fractions isolated from LPS-stimulated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and protein kinase A inhibitor peptides. This effect is similar to the effect produced by the commercially available phosphorylated and activated MAPKAP kinase-2 (MAP kinase activated protein kinase-2). Secondly, a 27 kDa protein that aligns with a protein recognized by anti-hsp27 antibody is phosphorylated upon LPS stimulation of intact human neutrophils prelabelled with radioactive phosphate. Lastly, immune complex protein kinase assays, using [gamma-32P]ATP and activating transcription factor 2 (ATF2) as substrates, showed increased p38 MAP kinase activity from LPS-stimulated human neutrophils. The phosphorylation and activation of this p38 MAP kinase can be affected by both G-protein-coupled receptors such as platelet-activating factor (PAF) and non-G-protein-coupled receptors such as the cytokine

  8. Inhibition of small G proteins by clostridium sordellii lethal toxin activates cdc2 and MAP kinase in Xenopus oocytes.

    PubMed

    Rime, H; Talbi, N; Popoff, M R; Suziedelis, K; Jessus, C; Ozon, R

    1998-12-15

    The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase that modifies and inhibits small G proteins of the Ras family, Ras and Rap, as well as Rac proteins. LT induces cdc2 kinase activation and germinal vesicle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes. Toxin B from Clostridium difficile, that glucosylates and inactivates Rac proteins, does not induce cdc2 activation, indicating that proteins of the Ras family, Ras and/or Rap, negatively regulate cdc2 kinase activation in Xenopus oocyte. In oocyte extracts, LT catalyzes the incorporation of [14C]glucose into a group of proteins of 23 kDa and into one protein of 27 kDa. The 23-kDa proteins are recognized by anti-Rap1 and anti-Rap2 antibodies, whereas the 27-kDa protein is recognized by several anti-Ras antibodies and probably corresponds to K-Ras. Microinjection of LT into oocytes together with UDP-[14C]glucose results in a glucosylation pattern similar to the in vitro glucosylation, indicating that the 23- and 27-kDa proteins are in vivo substrates of LT. In vivo time-course analysis reveals that the 27-kDa protein glucosylation is completed within 2 h, well before cdc2 kinase activation, whereas the 23-kDa proteins are partially glucosylated at GVBD. This observation suggests that the 27-kDa Ras protein could be the in vivo target of LT allowing cdc2 kinase activation. Interestingly, inactivation of Ras proteins does not prevent the phosphorylation of c-Raf1 and the activation of MAP kinase that occurs normally around GVBD. PMID:9882492

  9. Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) integrates developmental signals for eyelid closure

    PubMed Central

    Geh, Esmond; Meng, Qinghang; Mongan, Maureen; Wang, Jingcai; Takatori, Atsushi; Zheng, Yi; Puga, Alvaro; Lang, Richard A.; Xia, Ying

    2011-01-01

    Developmental eyelid closure is an evolutionarily conserved morphogenetic event requiring proliferation, differentiation, cytoskeleton reorganization, and migration of epithelial cells at the tip of the developing eyelid. Many signaling events take place during eyelid closure, but how the signals converge to regulate the morphogenetic process remains an open and intriguing question. Here we show that mitogen-activated protein kinase kinase kinase 1 (MAP3K1) highly expressed in the developing eyelid epithelium, forms with c-Jun, a regulatory axis that orchestrates morphogenesis by integrating two different networks of eyelid closure signals. A TGF-α/EGFR-RhoA module initiates one of these networks by inducing c-Jun expression which, in a phosphorylation-independent manner, binds to the Map3k1 promoter and causes an increase in MAP3K1 expression. RhoA knockout in the ocular surface epithelium disturbs this network by decreasing MAP3K1 expression, and causes delayed eyelid closure in Map3k1 hemizygotes. The second network is initiated by the enzymatic activity of MAP3K1, which phosphorylates and activates a JNK-c-Jun module, leading to AP-1 transactivation and induction of its downstream genes, such as Pai-1. MAP3K1 inactivation reduces AP-1 activity and PAI-1 expression both in cells and developing eyelids. MAP3K1 is therefore the nexus of an intracrine regulatory loop connecting the TGF-α/EGFR/RhoA-c-Jun and JNK-c-Jun-AP-1 pathways in developmental eyelid closure. PMID:21969564

  10. Active Fire Mapping Program

    MedlinePlus

    ... Incidents (Home) New Large Incidents Fire Detection Maps MODIS Satellite Imagery VIIRS Satellite Imagery Fire Detection GIS ... Data Web Services Latest Detected Fire Activity Other MODIS Products Frequently Asked Questions About Active Fire Maps ...

  11. The active gene that encodes human High Mobility Group 1 protein (HMG1) contains introns and maps to chromosome 13

    SciTech Connect

    Ferrari, S.; Finelli, P.; Rocchi, M.

    1996-07-15

    The human genome contains a large number of sequences related to the cDNA for High Mobility Group 1 protein (HMG1), which so far has hampered the cloning and mapping of the active HMG1 gene. We show that the human HMG1 gene contains introns, while the HMG1-related sequences do not and most likely are retrotransposed pseudogenes. We identified eight YACs from the ICI and CEPH libraries that contain the human HMG1 gene. The HMG1 gene is similar in structure to the previously characterized murine homologue and maps to human chromosome 13 and q12, as determined by in situ hybridization. The mouse Hmg1 gene maps to the telomeric region of murine Chromosome 5, which is syntenic to the human 13q12 band. 18 refs., 3 figs.

  12. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis*

    PubMed Central

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W.-Y.; Puga, Alvaro; Xia, Ying

    2015-01-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1+/− embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  13. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis.

    PubMed

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W-Y; Puga, Alvaro; Xia, Ying

    2015-08-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1(+/-) embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  14. New MAPS for misfolded proteins.

    PubMed

    Volkmar, Norbert; Fenech, Emma; Christianson, John C

    2016-06-28

    Clearing misfolded proteins from the cytoplasm is essential to maintain cellular homeostasis. Now, a parallel clearance system is described that uses the deubiquitylase USP19 to enable secretion of misfolded cytoplasmic proteins when conventional proteasomal degradation is compromised. Misfolding-associated protein secretion (MAPS) has important implications for protein quality control and prion-like transmission. PMID:27350445

  15. Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways

    SciTech Connect

    Menschikowski, Mario; Hagelgans, Albert; Eisenhofer, Graeme; Siegert, Gabriele

    2009-09-10

    The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}), but not interferon-{gamma} and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1{beta} and TNF-{alpha} correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl-L-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1{beta} and TNF-{alpha}, and downstream by MAP kinase signaling pathways and metalloproteinases.

  16. Protein folding using contact maps.

    PubMed

    Vendruscolo, M; Domany, E

    2000-01-01

    We discuss the problem of representations of protein structure and give the definition of contact maps. We present a method to obtain a three-dimensional polypeptide conformation from a contact map. We also explain how to deal with the case of nonphysical contact maps. We describe a stochastic method to perform dynamics in contact map space. We explain how the motion is restricted to physical regions of the space. First, we introduce the exact free energy of a contact map and discuss two simple approximations to it. Second, we present a method to derive energy parameters based on perception learning. We prove in an extensive number of situations that the pairwise contact approximation both when alone and when supplemented with a hydrophobic term is unsuitable for stabilizing proteins' native states. PMID:10668399

  17. A protein map of the yeast activated spliceosome as obtained by electron microscopy.

    PubMed

    Sun, Chengfu; Rigo, Norbert; Fabrizio, Patrizia; Kastner, Berthold; Lührmann, Reinhard

    2016-09-01

    We have elucidated the spatial arrangement of proteins and snRNP subunits within the purified spliceosomal B(act) complex from Saccharomyces cerevisiae, using negative-stain immunoelectron microscopy. The B(act) spliceosome exhibits a mushroom-like shape with a main body connected to a foot and a steep and a shallow slope. The U5 core components, including proteins Snu114 and Prp8, are located in the main body and foot, while Brr2 is on the shallow slope. U2 snRNP components and the RNA helicase Prp2 were predominantly located in the upper regions of both slopes. While several proteins of the "nineteen complex" are located on the steep slope, Prp19, Cef1, and the U6 snRNA-binding protein Cwc2 are on the main body. Our results also indicate that the catalytic core RNP of the spliceosome resides in its main body. We thus assign distinct domains of the B(act) complex to its snRNP and protein components, and we provide first structural insights into the remodeling events at the spliceosome during its transformation from the B to the B(act) complex. PMID:27368340

  18. Intercreativity: Mapping Online Activism

    NASA Astrophysics Data System (ADS)

    Meikle, Graham

    How do activists use the Internet? This article maps a wide range of activist practice and research by applying and developing Tim Berners-Lee's concept of ‘intercreativity' (1999). It identifies four dimensions of Net activism: intercreative texts, tactics, strategies and networks. It develops these through examples of manifestations of Net activism around one cluster of issues: support campaigns for refugees and asylum seekers.

  19. Evidence of two mechanisms for the activation of the glucose transporter GLUT1 by anisomycin: p38(MAP kinase) activation and protein synthesis inhibition in mammalian cells.

    PubMed Central

    Barros, L F; Young, M; Saklatvala, J; Baldwin, S A

    1997-01-01

    1. Inhibitors of protein synthesis stimulate sugar transport in mammalian cells through activation of plasma membrane GLUT1, the housekeeping isoform of the glucose transporter. However, it has been reported that some of these compounds, in addition to their effect on protein synthesis, also activate protein kinases. 2. In the present study we have explored the role of these two effects on GLUT1 activation. In 3T3-L1 adipocytes and Clone 9 cells, stimulation of sugar transport by puromycin, a translational inhibitor that does not activate kinases, was not detectable until 90 min after exposure. In contrast, stimulation by anisomycin, a potent Jun-NH2-terminal kinase (JNK) agonist, exhibited no lag phase. An intermediate response was observed to emetine and cycloheximide, weak activators of JNK. 3. The potency of anisomycin to stimulate transport acutely (30 min of exposure) was 5- to 10-fold greater than for its chronic stimulation of transport, measured after 4 h of exposure. The stimulation of transport by a low concentration of anisomycin (0.3 microM) was transient, peaked at 30-60 min and it was inhibited (IC50 < 1 microM) by SB203580, which indicates that its mediator is not JNK, but the homologous p38(MAP kinase) (p38(MAPK)). In contrast, the responses to 4 h exposure to 300 microM anisomycin or puromycin were refractory to SB203580. 4. Exposure to anisomycin resulted in rapid activation of p38(MAPK). Activation of both p38(MAPK) and GLUT1 by 0.3 microM anisomycin was cancelled by puromycin. 5. We conclude that the activation of GLUT1 in response to anisomycin includes two components: a delayed component involving translational inhibition and a fast, puromycin-inhibitable component that is secondary to activation of p38(MAPK). Images Figure 2 Figure 7 PMID:9401960

  20. Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

    PubMed Central

    Böcker, Dietmar

    2001-01-01

    MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [ P 32 ]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 ( IC 50 =51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [ H 3 ]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but

  1. Protein affinity map of chemical space.

    PubMed

    Kauvar, L M; Villar, H O; Sportsman, J R; Higgins, D L; Schmidt, D E

    1998-09-11

    Affinity fingerprinting is a quantitative method for mapping chemical space based on binding preferences of compounds for a reference panel of proteins. An effective reference panel of <20 proteins can be empirically selected which shows differential interaction with nearly all compounds. By using this map to iteratively sample the chemical space, identification of active ligands from a library of 30,000 candidate compounds has been accomplished for a wide spectrum of specific protein targets. In each case, <200 compounds were directly assayed against the target. Further, analysis of the fingerprint database suggests a strategy for effective selection of affinity chromatography ligands and scaffolds for combinatorial chemistry. With such a system, the large numbers of potential therapeutic targets emerging from genome research can be categorized according to ligand binding properties, complementing sequence based classification. PMID:9792501

  2. Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter.

    PubMed Central

    Bruening, W; Giasson, B; Mushynski, W; Durham, H D

    1998-01-01

    The immediate/early promoter/enhancer of cytomegalovirus (CMV promoter) is one of the most commonly used promoters for expression of transgenes in eukaryotic cells. In practice, the CMV promoter is often thought of as a constitutively active unregulated promoter. However, we have observed that transcription from the CMV promoter can be up-regulated by a variety of environmental stresses. Many forms of cellular stress stimulate MAP kinase signalling pathways, resulting in activation of stress-activated protein kinases [SAPKs, also called Jun N-terminal kinases (JNKs)] and p38 kinases. We have found that the same conditions that lead to activation of SAPK/JNKs and p38 kinases can also dramatically increase expression from the CMV promoter. Inhibitors of p38 kinases abolished basal transcription from the CMV promoter and completely blocked stress-induced up-regulation of the CMV promoter. Overexpression of a dominant negative JNK kinase had no effect on basal transcription, but significantly reduced up-regulation caused by stress. These results have grave implications for use of the CMV promoter. If the CMV promoter can be up-regulated by cellular stresses, inadvertent activation of the stress kinase pathways may complicate, if not invalidate, the interpretation of a wide range of experiments. PMID:9421504

  3. BDNF-mediated regulation of ethanol consumption requires the activation of the MAP kinase pathway and protein synthesis

    PubMed Central

    Jeanblanc, Jerome; Logrip, Marian L.; Janak, Patricia H.; Ron, Dorit

    2013-01-01

    We previously found that the brain-derived neurotrophic factor (BDNF) in the dorsolateral striatum (DLS) is part of a homeostatic pathway that gates ethanol self-administration [Jeanblanc et al. (2009). J Neurosci, 29, 13494–13502)]. Specifically, we showed that moderate levels (10%) of ethanol consumption increase BDNF expression within the DLS, and that direct infusion of BDNF into the DLS decreases operant self-administration of a 10% ethanol solution. BDNF binding to its receptor, TrkB, activates the mitogen-activated protein kinase (MAPK), phospholipase C-γ (PLC-γ) and phosphatidylinositol 3-kinase (PI3K) pathways. Thus, here, we set out to identify which of these intracellular pathway(s) plays a role in the regulation of ethanol consumption by BDNF. We found that inhibition of the MAPK, but not PLC-γ or PI3K, activity blocks the BDNF-mediated reduction of ethanol consumption. As activation of the MAPK pathway leads to the initiation of transcription and/or translation events, we tested whether the BDNF-mediated reduction of ethanol self-administration requires de novo protein synthesis. We found that the inhibitory effect of BDNF on ethanol intake is blocked by the protein synthesis inhibitor cycloheximide. Together, our results show that BDNF attenuates ethanol drinking via activation of the MAPK pathway in a protein synthesis-dependent manner within the DLS. PMID:23189980

  4. Induction of Macrophage Function in Human THP-1 Cells Is Associated with Rewiring of MAPK Signaling and Activation of MAP3K7 (TAK1) Protein Kinase

    PubMed Central

    Richter, Erik; Ventz, Katharina; Harms, Manuela; Mostertz, Jörg; Hochgräfe, Falko

    2016-01-01

    Macrophages represent the primary human host response to pathogen infection and link the immediate defense to the adaptive immune system. Mature tissue macrophages convert from circulating monocyte precursor cells by terminal differentiation in a process that is not fully understood. Here, we analyzed the protein kinases of the human monocytic cell line THP-1 before and after induction of macrophage differentiation by using kinomics and phosphoproteomics. When comparing the macrophage-like state with the monocytic precursor, 50% of the kinome was altered in expression and even 71% of covered kinase phosphorylation sites were affected. Kinome rearrangements are for example characterized by a shift of overrepresented cyclin-dependent kinases associated with cell cycle control in monocytes to calmodulin-dependent kinases and kinases involved in proinflammatory signaling. Eventually, we show that monocyte-to-macrophage differentiation is associated with major rewiring of mitogen-activated protein kinase signaling networks and demonstrate that protein kinase MAP3K7 (TAK1) acts as the key signaling hub in bacterial killing, chemokine production and differentiation. Our study proves the fundamental role of protein kinases and cellular signaling as major drivers of macrophage differentiation and function. The finding that MAP3K7 is central to macrophage function suggests MAP3K7 and its networking partners as promising targets in host-directed therapy for macrophage-associated disease. PMID:27066479

  5. cDNA cloning and chromosomal mapping of a novel human GAP (GAP1M), GTPase-activating protein of Ras

    SciTech Connect

    Li, Shaowei; Nakamura, Shun; Hattori, Seisuke

    1996-08-01

    We have previously isolated a novel Ras GTPase-activating protein (Ras GAP), Gapl{sup m}, from rat brain. Gap1{sup m} is considered to be a negative regulator of the Ras signaling pathways, like other Ras GAPs, neurofibromin, which is a gene product of the neurofibromatosis type I gene, and p120GAP. In this study we have isolated a human cDNA of this Gap and mapped the gene. The gene encodes a protein of 853 amino acids that shows 89% sequence identity to rat Gapl{sup m}. The human gene was mapped to chromosome 3 by PCR analysis on a panel of human-mouse hybrid cells. FISH analysis refined the location of the gene further to 3q22-q23. 11 refs., 2 figs.

  6. PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway, Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

    PubMed Central

    Teichert, Ines; Steffens, Eva Katharina; Schnaß, Nicole; Fränzel, Benjamin; Krisp, Christoph; Wolters, Dirk A.; Kück, Ulrich

    2014-01-01

    Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems. PMID:25188365

  7. A Protein Interaction Map of Drosophila melanogaster

    NASA Astrophysics Data System (ADS)

    Giot, L.; Bader, J. S.; Brouwer, C.; Chaudhuri, A.; Kuang, B.; Li, Y.; Hao, Y. L.; Ooi, C. E.; Godwin, B.; Vitols, E.; Vijayadamodar, G.; Pochart, P.; Machineni, H.; Welsh, M.; Kong, Y.; Zerhusen, B.; Malcolm, R.; Varrone, Z.; Collis, A.; Minto, M.; Burgess, S.; McDaniel, L.; Stimpson, E.; Spriggs, F.; Williams, J.; Neurath, K.; Ioime, N.; Agee, M.; Voss, E.; Furtak, K.; Renzulli, R.; Aanensen, N.; Carrolla, S.; Bickelhaupt, E.; Lazovatsky, Y.; DaSilva, A.; Zhong, J.; Stanyon, C. A.; Finley, R. L.; White, K. P.; Braverman, M.; Jarvie, T.; Gold, S.; Leach, M.; Knight, J.; Shimkets, R. A.; McKenna, M. P.; Chant, J.; Rothberg, J. M.

    2003-12-01

    Drosophila melanogaster is a proven model system for many aspects of human biology. Here we present a two-hybrid-based protein-interaction map of the fly proteome. A total of 10,623 predicted transcripts were isolated and screened against standard and normalized complementary DNA libraries to produce a draft map of 7048 proteins and 20,405 interactions. A computational method of rating two-hybrid interaction confidence was developed to refine this draft map to a higher confidence map of 4679 proteins and 4780 interactions. Statistical modeling of the network showed two levels of organization: a short-range organization, presumably corresponding to multiprotein complexes, and a more global organization, presumably corresponding to intercomplex connections. The network recapitulated known pathways, extended pathways, and uncovered previously unknown pathway components. This map serves as a starting point for a systems biology modeling of multicellular organisms, including humans.

  8. The mitogen-activated protein kinome from Anopheles gambiae: identification, phylogeny and functional characterization of the ERK, JNK and p38 MAP kinases

    PubMed Central

    2011-01-01

    Background Anopheles gambiae is the primary mosquito vector of human malaria parasites in sub-Saharan Africa. To date, three innate immune signaling pathways, including the nuclear factor (NF)-kappaB-dependent Toll and immune deficient (IMD) pathways and the Janus kinase/signal transducers and activators of transcription (Jak-STAT) pathway, have been extensively characterized in An. gambiae. However, in addition to NF-kappaB-dependent signaling, three mitogen-activated protein kinase (MAPK) pathways regulated by JNK, ERK and p38 MAPK are critical mediators of innate immunity in other invertebrates and in mammals. Our understanding of the roles of the MAPK signaling cascades in anopheline innate immunity is limited, so identification of the encoded complement of these proteins, their upstream activators, and phosphorylation profiles in response to relevant immune signals was warranted. Results In this study, we present the orthologs and phylogeny of 17 An. gambiae MAPKs, two of which were previously unknown and two others that were incompletely annotated. We also provide detailed temporal activation profiles for ERK, JNK, and p38 MAPK in An. gambiae cells in vitro to immune signals that are relevant to malaria parasite infection (human insulin, human transforming growth factor-beta1, hydrogen peroxide) and to bacterial lipopolysaccharide. These activation profiles and possible upstream regulatory pathways are interpreted in light of known MAPK signaling cascades. Conclusions The establishment of a MAPK "road map" based on the most advanced mosquito genome annotation can accelerate our understanding of host-pathogen interactions and broader physiology of An. gambiae and other mosquito species. Further, future efforts to develop predictive models of anopheline cell signaling responses, based on iterative construction and refinement of data-based and literature-based knowledge of the MAP kinase cascades and other networked pathways will facilitate identification of the

  9. Mapping membrane protein structure with fluorescence

    PubMed Central

    Taraska, Justin W.

    2012-01-01

    Membrane proteins regulate many cellular processes including signaling cascades, ion transport, membrane fusion, and cell-to-cell communications. Understanding the architecture and conformational fluctuations of these proteins is critical to understanding their regulation and functions. Fluorescence methods including intensity mapping, fluorescence resonance energy transfer, and photo-induced electron transfer, allow for targeted measurements of domains within membrane proteins. These methods can reveal how a protein is structured and how it transitions between different conformational states. Here, I will review recent work done using fluorescence to map the structures of membrane proteins, focusing on how each of these methods can be applied to understanding the dynamic nature of individual membrane proteins and protein complexes. PMID:22445227

  10. AMP-activated Protein Kinase Up-regulates Mitogen-activated Protein (MAP) Kinase-interacting Serine/Threonine Kinase 1a-dependent Phosphorylation of Eukaryotic Translation Initiation Factor 4E.

    PubMed

    Zhu, Xiaoqing; Dahlmans, Vivian; Thali, Ramon; Preisinger, Christian; Viollet, Benoit; Voncken, J Willem; Neumann, Dietbert

    2016-08-12

    AMP-activated protein kinase (AMPK) is a molecular energy sensor that acts to sustain cellular energy balance. Although AMPK is implicated in the regulation of a multitude of ATP-dependent cellular processes, exactly how these processes are controlled by AMPK as well as the identity of AMPK targets and pathways continues to evolve. Here we identify MAP kinase-interacting serine/threonine protein kinase 1a (MNK1a) as a novel AMPK target. Specifically, we show AMPK-dependent Ser(353) phosphorylation of the human MNK1a isoform in cell-free and cellular systems. We show that AMPK and MNK1a physically interact and that in vivo MNK1a-Ser(353) phosphorylation requires T-loop phosphorylation, in good agreement with a recently proposed structural regulatory model of MNK1a. Our data suggest a physiological role for MNK1a-Ser(353) phosphorylation in regulation of the MNK1a kinase, which correlates with increased eIF4E phosphorylation in vitro and in vivo. PMID:27413184

  11. A Protein Complex Map of Trypanosoma brucei

    PubMed Central

    Mehta, Vaibhav; Najafabadi, Hamed S.; Moshiri, Houtan; Jardim, Armando; Salavati, Reza

    2016-01-01

    The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org. PMID:26991453

  12. Emerging technologies to map the protein methylome

    PubMed Central

    Carlson, Scott M.; Gozani, Or

    2014-01-01

    Protein methylation plays an integral role in cellular signaling, most notably by modulating proteins bound at chromatin, and increasingly through regulation of non-histone proteins. One central challenge in understanding how methylation acts in signaling is identifying and measuring protein methylation. This includes locus-specific modification of histones, on individual non-histone proteins, and globally across the proteome. Protein methylation has traditionally been studied using candidate approaches such as methylation-specific antibodies, mapping of post-translational modifications by mass spectrometry, and radioactive labeling to characterize methylation on target proteins. Recent developments have provided new approaches to identify methylated proteins, measure methylation levels, identify substrates of methyltransferase enzymes, and match methylated proteins to methyl-specific reader domains. Methyl-binding protein domains and improved antibodies with broad specificity for methylated proteins are being used to characterize the “protein methylome”. They also have the potential to be used in high-throughput assays for inhibitor screens and drug development. These tools are often coupled to improvements in mass spectrometry to quickly identify methylated residues, and to protein microarrays, where they can be used to screen for methylated proteins. Finally, new chemical biology strategies are being used to probe the function of methyltransferases, demethylases and methyl-binding “reader” domains. These tools are creating a “system-level” understanding of protein methylation, and integrating protein methylation into broader signaling processes. PMID:24805349

  13. Isolation of Intrinsically Active (MEK-independent) Variants of the ERK Family of Mitogen-activated Protein (MAP) Kinases*S⃞♦

    PubMed Central

    Levin-Salomon, Vered; Kogan, Konstantin; Ahn, Natalie G.; Livnah, Oded; Engelberg, David

    2008-01-01

    MAPKs are key components of cell signaling pathways with a unique activation mechanism: i.e. dual phosphorylation of neighboring threonine and tyrosine residues. The ERK enzymes form a subfamily of MAPKs involved in proliferation, differentiation, development, learning, and memory. The exact role of each Erk molecule in these processes is not clear. An efficient strategy for addressing this question is to activate individually each molecule, for example, by expressing intrinsically active variants of them. However, such molecules were not produced so far. Here, we report on the isolation, via a specifically designed genetic screen, of six variants (each carries a point mutation) of the yeast MAPK Mpk1/Erk that are active, independent of upstream phosphorylation. One of the activating mutations, R68S, occurred in a residue conserved in the mammalian Erk1 (Arg-84) and Erk2 (Arg-65) and in the Drosophila ERK Rolled (Arg-80). Replacing this conserved Arg with Ser rendered these MAPKs intrinsically active to very high levels when tested in vitro as recombinant proteins. Combination of the Arg to Ser mutation with the sevenmaker mutation (producing Erk2R65S+D319N and RolledR80S+D334N) resulted in even higher activity (45 and 70%, respectively, in reference to fully active dually phosphorylated Erk2 or Rolled). Erk2R65S and Erk2R65S+D319N were found to be spontaneously active also when expressed in human HEK293 cells. We further revealed the mechanism of action of the mutants and show that it involves acquisition of autophosphorylation activity. Thus, a first generation of Erk molecules that are spontaneously active in vitro and in vivo has been obtained. PMID:18829462

  14. Protein interaction mapping: A Drosophila case study

    PubMed Central

    Formstecher, Etienne; Aresta, Sandra; Collura, Vincent; Hamburger, Alexandre; Meil, Alain; Trehin, Alexandra; Reverdy, Céline; Betin, Virginie; Maire, Sophie; Brun, Christine; Jacq, Bernard; Arpin, Monique; Bellaiche, Yohanns; Bellusci, Saverio; Benaroch, Philippe; Bornens, Michel; Chanet, Roland; Chavrier, Philippe; Delattre, Olivier; Doye, Valérie; Fehon, Richard; Faye, Gérard; Galli, Thierry; Girault, Jean-Antoine; Goud, Bruno; de Gunzburg, Jean; Johannes, Ludger; Junier, Marie-Pierre; Mirouse, Vincent; Mukherjee, Ashim; Papadopoulo, Dora; Perez, Franck; Plessis, Anne; Rossé, Carine; Saule, Simon; Stoppa-Lyonnet, Dominique; Vincent, Alain; White, Michael; Legrain, Pierre; Wojcik, Jérôme; Camonis, Jacques; Daviet, Laurent

    2005-01-01

    The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps. PMID:15710747

  15. Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2.

    PubMed

    Zhang, Yu-Hang; Shetty, Keerthi; Surleac, Marius D; Petrescu, Andrei J; Schatz, David G

    2015-05-01

    The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pulldown assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (KD ∼0.4 μM) (where RAG1 and RAG2 are recombination activating genes 1 or 2). Using the Hermes transposase as a guide, we constructed a 36-kDa "mini" RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and the residues N-terminal to it, but it lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (amino acids 997-1008) near the RAG1 C terminus and a region of RAG1 from amino acids 479 to 559. Two adjacent acidic amino acids in this region (Asp-546 and Glu-547) are important for both the RAG1-RAG2 interaction and recombination activity, with Asp-546 of particular importance. Structural modeling of Mini-RAG1 suggests that Asp-546/Glu-547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative Western blotting allowed us to estimate that mouse thymocytes contain on average ∼1,800 monomers of RAG1 and ∼15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the KD value for their interaction, which could help limit off-target RAG activity. PMID:25745109

  16. Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2*♦

    PubMed Central

    Zhang, Yu-Hang; Shetty, Keerthi; Surleac, Marius D.; Petrescu, Andrei J.; Schatz, David G.

    2015-01-01

    The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pulldown assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (KD ∼0.4 μm) (where RAG1 and RAG2 are recombination activating genes 1 or 2). Using the Hermes transposase as a guide, we constructed a 36-kDa “mini” RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and the residues N-terminal to it, but it lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (amino acids 997–1008) near the RAG1 C terminus and a region of RAG1 from amino acids 479 to 559. Two adjacent acidic amino acids in this region (Asp-546 and Glu-547) are important for both the RAG1-RAG2 interaction and recombination activity, with Asp-546 of particular importance. Structural modeling of Mini-RAG1 suggests that Asp-546/Glu-547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative Western blotting allowed us to estimate that mouse thymocytes contain on average ∼1,800 monomers of RAG1 and ∼15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the KD value for their interaction, which could help limit off-target RAG activity. PMID:25745109

  17. Quaternion maps of global protein structure.

    PubMed

    Hanson, Andrew J; Thakur, Sidharth

    2012-09-01

    The geometric structures of proteins are vital to the understanding of biochemical interactions. However, there is much yet to be understood about the spatial arrangements of the chains of amino acids making up any given protein. In particular, while conventional analysis tools like the Ramachandran plot supply some insight into the local relative orientation of pairs of amino acid residues, they provide little information about the global relative orientations of large groups of residues. We apply quaternion maps to families of coordinate frames defined naturally by amino acid residue structures as a way to expose global spatial relationships among residues within proteins. The resulting visualizations enable comparisons of absolute orientations as well as relative orientations, and thus generalize the framework of the Ramachandran plot. There are a variety of possible quaternion frames and visual representation strategies that can be chosen, and very complex quaternion maps can result. Just as Ramachandran plots are useful for addressing particular questions and not others, quaternion tools have characteristic domains of relevance. In particular, quaternion maps show great potential for answering specific questions about global residue alignment in crystallographic data and statistical orientation properties in Nuclear Magnetic Resonance (NMR) data that are very difficult to treat by other methods. PMID:23099777

  18. Mapping auroral activity with Twitter

    NASA Astrophysics Data System (ADS)

    Case, N. A.; MacDonald, E. A.; Heavner, M.; Tapia, A. H.; Lalone, N.

    2015-05-01

    Twitter is a popular, publicly accessible, social media service that has proven useful in mapping large-scale events in real time. In this study, for the first time, the use of Twitter as a measure of auroral activity is investigated. Peaks in the number of aurora-related tweets are found to frequently coincide with geomagnetic disturbances (detection rate of 91%). Additionally, the number of daily aurora-related tweets is found to strongly correlate with several auroral strength proxies (ravg≈0.7). An examination is made of the bias for location and time of day within Twitter data, and a first-order correction of these effects is presented. Overall, the results suggest that Twitter can provide both specific details about an individual aurora and accurate real-time indication of when, and even from where, an aurora is visible.

  19. Protein Flexibility in Docking and Surface Mapping

    PubMed Central

    Lexa, Katrina W.; Carlson, Heather A.

    2012-01-01

    Structure-based drug design has become an essential tool for rapid lead discovery and optimization. As available structural information has increased, researchers have become increasingly aware of the importance of protein flexibility for accurate description of the native state. Typical protein–ligand docking efforts still rely on a single rigid receptor, which is an incomplete representation of potential binding conformations of the protein. These rigid docking efforts typically show the best performance rates between 50 and 75%, while fully flexible docking methods can enhance pose prediction up to 80–95%. This review examines the current toolbox for flexible protein–ligand docking and receptor surface mapping. Present limitations and possibilities for future development are discussed. PMID:22569329

  20. Mitogen-Activated Protein Kinase 8 (MAP3K8) Mediates the Signaling Pathway of Estradiol Stimulating Progesterone Production Through G Protein-Coupled Receptor 30 (GPR30) in Mouse Corpus Luteum.

    PubMed

    Liu, Ying; Li, Yueqin; Zhang, Di; Liu, Jiali; Gou, Kemian; Cui, Sheng

    2015-05-01

    The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles, and the most important function is to synthesize and secrete progesterone (P(4)), a key hormone to maintain normal pregnancy and estrous cycle in most mammals. It is known that estrogen has a vital role in stimulating P(4) synthesis in CL, but it still remains unclear about the mechanism of estradiol (E(2)) regulating P(4) production in CL. Our results here first show that all of the CL cells express MAPK 8 (MAP3K8), and the MAP3K8 level is much higher at the midstage than at the early and late stages during CL development. The further functional studies show that the forced inhibition of endogenous MAP3K8 by using MAP3K8 small interfering RNA and MAP3K8 signaling inhibitor (MAP3K8i) in the luteal cells significantly block the P(4) synthesis and neutralize the enhancing effect of E(2) on P(4) production in the CL. In addition, our results here demonstrate that the stimulating effect of E(2) on P(4) synthesis relies on the estrogen no-classical protein-coupled receptor 30, and MAP3K8 is involved in mediating the protein-coupled receptor 30signaling of E(2) affecting P(4) synthesis via stimulating ERK phosphorylation. These novel findings are critical for our understanding the ovary physiology and pathological mechanism. PMID:25763610

  1. Rapid Regulation of Microtubule-Associated Proteins 2 (MAP2) in Dendrites of N. Laminaris of the Chick Following Deprivation of Afferent Activity

    PubMed Central

    Wang, Yuan; Rubel, Edwin W

    2009-01-01

    Differential innervation of segregated dendritic domains in the chick nucleus laminaris (NL), composed of third-order auditory neurons, provides a unique model to study synaptic regulation of dendritic structure. Altering the synaptic input to one dendritic domain affects the structure and length of the manipulated dendrites while leaving the other set of unmanipulated dendrites largely unchanged. Little is known about the effects of neuronal input on the cytoskeletal structure of NL dendrites and whether changes in the cytoskeleton are responsible for dendritic remodeling following manipulations of synaptic inputs. In this study, we investigate changes in the immunoreactivity of high-molecular weight microtubule associated protein 2 (MAP2) in NL dendrites following two different manipulations of their afferent input. Unilateral cochlea removal eliminates excitatory synaptic input to the ventral dendrites of the contralateral NL and the dorsal dendrites of the ipsilateral NL. This manipulation produced a dramatic decrease in MAP2 immunoreactivity in the deafferented dendrites. This decrease was detected as early as three hours following the surgery, well before any degeneration of afferent axons. A similar decrease in MAP2 immunoreactivity in deafferented NL dendrites was detected following a midline transection that silences the excitatory synaptic input to the ventral dendrites on both sides of the brain. These changes were most distinct in the caudal portion of the nucleus where individual deafferented dendritic branches contained less immunoreactivity than intact dendrites. Our results suggest that the cytoskeletal protein MAP2, which is distributed in dendrites, perikarya, and postsynaptic densities, may play a role in deafferentation-induced dendritic remodeling. PMID:18440716

  2. Characterization and epitope mapping of monoclonal antibodies raised against rat hepatitis E virus capsid protein: An evaluation of their neutralizing activity in a cell culture system.

    PubMed

    Kobayashi, Tominari; Takahashi, Masaharu; Tanggis; Mulyanto; Jirintai, Suljid; Nagashima, Shigeo; Nishizawa, Tsutomu; Okamoto, Hiroaki

    2016-07-01

    Hepatitis E virus (HEV) is the causative agent of acute hepatitis. Rat HEV is a recently discovered virus related to, but distinct from, human HEV. Since laboratory rats can be reproducibly infected with rat HEV and a cell culture system has been established for rat HEV, this virus may be used as a surrogate virus for human HEV, enabling studies on virus replication and mechanism of infection. However, monoclonal antibodies (MAbs) against rat HEV capsid (ORF2) protein are not available. In this study, 12 murine MAbs were generated against a recombinant ORF2 protein of rat HEV (rRatHEV-ORF2: amino acids 101-644) and were classified into at least six distinct groups by epitope mapping and a cross-reactivity analysis with human HEV ORF2 proteins. Two non-cross-reactive MAbs recognizing the protruding (P) domain detected both non-denatured and denatured rRatHEV-ORF2 protein and efficiently captured cell culture-produced rat HEV particles that had been treated with deoxycholate and trypsin, but not those without prior treatment. In addition, these two MAbs were able to efficiently neutralize replication of cell culture-generated rat HEV particles without lipid membranes (but not those with lipid membranes) in a cell culture system, similar to human HEV. PMID:26992654

  3. Building protein interaction maps for Down's syndrome.

    PubMed

    Gardiner, Katheleen; Davisson, Muriel T; Crnic, Linda S

    2004-08-01

    Now that the complete sequences for human chromosome 21 and the orthologous mouse genomic regions are known, reasonably complete, conserved, protein-coding gene catalogues are also available. The central issue now facing Down's syndrome researchers is the correlation of increased expression of specific, normal, chromosome 21 genes with the development of specific deficits in learning and memory. Because of the number of candidate genes involved, the number of alternative splice variants of individual genes and the number of pathways in which these genes function, a pathway analysis approach will be critical to success. Here, three examples, both gene specific and pathway related, that would benefit from pathway analysis are discussed: (1) the potential roles of eight chromosome 21 proteins in RNA processing pathways; (2) the chromosome 21 protein intersectin 1 and its domain composition, alternative splicing, protein interactions and functions; and (3) the interactions of ten chromosome 21 proteins with components of the mitogen-activated protein kinase and the calcineurin signalling pathways. A productive approach to developing gene-phenotype correlations in Down's syndrome will make use of known and predicted functions and interactions of chromosome 21 genes to predict pathways that may be perturbed by their increased levels of expression. Investigations may then be targeted in animal models to specific interactions, intermediate steps or end-points of such pathways and the downstream - perhaps amplified - consequences of gene dosage directly assessed. Once pathway perturbations have been identified, the potential for rational design of therapeutics becomes practical. PMID:15355596

  4. Non-Microtubular Localizations of Microtubule-Associated Protein 6 (MAP6)

    PubMed Central

    Gory-Fauré, Sylvie; Windscheid, Vanessa; Brocard, Jacques; Montessuit, Sylvie; Tsutsumi, Ryouhei; Denarier, Eric; Fukata, Yuko; Bosc, Christophe; Delaroche, Julie; Collomb, Nora; Fukata, Masaki; Martinou, Jean-Claude; Pernet-Gallay, Karin; Andrieux, Annie

    2014-01-01

    MAP6 proteins (MAP6s), which include MAP6-N (also called Stable Tubule Only Polypeptide, or STOP) and MAP6d1 (MAP6 domain-containing protein 1, also called STOP-Like protein 21 kD, or SL21), bind to and stabilize microtubules. MAP6 deletion in mice severely alters integrated brain functions and is associated with synaptic defects, suggesting that MAP6s may also have alternative cellular roles. MAP6s reportedly associate with the Golgi apparatus through palmitoylation of their N-terminal domain, and specific isoforms have been shown to bind actin. Here, we use heterologous systems to investigate several biochemical properties of MAP6 proteins. We demonstrate that the three N-terminal cysteines of MAP6d1 are palmitoylated by a subset of DHHC-type palmitoylating enzymes. Analysis of the subcellular localization of palmitoylated MAP6d1, including electron microscopic analysis, reveals possible localization to the Golgi and the plasma membrane but no association with the endoplasmic reticulum. Moreover, we observed localization of MAP6d1 to mitochondria, which requires the N-terminus of the protein but does not require palmitoylation. We show that endogenous MAP6d1 localized at mitochondria in mature mice neurons as well as at the outer membrane and in the intermembrane space of purified mouse mitochondria. Last, we found that MAP6d1 can multimerize via a microtubule-binding module. Interestingly, most of these properties of MAP6d1 are shared by MAP6-N. Together, these results describe several properties of MAP6 proteins, including their intercellular localization and multimerization activity, which may be relevant to neuronal differentiation and synaptic functions. PMID:25526643

  5. Computationally mapping sequence space to understand evolutionary protein engineering.

    PubMed

    Armstrong, Kathryn A; Tidor, Bruce

    2008-01-01

    Evolutionary protein engineering has been dramatically successful, producing a wide variety of new proteins with altered stability, binding affinity, and enzymatic activity. However, the success of such procedures is often unreliable, and the impact of the choice of protein, engineering goal, and evolutionary procedure is not well understood. We have created a framework for understanding aspects of the protein engineering process by computationally mapping regions of feasible sequence space for three small proteins using structure-based design protocols. We then tested the ability of different evolutionary search strategies to explore these sequence spaces. The results point to a non-intuitive relationship between the error-prone PCR mutation rate and the number of rounds of replication. The evolutionary relationships among feasible sequences reveal hub-like sequences that serve as particularly fruitful starting sequences for evolutionary search. Moreover, genetic recombination procedures were examined, and tradeoffs relating sequence diversity and search efficiency were identified. This framework allows us to consider the impact of protein structure on the allowed sequence space and therefore on the challenges that each protein presents to error-prone PCR and genetic recombination procedures. PMID:18020358

  6. Whole-brain activity mapping onto a zebrafish brain atlas

    PubMed Central

    Randlett, Owen; Wee, Caroline L.; Naumann, Eva A.; Nnaemeka, Onyeka; Schoppik, David; Fitzgerald, James E.; Portugues, Ruben; Lacoste, Alix M.B.; Riegler, Clemens; Engert, Florian; Schier, Alexander F.

    2015-01-01

    In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open source atlas containing molecular labels and anatomical region definitions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated-Extracellular signal-regulated kinase (ERK/MAPK) as a readout of neural activity, we have developed a system to create and contextualize whole brain maps of stimulus- and behavior-dependent neural activity. This MAP-Mapping (Mitogen Activated Protein kinase – Mapping) assay is technically simple, fast, inexpensive, and data analysis is completely automated. Since MAP-Mapping is performed on fish that are freely swimming, it is applicable to nearly any stimulus or behavior. We demonstrate the utility of our high-throughput approach using hunting/feeding, pharmacological, visual and noxious stimuli. The resultant maps outline hundreds of areas associated with behaviors. PMID:26778924

  7. The mepsMAP server. Mapping epitopes on protein surface: mining annotated proteins.

    PubMed

    Carrabino, D; D'Onorio De Meo, P; Sanna, N; Castrignanò, T; Orsini, M; Floris, M; Tramontano, A

    2007-06-01

    For a growing number of biologists DNA or protein data are typically retrieved and managed on the Web, and not in the laboratory. A large number of bioinformatics datasets from primary and (thousands of) secondary databases are scattered on the Web in various formats. A biologist end-user might need to access and use tens of databases and tools every day. For this reason, the bioinformatics community is developing more and more service-oriented architectures (SOAs): software architecture of loosely coupled software services that can be accessed without knowledge of, or control over, their internal architecture. Data-processing and analysis tasks can be automated by having free access to bioinformatics Web services (WSs) that are the building blocks of the SOAs. In this paper we introduce a new bioinformatics Web server, mepsMAP (mapping epitopes on protein surface: Mining Annotated Proteins), developed to identify the recognition sites between antibodies and their cognate antigens. In some cases, the recognition site is represented by a continuous segment of the antigen sequence, but much more often the epitope is "conformational," i.e., the antibody recognizes the location and type of exposed antigen side chains that are not necessarily contiguous in the antigen's sequence, but brought together by its three-dimensional structure. A facility on the server allows the user to search putative conformational epitopes on protein surface, querying the system for proteins with a given annotation. The mepsMAP server has been implemented as a SOA composed by a database and a set of four WSs. We present here the software architecture of the system with a detailed description of the WS dataflow that has been optimized to provide the best computing performance while maintaining the easiest end-user access to the system via a Web interface. PMID:17695751

  8. Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function.

    PubMed

    Floyd, Brendan J; Wilkerson, Emily M; Veling, Mike T; Minogue, Catie E; Xia, Chuanwu; Beebe, Emily T; Wrobel, Russell L; Cho, Holly; Kremer, Laura S; Alston, Charlotte L; Gromek, Katarzyna A; Dolan, Brendan K; Ulbrich, Arne; Stefely, Jonathan A; Bohl, Sarah L; Werner, Kelly M; Jochem, Adam; Westphall, Michael S; Rensvold, Jarred W; Taylor, Robert W; Prokisch, Holger; Kim, Jung-Ja P; Coon, Joshua J; Pagliarini, David J

    2016-08-18

    Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function. PMID:27499296

  9. Phosphodiesterase MoPdeH targets MoMck1 of the conserved mitogen-activated protein (MAP) kinase signalling pathway to regulate cell wall integrity in rice blast fungus Magnaporthe oryzae.

    PubMed

    Yin, Ziyi; Tang, Wei; Wang, Jingzhen; Liu, Xinyu; Yang, Lina; Gao, Chuyun; Zhang, Jinlong; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2016-06-01

    In the rice blast fungus Magnaporthe oryzae, the high-affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance through an unknown mechanism. By utilizing affinity purification, we found that MoPdeH interacts with MoMck1, one of the components of the mitogen-activated protein (MAP) kinase cascade that regulates CWI. Overexpression of MoMCK1 suppressed defects in autolysis and pathogenicity of the ΔMopdeH mutant, although partially, suggesting that MoPdeH plays a critical role in CWI maintenance mediated by the MAP kinase pathway. We found that MoMck1 and two other MAP kinase cascade components, MoMkk1 and MoMps1, modulate intracellular cAMP levels by regulating the expression of MoPDEH through a feedback loop. In addition, disruption of MoMKK1 resulted in less aerial hyphal formation, defective asexual development and attenuated pathogenicity. Moreover, MoMkk1 plays a role in the response to osmotic stress via regulation of MoOsm1 phosphorylation levels, whereas endoplasmic reticulum (ER) stress enhances MoMps1 phosphorylation and loss of the MAP kinase cascade component affects the unfolded protein response (UPR) pathway. Taken together, our findings demonstrate that MoPdeH functions upstream of the MoMck1-MoMkk1-MoMps1 MAP kinase pathway to regulate CWI, and that MoPdeH also mediates crosstalk between the cAMP signalling pathway, the osmotic sensing high osmolarity glycerol (HOG) pathway and the dithiothreitol (DTT)-induced UPR pathway in M. oryzae. PMID:27193947

  10. Whole-brain activity mapping onto a zebrafish brain atlas.

    PubMed

    Randlett, Owen; Wee, Caroline L; Naumann, Eva A; Nnaemeka, Onyeka; Schoppik, David; Fitzgerald, James E; Portugues, Ruben; Lacoste, Alix M B; Riegler, Clemens; Engert, Florian; Schier, Alexander F

    2015-11-01

    In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open-source atlas containing molecular labels and definitions of anatomical regions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated extracellular signal–regulated kinase (ERK) as a readout of neural activity, we have developed a system to create and contextualize whole-brain maps of stimulus- and behavior-dependent neural activity. This mitogen-activated protein kinase (MAP)-mapping assay is technically simple, and data analysis is completely automated. Because MAP-mapping is performed on freely swimming fish, it is applicable to studies of nearly any stimulus or behavior. Here we demonstrate our high-throughput approach using pharmacological, visual and noxious stimuli, as well as hunting and feeding. The resultant maps outline hundreds of areas associated with behaviors. PMID:26778924

  11. New NSO Solar Surface Activity Maps

    NASA Astrophysics Data System (ADS)

    Henney, C. J.; Harvey, J. W.

    2001-05-01

    Using NSO-Kitt Peak Vacuum Telescope (KPVT) synoptic data, we present several new solar surface activity maps. The motivation is to test conventional wisdom about conditions that are likely to produce solar activity such as flares, coronal mass ejections and high speed solar wind streams. The ultimate goal is to improve real-time, observation-based models for the purpose of predicting solar activity. A large number of maps will eventually be produced based on the wide range of ideas and models of the conditions thought to lead to solar activity events. When data from the new SOLIS instruments becomes available, the range of possible models that can be tested will be greatly expanded. At present, the daily maps include ones that show magnetic field complexity, emerging flux and high speed solar wind sources. As a proxy for local magnetic potential energy, each element of the magnetic complexity map is the distance-weighted rms of the opposing ambient magnetic field. The flux emergence map is the difference between the two most recent absolute magnetic flux images. The solar wind source map is produced from coronal hole area data. The new maps are available on the NSO-Kitt Peak World Wide Web page. This research was supported in part by the Office of Navel Research Grant N00014-91-J-1040. The NSO-Kitt Peak data used here are produced cooperatively by NSF/AURA, NASA/GSFC, and NOAA/SEC.

  12. Mapping recent chikungunya activity in the Americas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To better understand chikungunya activity in the America we mapped recent chikungunya activity in the Americas. This activity is needed to better understand that the relationships between climatic factors and disease outbreak patters are critical to the design and constructing of predictive models....

  13. [Mitogen-activated protein kinases in atherosclerosis].

    PubMed

    Bryk, Dorota; Olejarz, Wioletta; Zapolska-Downar, Danuta

    2014-01-01

    Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases) intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase), JNK (c-Jun N-terminal kinase) and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis. PMID:24491891

  14. Evolution and similarity evaluation of protein structures in contact map space.

    PubMed

    Gupta, Nitin; Mangal, Nitin; Biswas, Somenath

    2005-05-01

    Prediction of fold from amino acid sequence of a protein has been an active area of research in the past few years, but the limited accuracy of existing techniques emphasizes the need to develop newer approaches to tackle this task. In this study, we use contact map prediction as an intermediate step in fold prediction from sequence. Contact map is a reduced graph-theoretic representation of proteins that models the local and global inter-residue contacts in the structure. We start with a population of random contact maps for the protein sequence and "evolve" the population to a "high-feasibility" configuration using a genetic algorithm. A neural network is employed to assess the feasibility of contact maps based on their 4 physically relevant properties. We also introduce 5 parameters, based on algebraic graph theory and physical considerations, that can be used to judge the structural similarity between proteins through contact maps. To predict the fold of a given amino acid sequence, we predict a contact map that will sufficiently approximate the structure of the corresponding protein. Then we assess the similarity of this contact map with the representative contact map of each fold; the fold that corresponds to the closest match is our predicted fold for the input sequence. We have found that our feasibility measure is able to differentiate between feasible and infeasible contact maps. Further, this novel approach is able to predict the folds from sequences significantly better than a random predictor. PMID:15726585

  15. Echo Mapping of Active Galactic Nuclei

    NASA Technical Reports Server (NTRS)

    Peterson, B. M.; Horne, K.

    2004-01-01

    Echo mapping makes use of the intrinsic variability of the continuum source in active galactic nuclei to map out the distribution and kinematics of line-emitting gas from its light travel time-delayed response to continuum changes. Echo mapping experiments have yielded sizes for the broad line-emitting region in about three dozen AGNs. The dynamics of the line-emitting gas seem to be dominated by the gravity of the central black hole, enabling measurement of the black-hole masses in AGNs. We discuss requirements for future echo-mapping experiments that will yield the high quality velocity-delay maps of the broad-line region that are needed to determine its physical nature.

  16. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

    PubMed

    Lv, Qiang; Yang, Xu-Zhong; Fu, Long-Yun; Lu, Yv-Ting; Lu, Yan-Hua; Zhao, Jian; Wang, Fu-Jun

    2015-07-01

    MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area. PMID:25797209

  17. FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.

    PubMed Central

    Worthen, G S; Avdi, N; Buhl, A M; Suzuki, N; Johnson, G L

    1994-01-01

    Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants. Images PMID:8040337

  18. The PHD motif of Map3k1 activates cytokine-dependent MAPK signaling

    PubMed Central

    Gallagher, Ewen; Suddason, Tesha

    2015-01-01

    We generated a mutation in the gene encoding mitogen-activated protein kinase kinase kinase 1 (Map3k1) that results in a protein with an inactive plant homeodomain (PHD). Map3k1mPHD cells are defective in cytokine-mediated MAPK signaling. Protein array identified transforming growth factor (TGF-β)-activated kinase 1 binding protein 1 (Tab1) as a PHD substrate. The Map3k1 PHD transfers Lys63-linked poly-ubiquitin onto Tab1 to activate MAPKs. PMID:27308457

  19. Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions

    PubMed Central

    Kang, Bok Eum; Baker, Bradley J.

    2016-01-01

    An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response. PMID:27040905

  20. Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions.

    PubMed

    Kang, Bok Eum; Baker, Bradley J

    2016-01-01

    An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response. PMID:27040905

  1. Synthetic protein interactions reveal a functional map of the cell

    PubMed Central

    Berry, Lisa K; Ólafsson, Guðjón; Ledesma-Fernández, Elena; Thorpe, Peter H

    2016-01-01

    To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.13053.001 PMID:27098839

  2. Reverberation mapping of active galactic nuclei

    NASA Technical Reports Server (NTRS)

    Peterson, Bradley M.

    1993-01-01

    The broad emission lines in the spectra of active galactic nuclei respond to variations in the luminosity of the central continuum source with a delay due to light-travel time effects within the emission-line region. It is therefore possible through the process of 'reverberation mapping' to determine the geometry and kinematics of the emission-line region by careful monitoring of the continuum variations and the resulting emission-line response. In this review, I will discuss progress in application of the reverberation mapping technique. I will describe the underlying assumptions and limitations of the method, discuss how the results obtained to date are changing our understanding of active nuclei, and outline several new questions that might be addressed through further reverberation mapping programs.

  3. A proteome-wide protein interaction map for Campylobacter jejuni

    PubMed Central

    Parrish, Jodi R; Yu, Jingkai; Liu, Guozhen; Hines, Julie A; Chan, Jason E; Mangiola, Bernie A; Zhang, Huamei; Pacifico, Svetlana; Fotouhi, Farshad; DiRita, Victor J; Ideker, Trey; Andrews, Phillip; Finley, Russell L

    2007-01-01

    Background Data from large-scale protein interaction screens for humans and model eukaryotes have been invaluable for developing systems-level models of biological processes. Despite this value, only a limited amount of interaction data is available for prokaryotes. Here we report the systematic identification of protein interactions for the bacterium Campylobacter jejuni, a food-borne pathogen and a major cause of gastroenteritis worldwide. Results Using high-throughput yeast two-hybrid screens we detected and reproduced 11,687 interactions. The resulting interaction map includes 80% of the predicted C. jejuni NCTC11168 proteins and places a large number of poorly characterized proteins into networks that provide initial clues about their functions. We used the map to identify a number of conserved subnetworks by comparison to protein networks from Escherichia coli and Saccharomyces cerevisiae. We also demonstrate the value of the interactome data for mapping biological pathways by identifying the C. jejuni chemotaxis pathway. Finally, the interaction map also includes a large subnetwork of putative essential genes that may be used to identify potential new antimicrobial drug targets for C. jejuni and related organisms. Conclusion The C. jejuni protein interaction map is one of the most comprehensive yet determined for a free-living organism and nearly doubles the binary interactions available for the prokaryotic kingdom. This high level of coverage facilitates pathway mapping and function prediction for a large number of C. jejuni proteins as well as orthologous proteins from other organisms. The broad coverage also facilitates cross-species comparisons for the identification of evolutionarily conserved subnetworks of protein interactions. PMID:17615063

  4. Focused grid-based resampling for protein docking and mapping.

    PubMed

    Mamonov, Artem B; Moghadasi, Mohammad; Mirzaei, Hanieh; Zarbafian, Shahrooz; Grove, Laurie E; Bohnuud, Tanggis; Vakili, Pirooz; Ch Paschalidis, Ioannis; Vajda, Sandor; Kozakov, Dima

    2016-04-30

    The fast Fourier transform (FFT) sampling algorithm has been used with success in application to protein-protein docking and for protein mapping, the latter docking a variety of small organic molecules for the identification of binding hot spots on the target protein. Here we explore the local rather than global usage of the FFT sampling approach in docking applications. If the global FFT based search yields a near-native cluster of docked structures for a protein complex, then focused resampling of the cluster generally leads to a substantial increase in the number of conformations close to the native structure. In protein mapping, focused resampling of the selected hot spot regions generally reveals further hot spots that, while not as strong as the primary hot spots, also contribute to ligand binding. The detection of additional ligand binding regions is shown by the improved overlap between hot spots and bound ligands. © 2016 Wiley Periodicals, Inc. PMID:26837000

  5. Separating proteins with activated carbon.

    PubMed

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon. PMID:24898563

  6. Protein extraction from activated sludge.

    PubMed

    Denecke, M

    2006-01-01

    Two methods for the separation of protein originating from activated sludge were compared. In one method, the total protein was isolated out of the activated sludge (crude extract). These samples included all dissolved proteins originating from the bacterial cells and biofilm made up of extracellular polymeric substances (EPS). Every time polyacrylamide gel electrophoresis (PAGE) was done, the protein bands from samples of crude extract were covered by polymeric substances including carbohydrates, uronic acids or humic compounds. Using the immunoblot technique it was possible to demonstrate the presence of the heat shock protein HSP70 in crude extracts of activated sludge. The comparison of protein fingerprints required that clear and distinct bands appear on the PAGE analysis. To this end, a procedure to separates bacterial cells from the EPS was developed. Bacterial cells were separated by incubation with EDTA and subsequent filtration. The isolated cells were directly incubated in a sample buffer. PMID:16898150

  7. Mapping of the Tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo.

    PubMed

    Grdzelishvili, V Z; Chapman, S N; Dawson, W O; Lewandowski, D J

    2000-09-15

    The Tobacco mosaic virus movement protein (MP) and coat protein (CP) are expressed from 3'-coterminal subgenomic RNAs (sgRNAs). The transcription start site of the MP sgRNA, previously mapped to positions 4838 (Y. Watanabe, T. Meshi, and Y. Okada (1984), FEBS Lett. 173, 247-250) and 4828 (K. Lehto, G. L. Grantham, and W. O. Dawson (1990), Virology 174, 145-157) for the TMV OM and U1 strains, respectively, has been reexamined and mapped to position 4838 for strain U1. Sequences of the MP and CP sgRNA promoters were delineated by deletion analysis. The boundaries for minimal and full MP sgRNA promoter activity were localized between -35 and +10 and -95 and +40, respectively, relative to the transcription start site. The minimal CP sgRNA promoter was mapped between -69 and +12, whereas the boundaries of the fully active promoter were between -157 and +54. Computer analysis predicted two stem-loop structures (SL1 and SL2) upstream of the MP sgRNA transcription start site. Deletion analysis and site-directed mutagenesis suggested that SL1 secondary structure, but not its sequence, was required for MP sgRNA promoter activity, whereas a 39-nt deletion removing most of the SL2 region increased MP sgRNA accumulation fourfold. Computer-predicted folding of the fully active CP sgRNA promoter revealed one long stem-loop structure. Deletion analysis suggested that the upper part of this stem-loop, located upstream of the transcription start site, was essential for transcription and that the lower part of the stem had an enhancing role. PMID:11017798

  8. The Role of Mitogen-Activated Protein (MAP) Kinase Signaling Components in the Fungal Development, Stress Response and Virulence of the Fungal Cereal Pathogen Bipolaris sorokiniana

    PubMed Central

    Leng, Yueqiang; Zhong, Shaobin

    2015-01-01

    Mitogen-activated protein kinases (MAPKs) have been demonstrated to be involved in fungal development, sexual reproduction, pathogenicity and/or virulence in many filamentous plant pathogenic fungi, but genes for MAPKs in the fungal cereal pathogen Bipolaris sorokiniana have not been characterized. In this study, orthologues of three MAPK genes (CsSLT2, CsHOG1 and CsFUS3) and one MAPK kinase kinase (MAPKKK) gene (CsSTE11) were identified in the whole genome sequence of the B. sorokiniana isolate ND90Pr, and knockout mutants were generated for each of them. The ∆Csfus3 and ∆Csste11 mutants were defective in conidiation and formation of appressoria-like structures, showed hypersensitivity to oxidative stress and lost pathogenicity on non-wounded leaves of barley cv. Bowman. When inoculated on wounded leaves of Bowman, the ∆Csfus3 and ∆Csste11 mutants were reduced in virulence compared to the wild type. No morphological changes were observed in the ∆Cshog1 mutants in comparison with the wild type; however, they were slightly reduced in growth under oxidative stress and were hypersensitive to hyperosmotic stress. The ∆Cshog1 mutants formed normal appressoria-like structures but were reduced in virulence when inoculated on Bowman leaves. The ∆Csslt2 mutants produced more vegetative hyphae, had lighter pigmentation, were more sensitive to cell wall degrading enzymes, and were reduced in virulence on Bowman leaves, although they formed normal appressoria like the wild type. Root infection assays indicated that the ∆Cshog1 and ∆Csslt2 mutants were able to infect barley roots while the ∆Csfus3 and ∆Csste11 failed to cause any symptoms. However, no significant difference in virulence was observed for ∆Cshog1 mutants while ∆Csslt2 mutants showed significantly reduced virulence on barley roots in comparison with the wild type. Our results indicated that all of these MAPK and MAPKKK genes are involved in the regulation of fungal development under

  9. Global, quantitative and dynamic mapping of protein subcellular localization

    PubMed Central

    Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

    2016-01-01

    Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

  10. Uav Data Processing for Rapid Mapping Activities

    NASA Astrophysics Data System (ADS)

    Tampubolon, W.; Reinhardt, W.

    2015-08-01

    During disaster and emergency situations, geospatial data plays an important role to serve as a framework for decision support system. As one component of basic geospatial data, large scale topographical maps are mandatory in order to enable geospatial analysis within quite a number of societal challenges. The increasing role of geo-information in disaster management nowadays consequently needs to include geospatial aspects on its analysis. Therefore different geospatial datasets can be combined in order to produce reliable geospatial analysis especially in the context of disaster preparedness and emergency response. A very well-known issue in this context is the fast delivery of geospatial relevant data which is expressed by the term "Rapid Mapping". Unmanned Aerial Vehicle (UAV) is the rising geospatial data platform nowadays that can be attractive for modelling and monitoring the disaster area with a low cost and timely acquisition in such critical period of time. Disaster-related object extraction is of special interest for many applications. In this paper, UAV-borne data has been used for supporting rapid mapping activities in combination with high resolution airborne Interferometric Synthetic Aperture Radar (IFSAR) data. A real disaster instance from 2013 in conjunction with Mount Sinabung eruption, Northern Sumatra, Indonesia, is used as the benchmark test for the rapid mapping activities presented in this paper. On this context, the reliable IFSAR dataset from airborne data acquisition in 2011 has been used as a comparable dataset for accuracy investigation and assessment purpose in 3 D reconstructions. After all, this paper presents a proper geo-referencing and feature extraction method of UAV data to support rapid mapping activities.

  11. Intracellular protein interaction mapping with FRET hybrids

    PubMed Central

    You, Xia; Nguyen, Annalee W.; Jabaiah, Abeer; Sheff, Mark A.; Thorn, Kurt S.; Daugherty, Patrick S.

    2006-01-01

    A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types. PMID:17130455

  12. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein

    PubMed Central

    Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

    2016-01-01

    In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents. PMID:27459300

  13. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein.

    PubMed

    Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

    2016-01-01

    In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents. PMID:27459300

  14. Activation of protein kinase C induces mitogen-activated protein kinase dephosphorylation and pronucleus formation in rat oocytes.

    PubMed

    Lu, Qing; Smith, Gary D; Chen, Da-Yuan; Han, Zhi-Ming; Sun, Qing-Yuan

    2002-07-01

    Mammalian oocytes are arrested at metaphase of the second meiotic division (MII) before fertilization. When oocytes are stimulated by spermatozoa, they exit MII stage and complete meiosis. It has been suggested that an immediate increase in intracellular free calcium concentration and inactivation of maturation promoting factor (MPF) are required for oocyte activation. However, the underlying mechanism is still unclear. In the present study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, and their interplay in rat oocyte activation. We found that MAP kinase became dephosphorylated in correlation with pronucleus formation after fertilization. Protein kinase C activators, phorbol 12-myriatate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8), triggered dephosphorylation of MAP kinase and pronucleus formation in a dose-dependent and time-dependent manner. Dephosphorylation of MAP kinase was also correlated with pronucleus formation when oocytes were treated with PKC activators. Effects of PKC activators were abolished by the PKC inhibitors, calphostin C and staurosporine, as well as a protein phosphatase blocker, okadaic acid (OA). These results suggest that PKC activation may cause rat oocyte pronucleus formation via MAP kinase dephosphorylation, which is probably mediated by OA-sensitive protein phosphatases. We also provide evidence supporting the involvement of such a process in fertilization. PMID:12080000

  15. Systematic spatial mapping of proteins at exocytic and endocytic structures

    PubMed Central

    Larson, Ben T.; Sochacki, Kem A.; Kindem, Jonathan M.; Taraska, Justin W.

    2014-01-01

    Vesicular secretion (exocytosis) involves the release and then compensatory recycling of vesicle components through endocytosis. This fundamental cellular process is controlled by the coordinated assembly and interactions of dozens of proteins at the plasma membrane. Understanding the molecular composition of individual exocytic and endocytic structures and their organization across the plasma membrane is critical to understanding the behavior and regulation of these two cellular processes. Here we develop a high-resolution and high-throughput fluorescence imaging–based approach for the unbiased mapping of 78 proteins at single exocytic vesicles and endocytic structures in neuroendocrine PC12 cells. This analysis uses two-color single-frame images to provide a systems-level map of the steady-state distributions of proteins at individual exocytic and endocytic structures in the cell. Along with this quantitative map, we find that both calcium-regulated exocytic vesicles (dense core vesicles) and endocytic structures (clathrin-coated structures) and the proteins associated with these structures exhibit a random spatial distribution in unstimulated neuroendocrine PC12 cells. This approach is broadly applicable for quantitatively mapping the molecular composition and spatial organization of discrete cellular processes with central molecular hubs. PMID:24807904

  16. RRDistMaps: a UCSF Chimera tool for viewing and comparing protein distance maps

    PubMed Central

    Chen, Jonathan E.; Huang, Conrad C.; Ferrin, Thomas E.

    2015-01-01

    Motivation: Contact maps are a convenient method for the structural biologists to identify structural features through two-dimensional simplification. Binary (yes/no) contact maps with a single cutoff distance can be generalized to show continuous distance ranges. We have developed a UCSF Chimera tool, RRDistMaps, to compute such generalized maps in order to analyze pairwise variations in intramolecular contacts. An interactive utility, RRDistMaps, visualizes conformational changes, both local (e.g. binding-site residues) and global (e.g. hinge motion), between unbound and bound proteins through distance patterns. Users can target residue pairs in RRDistMaps for further navigation in Chimera. The interface contains the unique features of identifying long-range residue motion and aligning sequences to simultaneously compare distance maps. Availability and implementation: RRDistMaps was developed as part of UCSF Chimera release 1.10, which is freely available at http://rbvi.ucsf.edu/chimera/download.html, and operates on Linux, Windows, and Mac OS. Contact: conrad@cgl.ucsf.edu PMID:25540183

  17. Recent advances in large-scale protein interactome mapping.

    PubMed

    Mehta, Virja; Trinkle-Mulcahy, Laura

    2016-01-01

    Protein-protein interactions (PPIs) underlie most, if not all, cellular functions. The comprehensive mapping of these complex networks of stable and transient associations thus remains a key goal, both for systems biology-based initiatives (where it can be combined with other 'omics' data to gain a better understanding of functional pathways and networks) and for focused biological studies. Despite the significant challenges of such an undertaking, major strides have been made over the past few years. They include improvements in the computation prediction of PPIs and the literature curation of low-throughput studies of specific protein complexes, but also an increase in the deposition of high-quality data from non-biased high-throughput experimental PPI mapping strategies into publicly available databases. PMID:27158474

  18. Recent advances in large-scale protein interactome mapping

    PubMed Central

    Mehta, Virja; Trinkle-Mulcahy, Laura

    2016-01-01

    Protein-protein interactions (PPIs) underlie most, if not all, cellular functions. The comprehensive mapping of these complex networks of stable and transient associations thus remains a key goal, both for systems biology-based initiatives (where it can be combined with other ‘omics’ data to gain a better understanding of functional pathways and networks) and for focused biological studies. Despite the significant challenges of such an undertaking, major strides have been made over the past few years. They include improvements in the computation prediction of PPIs and the literature curation of low-throughput studies of specific protein complexes, but also an increase in the deposition of high-quality data from non-biased high-throughput experimental PPI mapping strategies into publicly available databases. PMID:27158474

  19. Bacterial Heat Shock Protein Activity

    PubMed Central

    Maleki, Farajollah; Khosravi, Afra; Nasser, Ahmad; Taghinejad, Hamid

    2016-01-01

    Bacteria are exposed to different types of stress in their growth conditions. They have developed appropriate responses, modulated by the re-modeling of protein complexes and by phosphorylation dependent signal transduction systems, to adapt and to survive in a variety range of nature. Proteins are essential components for biologic activity in the eukaryotic and prokaryotic cell. Heat Shock Proteins (HSP) have been identified from various organisms and have critical role in cell hemostasis. Chaperone can sense environment and have different potential role in the organism evolution. PMID:27134861

  20. MAP1S Protein Regulates the Phagocytosis of Bacteria and Toll-like Receptor (TLR) Signaling.

    PubMed

    Shi, Ming; Zhang, Yifan; Liu, Leyuan; Zhang, Tingting; Han, Fang; Cleveland, Joseph; Wang, Fen; McKeehan, Wallace L; Li, Yu; Zhang, Dekai

    2016-01-15

    Phagocytosis is a critical cellular process for innate immune defense against microbial infection. The regulation of phagocytosis process is complex and has not been well defined. An intracellular molecule might regulate cell surface-initiated phagocytosis, but the underlying molecular mechanism is poorly understood (1). In this study, we found that microtubule-associated protein 1S (MAP1S), a protein identified recently that is involved in autophagy (2), is expressed primarily in macrophages. MAP1S-deficient macrophages are impaired in the phagocytosis of bacteria. Furthermore, we demonstrate that MAP1S interacts directly with MyD88, a key adaptor of Toll-like receptors (TLRs), upon TLR activation and affects the TLR signaling pathway. Intriguingly, we also observe that, upon TLR activation, MyD88 participates in autophagy processing in a MAP1S-dependent manner by co-localizing with MAP1 light chain 3 (MAP1-LC3 or LC3). Therefore, we reveal that an intracellular autophagy-related molecule of MAP1S controls bacterial phagocytosis through TLR signaling. PMID:26565030

  1. Roles of MAP1152 and MAP1156 Proteins in the Immunopathogenesis of Mycobacterium paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Mycobacterium avium subsp. paratuberculosis (MAP) K-10 genome is a circular chromosome of about 4.8 Mb encoding 4,344 open reading frames (ORFs). Mycobacterial genomes are characterized by the presence of gene clusters encoding conserved proteins with PE and PPE motifs. Members of these families...

  2. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

    PubMed Central

    Choi, Yun-Hee; Yang, Dong Joo; Kulkarni, Atul; Moh, Sang Hyun; Kim, Ki Woo

    2015-01-01

    Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies. PMID:26703626

  3. Robots, Bulldozers, and Other Map Activities for the Primary Grades.

    ERIC Educational Resources Information Center

    Pritchard, Sandra F.

    1984-01-01

    Described are activities which will increase fluency in the use of north, south, east, west, up, and down; reinforce the proper positioning of the cardinal directions on a map grid; relate map symbols to familiar surface features; give practice in identifying map symbols; and provide an opportunity to construct maps. (RM)

  4. Functional Mapping of Protein-Protein Interactions in an Enzyme Complex by Directed Evolution

    PubMed Central

    Roderer, Kathrin; Neuenschwander, Martin; Codoni, Giosiana; Sasso, Severin; Gamper, Marianne; Kast, Peter

    2014-01-01

    The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84–90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84–86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes. PMID:25551646

  5. Brassinosteroid-regulated GSK3/Shaggy-like Kinases Phosphorylate Mitogen-activated Protein (MAP) Kinase Kinases, Which Control Stomata Development in Arabidopsis thaliana*

    PubMed Central

    Khan, Mamoona; Rozhon, Wilfried; Bigeard, Jean; Pflieger, Delphine; Husar, Sigrid; Pitzschke, Andrea; Teige, Markus; Jonak, Claudia; Hirt, Heribert; Poppenberger, Brigitte

    2013-01-01

    Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module. PMID:23341468

  6. Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle.

    PubMed

    Trappanese, Danielle M; Sivilich, Sarah; Ets, Hillevi K; Kako, Farah; Autieri, Michael V; Moreland, Robert S

    2016-06-01

    Vascular smooth muscle contraction is primarily regulated by phosphorylation of myosin light chain. There are also modulatory pathways that control the final level of force development. We tested the hypothesis that protein kinase C (PKC) and mitogen-activated protein (MAP) kinase modulate vascular smooth muscle activity via effects on MAP kinase phosphatase-1 (MKP-1). Swine carotid arteries were mounted for isometric force recording and subjected to histamine stimulation in the presence and absence of inhibitors of PKC [bisindolylmaleimide-1 (Bis)], MAP kinase kinase (MEK) (U0126), and MKP-1 (sanguinarine) and flash frozen for measurement of MAP kinase, PKC-potentiated myosin phosphatase inhibitor 17 (CPI-17), and caldesmon phosphorylation levels. CPI-17 was phosphorylated in response to histamine and was inhibited in the presence of Bis. Caldesmon phosphorylation levels increased in response to histamine stimulation and were decreased in response to MEK inhibition but were not affected by the addition of Bis. Inhibition of PKC significantly increased p42 MAP kinase, but not p44 MAP kinase. Inhibition of MEK with U0126 inhibited both p42 and p44 MAP kinase activity. Inhibition of MKP-1 with sanguinarine blocked the Bis-dependent increase of MAP kinase activity. Sanguinarine alone increased MAP kinase activity due to its effects on MKP-1. Sanguinarine increased MKP-1 phosphorylation, which was inhibited by inhibition of MAP kinase. This suggests that MAP kinase has a negative feedback role in inhibiting MKP-1 activity. Therefore, PKC catalyzes MKP-1 phosphorylation, which is reversed by MAP kinase. Thus the fine tuning of vascular contraction is due to the concerted effort of PKC, MAP kinase, and MKP-1. PMID:27053523

  7. Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

    SciTech Connect

    Markillie, Lye Meng; Lin, Chiann Tso; Adkins, Joshua N.; Auberry, Deanna L.; Hill, Eric A.; Hooker, Brian S.; Moore, Priscilla A.; Moore, Ronald J.; Shi, Liang; Wiley, H. S.; Kery, Vladimir

    2005-04-11

    Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.

  8. Mapping Homing Endonuclease Cleavage Sites Using In Vitro Generated Protein

    PubMed Central

    Belfort, Marlene

    2015-01-01

    Mapping the precise position of endonucleolytic cleavage sites is a fundamental experimental technique used to describe the function of a homing endonuclease. However, these proteins are often recalcitrant to cloning and over-expression in biological systems because of toxicity induced by spurious DNA cleavage events. In this chapter we outline the steps to successfully express a homing endonuclease in vitro and use this product in nucleotide-resolution cleavage assays. PMID:24510259

  9. Antiviral activities of whey proteins.

    PubMed

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Wang, Yan; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Xia, Jiang

    2015-09-01

    Milk contains an array of proteins with useful bioactivities. Many milk proteins encompassing native or chemically modified casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin demonstrated antiviral activities. Casein and alpha-lactalbumin gained anti-HIV activity after modification with 3-hydroxyphthalic anhydride. Many milk proteins inhibited HIV reverse transcriptase. Bovine glycolactin, angiogenin-1, lactogenin, casein, alpha-lactalbumin, beta-lactoglobulin, bovine lactoferrampin, and human lactoferrampin inhibited HIV-1 protease and integrase. Several mammalian lactoferrins prevented hepatitis C infection. Lactoferrin, methylated alpha-lactalbumin and methylated beta-lactoglobulin inhibited human cytomegalovirus. Chemically modified alpha-lactalbumin, beta-lactoglobulin and lysozyme, lactoferrin and lactoferricin, methylated alpha-lactalbumin, methylated and ethylated beta-lactoglobulins inhibited HSV. Chemically modified bovine beta-lactoglobulin had antihuman papillomavirus activity. Beta-lactoglobulin, lactoferrin, esterified beta-lactoglobulin, and esterified lactoferrindisplayed anti-avian influenza A (H5N1) activity. Lactoferrin inhibited respiratory syncytial virus, hepatitis B virus, adenovirus, poliovirus, hantavirus, sindbis virus, semliki forest virus, echovirus, and enterovirus. Milk mucin, apolactoferrin, Fe(3+)-lactoferrin, beta-lactoglobulin, human lactadherin, bovine IgG, and bovine kappa-casein demonstrated antihuman rotavirus activity. PMID:26198883

  10. The plant cytoskeleton: recent advances in the study of the plant microtubule-associated proteins MAP-65, MAP-190 and the Xenopus MAP215-like protein, MOR1.

    PubMed

    Hussey, Patrick J; Hawkins, Timothy J; Igarashi, Hisako; Kaloriti, Despina; Smertenko, Andrei

    2002-12-01

    The microtubule cytoskeleton is a dynamic filamentous structure involved in many key processes in plant cell morphogenesis including nuclear and cell division, deposition of cell wall, cell expansion, organelle movement and secretion. The principal microtubule protein is tubulin, which associates to form the wall of the tubule. In addition, various associated proteins bind microtubules either to anchor, cross-link or regulate the microtubule network within cells. Biochemical, molecular biological and genetic approaches are being successfully used to identify these microtubule-associated proteins (MAPs) in plants, and we describe recent progress on three of these proteins. PMID:12516862

  11. From Ramachandran Maps to Tertiary Structures of Proteins.

    PubMed

    DasGupta, Debarati; Kaushik, Rahul; Jayaram, B

    2015-08-27

    Sequence to structure of proteins is an unsolved problem. A possible coarse grained resolution to this entails specification of all the torsional (Φ, Ψ) angles along the backbone of the polypeptide chain. The Ramachandran map quite elegantly depicts the allowed conformational (Φ, Ψ) space of proteins which is still very large for the purposes of accurate structure generation. We have divided the allowed (Φ, Ψ) space in Ramachandran maps into 27 distinct conformations sufficient to regenerate a structure to within 5 Å from the native, at least for small proteins, thus reducing the structure prediction problem to a specification of an alphanumeric string, i.e., the amino acid sequence together with one of the 27 conformations preferred by each amino acid residue. This still theoretically results in 27(n) conformations for a protein comprising "n" amino acids. We then investigated the spatial correlations at the two-residue (dipeptide) and three-residue (tripeptide) levels in what may be described as higher order Ramachandran maps, with the premise that the allowed conformational space starts to shrink as we introduce neighborhood effects. We found, for instance, for a tripeptide which potentially can exist in any of the 27(3) "allowed" conformations, three-fourths of these conformations are redundant to the 95% confidence level, suggesting sequence context dependent preferred conformations. We then created a look-up table of preferred conformations at the tripeptide level and correlated them with energetically favorable conformations. We found in particular that Boltzmann probabilities calculated from van der Waals energies for each conformation of tripeptides correlate well with the observed populations in the structural database (the average correlation coefficient is ∼0.8). An alpha-numeric string and hence the tertiary structure can be generated for any sequence from the look-up table within minutes on a single processor and to a higher level of accuracy

  12. A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells

    PubMed Central

    Rafiq, Rather A.; Quadri, Afnan; Nazir, Lone A.; Peerzada, Kaiser; Ganai, Bashir A.; Tasduq, Sheikh A.

    2015-01-01

    Ultraviolet (UV) radiation–induced skin damage contributes strongly to the formation of melanoma, a highly lethal form of skin cancer. Quercetin (Qu), the most widely consumed dietary bioflavonoid and well known inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, has been reported to be chemopreventive in several forms of non-melanoma skin cancers. Here, we report that the treatment of ultraviolet (UV)-B-irradiated B16F10 melanoma cells with quercetin resulted in a dose dependent reduction in cell viability and increased apoptosis. The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM). Promotion of UVB-induced cell death by quercetin was further revealed by cleavage of chromosomal DNA, caspase activation, poly (ADP) ribose polymerase (PARP) cleavage, and an increase in sub-G1 cells. Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation. Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF). Overall, these results suggest the possibility of using quercetin in combination with UVB as a possible treatment option for melanoma in future. PMID:26148186

  13. A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells.

    PubMed

    Rafiq, Rather A; Quadri, Afnan; Nazir, Lone A; Peerzada, Kaiser; Ganai, Bashir A; Tasduq, Sheikh A

    2015-01-01

    Ultraviolet (UV) radiation-induced skin damage contributes strongly to the formation of melanoma, a highly lethal form of skin cancer. Quercetin (Qu), the most widely consumed dietary bioflavonoid and well known inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, has been reported to be chemopreventive in several forms of non-melanoma skin cancers. Here, we report that the treatment of ultraviolet (UV)-B-irradiated B16F10 melanoma cells with quercetin resulted in a dose dependent reduction in cell viability and increased apoptosis. The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM). Promotion of UVB-induced cell death by quercetin was further revealed by cleavage of chromosomal DNA, caspase activation, poly (ADP) ribose polymerase (PARP) cleavage, and an increase in sub-G1 cells. Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation. Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF). Overall, these results suggest the possibility of using quercetin in combination with UVB as a possible treatment option for melanoma in future. PMID:26148186

  14. Mapping gene activity of Arabidopsis root hairs

    PubMed Central

    2013-01-01

    Background Quantitative information on gene activity at single cell-type resolution is essential for the understanding of how cells work and interact. Root hairs, or trichoblasts, tubular-shaped outgrowths of specialized cells in the epidermis, represent an ideal model for cell fate acquisition and differentiation in plants. Results Here, we provide an atlas of gene and protein expression in Arabidopsis root hair cells, generated by paired-end RNA sequencing and LC/MS-MS analysis of protoplasts from plants containing a pEXP7-GFP reporter construct. In total, transcripts of 23,034 genes were detected in root hairs. High-resolution proteome analysis led to the reliable identification of 2,447 proteins, 129 of which were differentially expressed between root hairs and non-root hair tissue. Dissection of pre-mRNA splicing patterns showed that all types of alternative splicing were cell type-dependent, and less complex in EXP7-expressing cells when compared to non-root hair cells. Intron retention was repressed in several transcripts functionally related to root hair morphogenesis, indicative of a cell type-specific control of gene expression by alternative splicing of pre-mRNA. Concordance between mRNA and protein expression was generally high, but in many cases mRNA expression was not predictive for protein abundance. Conclusions The integrated analysis shows that gene activity in root hairs is dictated by orchestrated, multilayered regulatory mechanisms that allow for a cell type-specific composition of functional components. PMID:23800126

  15. Reverberation Mapping Campaign of Active Galactic Nuclei

    NASA Astrophysics Data System (ADS)

    Bhattacharjee, Anirban

    In this dissertation, I present results of black hole mass (M BH) measurements of four active galactic nuclei (AGN). AGN activity plays a key part in galaxy formation and evolution as evidenced by relationships like MBH-sigmastar. Accurate measurements of MBH is thus required to better understand these relationships. Luminosity of AGNs is also related to the radius of the broad line region (BLR). I have used reverberation mapping (RM) to obtain measurements of the radius of BLR and MBH of four AGNs. Reverberation data were collected over a period of 180-day span in 2012. None of these objects have been reverberation mapped before. We have also placed our objects on the Radius-Luminosity relationship and three out of four fall on the relationship. The fourth object lies above the Radius-Luminosity relationship and is a minor outlier. Two of these objects are Radio-Loud, which have orientation information available. This has increased the sample of radio-loud AGNs, which have RM from 5 to 7. We have increased the overall sample size of AGNs that have mass measurements from 62 to 66. We obtain masses for these following objects 3C 382 (MBH)= 30.1 -8.7+12.61 x 107 M O, PG2209+184 (MBH)=14.53-8.7 +5.79 x 107 MO, MARK 1040 (MBH)= 30.1-8.7+12.61 x 107 MO and 1ES0206+52(MBH)= 517.3-280+214 x 107 M O.

  16. Nanotools for Neuroscience and Brain Activity Mapping

    PubMed Central

    Alivisatos, A. Paul; Andrews, Anne M.; Boyden, Edward S.; Chun, Miyoung; Church, George M.; Deisseroth, Karl; Donoghue, John P.; Fraser, Scott E.; Lippincott-Schwartz, Jennifer; Looger, Loren L.; Masmanidis, Sotiris; McEuen, Paul L.; Nurmikko, Arto V.; Park, Hongkun; Peterka, Darcy S.; Reid, Clay; Roukes, Michael L.; Scherer, Axel; Schnitzer, Mark; Sejnowski, Terrence J.; Shepard, Kenneth L.; Tsao, Doris; Turrigiano, Gina; Weiss, Paul S.; Xu, Chris; Yuste, Rafael; Zhuang, Xiaowei

    2013-01-01

    Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function. PMID:23514423

  17. CapsidMaps: Protein-protein interaction pattern discovery platform for the structural analysis of virus capsids using Google Maps

    PubMed Central

    Carrillo-Tripp, Mauricio; Montiel-García, Daniel Jorge; Brooks, Charles L.; Reddy, Vijay

    2016-01-01

    Structural analysis and visualization of protein-protein interactions is a challenging task since it is difficult to appreciate easily the extent of all contacts made by the residues forming the interfaces. In the case of viruses, structural analysis becomes even more demanding because several interfaces coexist and, in most cases, these are formed by hundreds of contacting residues that belong to multiple interacting coat proteins. CapsidMaps is an interactive analysis and visualization tool that is designed to benefit the structural virology community. Developed as an improved extension of the φ-ψ Explorer, here we describe the details of its design and implementation. We present results of analysis of a spherical virus to showcase the features and utility of the new tool. CapsidMaps also facilitates the comparison of quaternary interactions between two spherical virus particles by computing a similarity (S)-score. The tool can also be used to identify residues that are solvent exposed and in the process of locating antigenic epitope regions as well as residues forming the inside surface of the capsid that interact with the nucleic acid genome. CapsidMaps is part of the VIPERdb Science Gateway, and is freely available as a web-based and cross-browser compliant application at http://viperdb.scripps.edu. PMID:25697908

  18. Mapping the Topography of a Protein Energy Landscape.

    PubMed

    Hutton, Richard D; Wilkinson, James; Faccin, Mauro; Sivertsson, Elin M; Pelizzola, Alessandro; Lowe, Alan R; Bruscolini, Pierpaolo; Itzhaki, Laura S

    2015-11-25

    Protein energy landscapes are highly complex, yet the vast majority of states within them tend to be invisible to experimentalists. Here, using site-directed mutagenesis and exploiting the simplicity of tandem-repeat protein structures, we delineate a network of these states and the routes between them. We show that our target, gankyrin, a 226-residue 7-ankyrin-repeat protein, can access two alternative (un)folding pathways. We resolve intermediates as well as transition states, constituting a comprehensive series of snapshots that map early and late stages of the two pathways and show both to be polarized such that the repeat array progressively unravels from one end of the molecule or the other. Strikingly, we find that the protein folds via one pathway but unfolds via a different one. The origins of this behavior can be rationalized using the numerical results of a simple statistical mechanics model that allows us to visualize the equilibrium behavior as well as single-molecule folding/unfolding trajectories, thereby filling in the gaps that are not accessible to direct experimental observation. Our study highlights the complexity of repeat-protein folding arising from their symmetrical structures; at the same time, however, this structural simplicity enables us to dissect the complexity and thereby map the precise topography of the energy landscape in full breadth and remarkable detail. That we can recapitulate the key features of the folding mechanism by computational analysis of the native structure alone will help toward the ultimate goal of designed amino-acid sequences with made-to-measure folding mechanisms-the Holy Grail of protein folding. PMID:26561984

  19. Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

    PubMed Central

    Roth Flach, Rachel J.; Skoura, Athanasia; Matevossian, Anouch; Danai, Laura V.; Zheng, Wei; Cortes, Christian; Bhattacharya, Samit K.; Aouadi, Myriam; Hagan, Nana; Yawe, Joseph C.; Vangala, Pranitha; Menendez, Lorena Garcia; Cooper, Marcus P.; Fitzgibbons, Timothy P.; Buckbinder, Leonard; Czech, Michael P.

    2015-01-01

    Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe−/− mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe−/− and Ldlr−/− mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFκB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis. PMID:26688060

  20. Dependency Map of Proteins in the Small Ribosomal Subunit

    PubMed Central

    Hamacher, Kay; Trylska, Joanna; McCammon, J. Andrew

    2006-01-01

    The assembly of the ribosome has recently become an interesting target for antibiotics in several bacteria. In this work, we extended an analytical procedure to determine native state fluctuations and contact breaking to investigate the protein stability dependence in the 30S small ribosomal subunit of Thermus thermophilus. We determined the causal influence of the presence and absence of proteins in the 30S complex on the binding free energies of other proteins. The predicted dependencies are in overall agreement with the experimentally determined assembly map for another organism, Escherichia coli. We found that the causal influences result from two distinct mechanisms: one is pure internal energy change, the other originates from the entropy change. We discuss the implications on how to target the ribosomal assembly most effectively by suggesting six proteins as targets for mutations or other hindering of their binding. Our results show that by blocking one out of this set of proteins, the association of other proteins is eventually reduced, thus reducing the translation efficiency even more. We could additionally determine the binding dependency of THX—a peptide not present in the ribosome of E. coli—and suggest its assembly path. PMID:16485038

  1. Spatial recognition and mapping of proteins using DNA aptamers

    NASA Astrophysics Data System (ADS)

    Wang, Congzhou; Yadavalli, Vamsi K.

    2014-11-01

    Atomic force microscopy-based adhesion force measurements have emerged as a powerful tool for the biophysical analyses of biological systems. Such measurements can now be extended to detection and mapping of biomolecules on surfaces via integrated imaging and force spectroscopy techniques. Critical to these experiments is the choice of the biomolecular recognition probe. In this study, we demonstrate how oligonucleotide aptamers can be used as versatile probes to simultaneously image and spatially locate targets on surfaces. We focus on two structurally distinct proteins relevant to the clotting cascade—human α-thrombin and vascular endothelial growth factor. Via AFM-recognition mapping using specific DNA aptamers on a commercially available instrument, we show a clear consistency between height and force measurements obtained simultaneously. Importantly, we are able to observe changes in binding due to changes in the external microenvironment, which demonstrate the ability to study fluctuating biological systems in real time. The aptamer specificity and the ability to distinguish their targets are shown through positive and negative controls. It is therefore possible to generate high resolution maps to spatially and temporally identify proteins at the molecular level on complex surfaces.

  2. How round is a protein? Exploring protein structures for globularity using conformal mapping

    PubMed Central

    Hass, Joel; Koehl, Patrice

    2014-01-01

    We present a new algorithm that automatically computes a measure of the geometric difference between the surface of a protein and a round sphere. The algorithm takes as input two triangulated genus zero surfaces representing the protein and the round sphere, respectively, and constructs a discrete conformal map f between these surfaces. The conformal map is chosen to minimize a symmetric elastic energy ES(f) that measures the distance of f from an isometry. We illustrate our approach on a set of basic sample problems and then on a dataset of diverse protein structures. We show first that ES(f) is able to quantify the roundness of the Platonic solids and that for these surfaces it replicates well traditional measures of roundness such as the sphericity. We then demonstrate that the symmetric elastic energy ES(f) captures both global and local differences between two surfaces, showing that our method identifies the presence of protruding regions in protein structures and quantifies how these regions make the shape of a protein deviate from globularity. Based on these results, we show that ES(f) serves as a probe of the limits of the application of conformal mapping to parametrize protein shapes. We identify limitations of the method and discuss its extension to achieving automatic registration of protein structures based on their surface geometry. PMID:25988167

  3. Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

    PubMed

    Cheng, Jonathan D; Valianou, Matthildi; Canutescu, Adrian A; Jaffe, Eileen K; Lee, Hyung-Ok; Wang, Hao; Lai, Jack H; Bachovchin, William W; Weiner, Louis M

    2005-03-01

    Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting

  4. AlphaSpace: Fragment-Centric Topographical Mapping To Target Protein-Protein Interaction Interfaces.

    PubMed

    Rooklin, David; Wang, Cheng; Katigbak, Joseph; Arora, Paramjit S; Zhang, Yingkai

    2015-08-24

    Inhibition of protein-protein interactions (PPIs) is emerging as a promising therapeutic strategy despite the difficulty in targeting such interfaces with drug-like small molecules. PPIs generally feature large and flat binding surfaces as compared to typical drug targets. These features pose a challenge for structural characterization of the surface using geometry-based pocket-detection methods. An attractive mapping strategy--that builds on the principles of fragment-based drug discovery (FBDD)--is to detect the fragment-centric modularity at the protein surface and then characterize the large PPI interface as a set of localized, fragment-targetable interaction regions. Here, we introduce AlphaSpace, a computational analysis tool designed for fragment-centric topographical mapping (FCTM) of PPI interfaces. Our approach uses the alpha sphere construct, a geometric feature of a protein's Voronoi diagram, to map out concave interaction space at the protein surface. We introduce two new features--alpha-atom and alpha-space--and the concept of the alpha-atom/alpha-space pair to rank pockets for fragment-targetability and to facilitate the evaluation of pocket/fragment complementarity. The resulting high-resolution interfacial map of targetable pocket space can be used to guide the rational design and optimization of small molecule or biomimetic PPI inhibitors. PMID:26225450

  5. Molecular cloning of a human protein that binds to the retinoblastoma protein and chromosomal mapping

    SciTech Connect

    Saijo, M.; Sakai, Y.; Taya, Y.

    1995-06-10

    We have isolated distinct clones for cellular proteins that bind to the retinoblastoma protein by direct screening of cDNA expression libraries using purified pRB as a probe. The total nucleotide sequence of one of these clones, RBQ-3, was determined and found to encode a protein of 66 kDa localized in the nucleus. The RBQ-3 preferentially binds to underphosphory-lated pRB. The region used for binding to this protein was mapped to the E1A-binding pocket B of pRB, which has sequence similarity to the general transcription factor TFIIB. We have mapped the gene to 1q32 using polymerase chain reaction analysis on a human-hamster hybrid cell panel and chromosomal fluorescence in situ hybridization. 64 refs., 7 figs.

  6. Exploring representations of protein structure for automated remote homology detection and mapping of protein structure space

    PubMed Central

    2014-01-01

    Background Due to rapid sequencing of genomes, there are now millions of deposited protein sequences with no known function. Fast sequence-based comparisons allow detecting close homologs for a protein of interest to transfer functional information from the homologs to the given protein. Sequence-based comparison cannot detect remote homologs, in which evolution has adjusted the sequence while largely preserving structure. Structure-based comparisons can detect remote homologs but most methods for doing so are too expensive to apply at a large scale over structural databases of proteins. Recently, fragment-based structural representations have been proposed that allow fast detection of remote homologs with reasonable accuracy. These representations have also been used to obtain linearly-reducible maps of protein structure space. It has been shown, as additionally supported from analysis in this paper that such maps preserve functional co-localization of the protein structure space. Methods Inspired by a recent application of the Latent Dirichlet Allocation (LDA) model for conducting structural comparisons of proteins, we propose higher-order LDA-obtained topic-based representations of protein structures to provide an alternative route for remote homology detection and organization of the protein structure space in few dimensions. Various techniques based on natural language processing are proposed and employed to aid the analysis of topics in the protein structure domain. Results We show that a topic-based representation is just as effective as a fragment-based one at automated detection of remote homologs and organization of protein structure space. We conduct a detailed analysis of the information content in the topic-based representation, showing that topics have semantic meaning. The fragment-based and topic-based representations are also shown to allow prediction of superfamily membership. Conclusions This work opens exciting venues in designing novel

  7. Refined mapping of the G[sub M2] activator protein (GM2A) locus to 5q31. 3-q33. 1, distal to the spinal muscular atrophy locus

    SciTech Connect

    Heng, H.H.Q.; Xie, B.; Shi, X.M.; Tsui, L.C.; Mahuran, D.J. )

    1993-11-01

    The G[sub M2] activator locus (GM2A) had previously been considered as a candidate gene for some forms of spinal muscular atrophy (SMA; mapped to 5q11.2-q13.3). It was eliminated as a possible candidate because PCR-based mapping failed to localize the gene to chromosome 5, as was previously reported using an ELISA-based methodology. However, the authors demonstrated that the PCR primers used preferentially amplified a processed pseudogene (GM2AP) that was mapped to chromosome 3 and that GM2A was located on chromosome 5. In this report, they reconsider the candidacy of GM2A by refining its localization on chromosome 5 using fluorescence in situ hybridization. They localize GM2A to 5q31.3-q33.1; thus, it is not a candidate gene for SMA. 11 refs., 2 figs.

  8. REGION 10 (EXCEPT ALASKA) GROUNDWATER ACTIVITIES MAP

    EPA Science Inventory

    This is a map of Washington, Oregon and Idaho, with inset blow ups of the Puget Sound area, Portland, and Spokane. The map shows RCRA treatment, storage, disposal, and incineration sites, CERCLA sites, TRI sites, NPDES majors, and Sole Source Aquifers Streams and county boundarie...

  9. Correlation of scintigraphic phase maps with intraoperative epicardial/endocardial maps in patients with activation disturbances

    SciTech Connect

    Dae, M.W.; Botvinick, E.H.; Scheinmann, M.H.; Morady, F.J.; Davis, J.A.; Schechtmann, N.; Frais, M.; Faulkner, D.; O'Connell, W.

    1984-01-01

    To assess the true accuracy of scintigraphic findings, 8 patients (PTS), 6 with pre-excitation (PEX) syndrome and 2 with intractable ventricular tachycardia (VT), were studied by phase analysis, prior to corrective surgery. Sites of earliest phase angle were determined in multiple projections during the conduction disturbance, compared to sites of early ventricular activation determined by epicardial mapping during PEX and, when performed, by endocardial mapping during VT, and to maps previously generated at conventional electrophysiologic study (EPS). Among PEX PTS, Rt and Lt lateral, Lt anterolateral, Rt and Lt posterolateral and posteroseptal bypass pathways mapped at surgery correlated with phase localization. While localization from EPS also correlated well with surgical maps in 4 PTS, 1 PT could not be mapped by EPS and another presented ambiguities. Scintigraphic localization also correlated well with surgical mapping in a PT with a RV VT focus while EPS was suggestive but uncertain. A second PT with VT mapped scintigraphically to originate in a Lt lateral focus, demonstrated a similar localization on EPS, and during surgical mapping, an incision made through the scintigraphic focus terminated VT. Incision in regions of earliest activity in the first VT PT and in PTS with PEX resolved the arrhythmia or interrupted the bypass tract. Phase mapping correlated closely with surface mapping at surgery while providing an accurate, independent method for noninvasive assessment of conduction disturbances and a complementary tool to standard EPS.

  10. Phosphotyrosine-dependent targeting of mitogen-activated protein kinase in differentiated contractile vascular cells.

    PubMed

    Khalil, R A; Menice, C B; Wang, C L; Morgan, K G

    1995-06-01

    Tyrosine phosphorylation has been linked to plasmalemmal targeting of src homology-2-containing proteins, activation of mitogen-activated protein (MAP) kinase, nuclear signaling, and proliferation of cultured cells. Significant tyrosine phosphorylation and MAP kinase activities have also been reported in differentiated cells, but the signaling role of tyrosine-phosphorylated MAP kinase in these cells is unclear. The spatial and temporal relation between phosphotyrosine and MAP kinase immunoreactivity was quantified in differentiated contractile vascular smooth muscle cells by using digital imaging microscopy. An initial association of MAP kinase with the plasmalemma required upstream protein kinase C activity but occurred in a tyrosine phosphorylation-independent manner. Subsequent to membrane association, a delayed redistribution of MAP kinase, colocalizing with the actin-binding protein caldesmon, occurred in a tyrosine phosphorylation-dependent manner. The apparent association of MAP kinase with the contractile proteins coincided with contractile activation. Thus, tyrosine phosphorylation appears to target MAP kinase to cytoskeletal proteins in contractile vascular cells. This targeting mechanism may determine the specific destination and thereby the specialized function of MAP kinase in other phenotypes. PMID:7538916

  11. Teaching Basic Geographical Skills: Map and Compass Activities.

    ERIC Educational Resources Information Center

    Trussell, Margaret Edith

    1986-01-01

    Presents a unit on map and compass activities which introduces compass direction, magnetic declination and conversion of map measurement to familiar units. Requires four, one-hour class meetings and may be followed by a half-day orienteering activity. (Author/JDH)

  12. Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

    PubMed Central

    Graham, A; McLees, A; Malarkey, K; Gould, G W; Plevin, R

    1996-01-01

    We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase. PMID:8615830

  13. Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

    PubMed

    Graham, A; McLees, A; Malarkey, K; Gould, G W; Plevin, R

    1996-04-15

    We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase. PMID:8615830

  14. Empirically controlled mapping of the Caenorhabditis elegans protein-protein interactome network.

    PubMed

    Simonis, Nicolas; Rual, Jean-François; Carvunis, Anne-Ruxandra; Tasan, Murat; Lemmens, Irma; Hirozane-Kishikawa, Tomoko; Hao, Tong; Sahalie, Julie M; Venkatesan, Kavitha; Gebreab, Fana; Cevik, Sebiha; Klitgord, Niels; Fan, Changyu; Braun, Pascal; Li, Ning; Ayivi-Guedehoussou, Nono; Dann, Elizabeth; Bertin, Nicolas; Szeto, David; Dricot, Amélie; Yildirim, Muhammed A; Lin, Chenwei; de Smet, Anne-Sophie; Kao, Huey-Ling; Simon, Christophe; Smolyar, Alex; Ahn, Jin Sook; Tewari, Muneesh; Boxem, Mike; Milstein, Stuart; Yu, Haiyuan; Dreze, Matija; Vandenhaute, Jean; Gunsalus, Kristin C; Cusick, Michael E; Hill, David E; Tavernier, Jan; Roth, Frederick P; Vidal, Marc

    2009-01-01

    To provide accurate biological hypotheses and elucidate global properties of cellular networks, systematic identification of protein-protein interactions must meet high quality standards.We present an expanded C. elegans protein-protein interaction network, or 'interactome' map, derived from testing a matrix of approximately 10,000 x approximately 10,000 proteins using a highly specific, high-throughput yeast two-hybrid system. Through a new empirical quality control framework, we show that the resulting data set (Worm Interactome 2007, or WI-2007) was similar in quality to low-throughput data curated from the literature. We filtered previous interaction data sets and integrated them with WI-2007 to generate a high-confidence consolidated map (Worm Interactome version 8, or WI8). This work allowed us to estimate the size of the worm interactome at approximately 116,000 interactions. Comparison with other types of functional genomic data shows the complementarity of distinct experimental approaches in predicting different functional relationships between genes or proteins PMID:19123269

  15. A protein interaction map for cell polarity development.

    PubMed

    Drees, B L; Sundin, B; Brazeau, E; Caviston, J P; Chen, G C; Guo, W; Kozminski, K G; Lau, M W; Moskow, J J; Tong, A; Schenkman, L R; McKenzie, A; Brennwald, P; Longtine, M; Bi, E; Chan, C; Novick, P; Boone, C; Pringle, J R; Davis, T N; Fields, S; Drubin, D G

    2001-08-01

    Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed. PMID:11489916

  16. Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins.

    PubMed

    Maeda, Kenji; Anand, Kanchan; Chiapparino, Antonella; Kumar, Arun; Poletto, Mattia; Kaksonen, Marko; Gavin, Anne-Claude

    2013-09-12

    The internal organization of eukaryotic cells into functionally specialized, membrane-delimited organelles of unique composition implies a need for active, regulated lipid transport. Phosphatidylserine (PS), for example, is synthesized in the endoplasmic reticulum and then preferentially associates--through mechanisms not fully elucidated--with the inner leaflet of the plasma membrane. Lipids can travel via transport vesicles. Alternatively, several protein families known as lipid-transfer proteins (LTPs) can extract a variety of specific lipids from biological membranes and transport them, within a hydrophobic pocket, through aqueous phases. Here we report the development of an integrated approach that combines protein fractionation and lipidomics to characterize the LTP-lipid complexes formed in vivo. We applied the procedure to 13 LTPs in the yeast Saccharomyces cerevisiae: the six Sec14 homology (Sfh) proteins and the seven oxysterol-binding homology (Osh) proteins. We found that Osh6 and Osh7 have an unexpected specificity for PS. In vivo, they participate in PS homeostasis and the transport of this lipid to the plasma membrane. The structure of Osh6 bound to PS reveals unique features that are conserved among other metazoan oxysterol-binding proteins (OSBPs) and are required for PS recognition. Our findings represent the first direct evidence, to our knowledge, for the non-vesicular transfer of PS from its site of biosynthesis (the endoplasmic reticulum) to its site of biological activity (the plasma membrane). We describe a new subfamily of OSBPs, including human ORP5 and ORP10, that transfer PS and propose new mechanisms of action for a protein family that is involved in several human pathologies such as cancer, dyslipidaemia and metabolic syndrome. PMID:23934110

  17. Computer-Based Concept Mapping: Active Studying for Active Learners.

    ERIC Educational Resources Information Center

    Anderson-Inman, Lynne; Zeitz, Leigh

    1993-01-01

    Describes the use of computer-generated concept maps using a new genre of graphics programs that allows students to create, alter and expand maps as they acquire more subject matter knowledge. Four examples of increasingly complex concept maps developed by a high school student studying cellular biology are discussed and illustrated. (Contains 19…

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

    PubMed

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

    2015-03-23

    Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-γ (PPARγ) NRs and the metabotropic glutamate (mGluR) and β2-adreneric (β2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the β2AR LBP were used in virtual screening to identify high efficacy agonists targeting β2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents. PMID:25692383

  19. Mapping Functional Group Free Energy Patterns at Protein Occluded Sites: Nuclear Receptors and G-Protein Coupled Receptors

    PubMed Central

    2015-01-01

    Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-γ (PPARγ) NRs and the metabotropic glutamate (mGluR) and β2-adreneric (β2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the β2AR LBP were used in virtual screening to identify high efficacy agonists targeting β2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents. PMID:25692383

  20. Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria

    PubMed Central

    Farasat, Iman; Kushwaha, Manish; Collens, Jason; Easterbrook, Michael; Guido, Matthew; Salis, Howard M

    2014-01-01

    Developing predictive models of multi-protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi-protein expression space across a > 10,000-fold range with tailored search parameters and well-predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram-positive and gram-negative bacterial hosts. We then combined the search algorithm with system-level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence-expression-activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate-limiting steps in metabolism. Creating sequence-expression-activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs. PMID:24952589

  1. Mitogen activated protein kinase at the nuclear pore complex

    PubMed Central

    Faustino, Randolph S; Maddaford, Thane G; Pierce, Grant N

    2011-01-01

    Abstract Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD-98059, two MAP kinase antagonists as well as with SB-202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N-terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function. PMID:20497490

  2. Mapping energy transfer channels in fucoxanthin-chlorophyll protein complex.

    PubMed

    Gelzinis, Andrius; Butkus, Vytautas; Songaila, Egidijus; Augulis, Ramūnas; Gall, Andrew; Büchel, Claudia; Robert, Bruno; Abramavicius, Darius; Zigmantas, Donatas; Valkunas, Leonas

    2015-02-01

    Fucoxanthin-chlorophyll protein (FCP) is the key molecular complex performing the light-harvesting function in diatoms, which, being a major group of algae, are responsible for up to one quarter of the total primary production on Earth. These photosynthetic organisms contain an unusually large amount of the carotenoid fucoxanthin, which absorbs the light in the blue-green spectral region and transfers the captured excitation energy to the FCP-bound chlorophylls. Due to the large number of fucoxanthins, the excitation energy transfer cascades in these complexes are particularly tangled. In this work we present the two-color two-dimensional electronic spectroscopy experiments on FCP. Analysis of the data using the modified decay associated spectra permits a detailed mapping of the excitation frequency dependent energy transfer flow with a femtosecond time resolution. PMID:25445318

  3. Protein Needs of Physically Active Children.

    PubMed

    Volterman, Kimberly A; Atkinson, Stephanie A

    2016-05-01

    Current Dietary Reference Intakes (DRI) for protein for children and youth require revision as they were derived primarily on nitrogen balance data in young children or extrapolated from adult values; did not account for the possible influence of above average physical activity; and did not set an upper tolerable level of intake. Revision of the protein DRIs requires new research that investigates: 1) long-term dose-response to identify protein and essential amino acid requirements of both sexes at various pubertal stages and under differing conditions of physical activity; 2) the acute protein needs (quantity and timing) following a single bout of exercise; 3) the potential adverse effects of chronic high intakes of protein; and 4) new measurement techniques (i.e., IAAO or stable isotope methodologies) to improve accuracy of protein needs. While active individuals may require protein in excess of current DRIs, most active Canadian children and youth have habitual protein intakes that exceed current recommendations. PMID:27137165

  4. Free-Hand Small-Scale Maps: Activities for Cognitive Mapping.

    ERIC Educational Resources Information Center

    Saveland, Robert N.

    1978-01-01

    Suggests that small-scale mapping activities can be used in geography courses to help students understand geographic concepts such as latitude, longitude, situation, relative location, and national boundaries. (Author/DB)

  5. Global tectonic activity map with orbital photographic supplement

    SciTech Connect

    Lowman, P.D. Jr.

    1981-01-01

    A three part map showing equatorial and polar regions was compiled showing tectonic and volcanic activity of the past one million years, including the present. Features shown include actively spreading ridges, spreading rates, major active faults, subduction zones, well defined plates, and volcanic areas active within the past one million years. Activity within this period was inferred from seismicity (instrumental and historic), physiography, and published literature. The tectonic activity map was used for planning global geodetic programs of satellite laser ranging and very long base line interferometry and for geologic education.

  6. A global tectonic activity map with orbital photographic supplement

    NASA Technical Reports Server (NTRS)

    Lowman, P. D., Jr.

    1981-01-01

    A three part map showing equatorial and polar regions was compiled showing tectonic and volcanic activity of the past one million years, including the present. Features shown include actively spreading ridges, spreading rates, major active faults, subduction zones, well defined plates, and volcanic areas active within the past one million years. Activity within this period was inferred from seismicity (instrumental and historic), physiography, and published literature. The tectonic activity map was used for planning global geodetic programs of satellite laser ranging and very long base line interferometry and for geologic education.

  7. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  8. A protein interaction map for cell polarity development

    PubMed Central

    Drees, Becky L.; Sundin, Bryan; Brazeau, Elizabeth; Caviston, Juliane P.; Chen, Guang-Chao; Guo, Wei; Kozminski, Keith G.; Lau, Michelle W.; Moskow, John J.; Tong, Amy; Schenkman, Laura R.; McKenzie, Amos; Brennwald, Patrick; Longtine, Mark; Bi, Erfei; Chan, Clarence; Novick, Peter; Boone, Charles; Pringle, John R.; Davis, Trisha N.; Fields, Stanley; Drubin, David G.

    2001-01-01

    Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed. PMID:11489916

  9. Systematic characterization of protein folding pathways using diffusion maps: Application to Trp-cage miniprotein

    SciTech Connect

    Kim, Sang Beom; Dsilva, Carmeline J.; Debenedetti, Pablo G.; Kevrekidis, Ioannis G.

    2015-02-28

    Understanding the mechanisms by which proteins fold from disordered amino-acid chains to spatially ordered structures remains an area of active inquiry. Molecular simulations can provide atomistic details of the folding dynamics which complement experimental findings. Conventional order parameters, such as root-mean-square deviation and radius of gyration, provide structural information but fail to capture the underlying dynamics of the protein folding process. It is therefore advantageous to adopt a method that can systematically analyze simulation data to extract relevant structural as well as dynamical information. The nonlinear dimensionality reduction technique known as diffusion maps automatically embeds the high-dimensional folding trajectories in a lower-dimensional space from which one can more easily visualize folding pathways, assuming the data lie approximately on a lower-dimensional manifold. The eigenvectors that parametrize the low-dimensional space, furthermore, are determined systematically, rather than chosen heuristically, as is done with phenomenological order parameters. We demonstrate that diffusion maps can effectively characterize the folding process of a Trp-cage miniprotein. By embedding molecular dynamics simulation trajectories of Trp-cage folding in diffusion maps space, we identify two folding pathways and intermediate structures that are consistent with the previous studies, demonstrating that this technique can be employed as an effective way of analyzing and constructing protein folding pathways from molecular simulations.

  10. Systematic characterization of protein folding pathways using diffusion maps: Application to Trp-cage miniprotein

    NASA Astrophysics Data System (ADS)

    Kim, Sang Beom; Dsilva, Carmeline J.; Kevrekidis, Ioannis G.; Debenedetti, Pablo G.

    2015-02-01

    Understanding the mechanisms by which proteins fold from disordered amino-acid chains to spatially ordered structures remains an area of active inquiry. Molecular simulations can provide atomistic details of the folding dynamics which complement experimental findings. Conventional order parameters, such as root-mean-square deviation and radius of gyration, provide structural information but fail to capture the underlying dynamics of the protein folding process. It is therefore advantageous to adopt a method that can systematically analyze simulation data to extract relevant structural as well as dynamical information. The nonlinear dimensionality reduction technique known as diffusion maps automatically embeds the high-dimensional folding trajectories in a lower-dimensional space from which one can more easily visualize folding pathways, assuming the data lie approximately on a lower-dimensional manifold. The eigenvectors that parametrize the low-dimensional space, furthermore, are determined systematically, rather than chosen heuristically, as is done with phenomenological order parameters. We demonstrate that diffusion maps can effectively characterize the folding process of a Trp-cage miniprotein. By embedding molecular dynamics simulation trajectories of Trp-cage folding in diffusion maps space, we identify two folding pathways and intermediate structures that are consistent with the previous studies, demonstrating that this technique can be employed as an effective way of analyzing and constructing protein folding pathways from molecular simulations.

  11. Mapping protein-RNA interactions by RCAP, RNA-cross-linking and peptide fingerprinting.

    PubMed

    Vaughan, Robert C; Kao, C Cheng

    2015-01-01

    RNA nanotechnology often feature protein RNA complexes. The interaction between proteins and large RNAs are difficult to study using traditional structure-based methods like NMR or X-ray crystallography. RCAP, an approach that uses reversible-cross-linking affinity purification method coupled with mass spectrometry, has been developed to map regions within proteins that contact RNA. This chapter details how RCAP is applied to map protein-RNA contacts within virions. PMID:25896007

  12. Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase.

    PubMed Central

    Pleiman, C M; Clark, M R; Gauen, L K; Winitz, S; Coggeshall, K M; Johnson, G L; Shaw, A S; Cambier, J C

    1993-01-01

    Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly. Images PMID:8395016

  13. WHERE MULTIFUNCTIONAL DNA REPAIR PROTEINS MEET: MAPPING THE INTERACTION DOMAINS BETWEEN XPG AND WRN

    SciTech Connect

    Rangaraj, K.; Cooper, P.K.; Trego, K.S.

    2009-01-01

    The rapid recognition and repair of DNA damage is essential for the maintenance of genomic integrity and cellular survival. Multiple complex and interconnected DNA damage responses exist within cells to preserve the human genome, and these repair pathways are carried out by a specifi c interplay of protein-protein interactions. Thus a failure in the coordination of these processes, perhaps brought about by a breakdown in any one multifunctional repair protein, can lead to genomic instability, developmental and immunological abnormalities, cancer and premature aging. This study demonstrates a novel interaction between two such repair proteins, Xeroderma pigmentosum group G protein (XPG) and Werner syndrome helicase (WRN), that are both highly pleiotropic and associated with inherited genetic disorders when mutated. XPG is a structure-specifi c endonuclease required for the repair of UV-damaged DNA by nucleotide excision repair (NER), and mutations in XPG result in the diseases Xeroderma pigmentosum (XP) and Cockayne syndrome (CS). A loss of XPG incision activity results in XP, whereas a loss of non-enzymatic function(s) of XPG causes CS. WRN is a multifunctional protein involved in double-strand break repair (DSBR), and consists of 3’–5’ DNA-dependent helicase, 3’–5’ exonuclease, and single-strand DNA annealing activities. Nonfunctional WRN protein leads to Werner syndrome, a premature aging disorder with increased cancer incidence. Far Western analysis was used to map the interacting domains between XPG and WRN by denaturing gel electrophoresis, which separated purifi ed full length and recombinant XPG and WRN deletion constructs, based primarily upon the length of each polypeptide. Specifi c interacting domains were visualized when probed with the secondary protein of interest which was then detected by traditional Western analysis using the antibody of the secondary protein. The interaction between XPG and WRN was mapped to the C-terminal region of

  14. Mapping membrane protein interactions in cell signaling systems.

    SciTech Connect

    Light, Yooli Kim; Hadi, Masood Z.; Lane, Pamela; Jacobsen, Richard B.; Hong, Joohee; Ayson, Marites J.; Wood, Nichole L.; Schoeniger, Joseph S.; Young, Malin M.

    2003-12-01

    We proposed to apply a chemical cross-linking, mass spectrometry and modeling method called MS3D to the structure determination of the rhodopsin-transducin membrane protein complex (RTC). Herein we describe experimental progress made to adapt the MS3D approach for characterizing membrane protein systems, and computational progress in experimental design, data analysis and protein structure modeling. Over the past three years, we have developed tailored experimental methods for all steps in the MS3D method for rhodopsin, including protein purification, a functional assay, cross-linking, proteolysis and mass spectrometry. In support of the experimental effort. we have out a data analysis pipeline in place that automatically selects the monoisotopic peaks in a mass spectrometric spectrum, assigns them and stores the results in a database. Theoretical calculations using 24 experimentally-derived distance constraints have resulted in a backbone-level model of the activated form of rhodopsin, which is a critical first step towards building a model of the RTC. Cross-linked rhodopsin-transducin complexes have been isolated via gel electrophoresis and further mass spectrometric characterization of the cross-links is underway.

  15. Using Brain Electrical Activity Mapping to Diagnose Learning Disabilities.

    ERIC Educational Resources Information Center

    Torello, Michael, W.; Duffy, Frank H.

    1985-01-01

    Cognitive neuroscience assumes that measurement of brain electrical activity should relate to cognition. Brain Electrical Activity Mapping (BEAM), a non-invasive technique, is used to record changes in activity from one brain area to another and is 80 to 90 percent successful in classifying subjects as dyslexic or normal. (MT)

  16. Orexin-stimulated MAP kinase cascades are activated through multiple G-protein signalling pathways in human H295R adrenocortical cells: diverse roles for orexins A and B.

    PubMed

    Ramanjaneya, Manjunath; Conner, Alex C; Chen, Jing; Kumar, Prashanth; Brown, James E P; Jöhren, Olaf; Lehnert, Hendrik; Stanfield, Peter R; Randeva, Harpal S

    2009-08-01

    Orexins A and B (ORA and ORB) are neuropeptide hormones found throughout the central nervous system and periphery. They are required for a host of physiological processes including mitogen-activated protein kinase (MAPK) regulation, steroidogenesis, appetite control and energy regulation. While some signalling mechanisms have been proposed for individual recombinant orexin receptors in generic mammalian cell types, it is clear that the peripheral effects of orexin are spatially and temporally complex. This study dissects the different G-protein signalling and MAPK pathways activated in a pluripotent human adrenal H295R cell line capable of all the physiological steps involved in steroidogenesis. Both extracellular receptor kinase 1/2 (ERK1/2) and p38 were phosphorylated rapidly with a subsequent decline, in a time- and dose-dependent manner, in response to both ORA and ORB. Conversely, there was little or no direct activation of the ERK5 or JNK pathway. Analysis using signalling and MAPK inhibitors as well as receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1/2 and p38 activation were predominantly G(q)- and to a lesser extent G(s)-mediated; p38 activation even had a small G(i)-component. Effects were broadly comparable for both orexin sub-types ORA and ORB and although most of the effects were transmitted through the orexin receptor-1 subtype, we did observe a role for orexin receptor-2-mediated activation of both ERK1/2 and p38. Cortisol secretion also differed in response to ORA and ORB. These data suggest multiple roles for orexin-mediated MAPK activation in an adrenal cell-line, this complexity may help to explain the diverse biological actions of orexins with wide-ranging consequences for our understanding of the mechanisms initiated by these steroidogenic molecules. PMID:19460850

  17. A Digital Tectonic Activity Map of the Earth

    NASA Technical Reports Server (NTRS)

    Lowman, Paul; Masuoka, Penny; Montgomery, Brian; OLeary, Jay; Salisbury, Demetra; Yates, Jacob

    1999-01-01

    The subject of neotectonics, covering the structures and structural activity of the last 5 million years (i.e., post-Miocene) is a well-recognized field, including "active tectonics," focussed on the last 500,000 years in a 1986 National Research Council report of that title. However, there is a cartographic gap between tectonic maps, generally showing all features regardless of age, and maps of current seismic or volcanic activity. We have compiled a map intended to bridge this gap, using modern data bases and computer-aided cartographic techniques. The maps presented here are conceptually descended from an earlier map showing tectonic and volcanic activity of the last one million years. Drawn by hand with the National Geographic Society's 1975 "The Physical World" map as a base, the 1981 map in various revisions has been widely reproduced in textbooks and various technical publications. However, two decades of progress call for a completely new map that can take advantage of new knowledge and cartographic techniques. The digital tectonic activity map (DTM), presented in shaded relief (Fig. 1) and schematic (Fig. 2) versions, is the result. The DTM is intended to show tectonism and volcanism of the last one million years, a period long enough to be representative of global activity, but short enough that features such as fault scarps and volcanos are still geomorphically recognizable. Data Sources and Cartographic Methods The DTM is based on a wide range of sources, summarized in Table 1. The most important is the digital elevation model, used to construct a shaded relief map. The bathymetry is largely from satellite altimetry, specifically the marine gravity compilations by Smith and Sandwell (1996). The shaded relief map was designed to match the new National Geographic Society world physical map (1992), although drawn independently, from the digital elevation model. The Robinson Projection is used instead of the earlier Van der Grinten one. Although neither

  18. High-Speed AFM Images of Thermal Motion Provide Stiffness Map of Interfacial Membrane Protein Moieties

    PubMed Central

    2014-01-01

    The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges. PMID:25516527

  19. Long Wavelength Monitoring of Protein Kinase Activity

    PubMed Central

    Oien, Nathan P.; Nguyen, Luong T.; Jernigan, Finith E.; Priestman, Melanie A.

    2014-01-01

    A family of long wavelength protein kinase fluorescent reporters is described in which the probing wavelength is pre-programmed using readily available fluorophores. These agents can assess protein kinase activity within the optical window of tissue, as exemplified by monitoring endogenous cAMP-dependent protein kinase activity (1) in erythrocyte lysates and (2) in intact erythrocytes using a light-activatable reporter. PMID:24604833

  20. Dependence of Mos-induced Cdc2 activation on MAP kinase function in a cell-free system.

    PubMed Central

    Huang, C Y; Ferrell, J E

    1996-01-01

    The progression of G2-arrested Xenopus laevis oocytes into meiotic M-phase is accompanied by the nearly simultaneous activation of p42 MAP kinase and Cdc2/cyclin B. This timing raises the possibility that the activation of one kinase might depend upon the other. Here we have examined whether Cdc2 activation requires p42 MAP kinase function. We have reconstituted Mos-induced Cdc2 activation in cell-free Xenopus oocyte extracts, and have found that Mos-induced Cdc2 activation requires active p42 MAP kinase, is inhibited by a MAP kinase phosphatase and is independent of protein synthesis. These findings indicate that p42 MAP kinase is an essential component of the M phase trigger in this system. Images PMID:8641282

  1. Salicylic acid activates a 48-kD MAP kinase in tobacco.

    PubMed Central

    Zhang, S; Klessig, D F

    1997-01-01

    The involvement of phosphorylation/dephosphorylation in the salicylic acid (SA) signal transduction pathway leading to pathogenesis-related gene induction has previously been demonstrated using kinase and phosphatase inhibitors. Here, we show that in tobacco suspension cells, SA induced a rapid and transient activation of a 48-kD kinase that uses myelin basic protein as a substrate. This kinase is called the p48 SIP kinase (for SA-Induced Protein kinase). Biologically active analogs of SA, which induce pathogenesis-related genes and enhanced resistance, also activated this kinase, whereas inactive analogs did not. Phosphorylation of a tyrosine residue(s) in the SIP kinase was associated with its activation. The SIP kinase was purified to homogeneity from SA-treated tobacco suspension culture cells. The purified SIP kinase is strongly phosphorylated on a tyrosine residue(s), and treatment with either protein tyrosine or serine/threonine phosphatases abolished its activity. Using primers corresponding to the sequences of internal tryptic peptides, we cloned the SIP kinase gene. Analysis of the SIP kinase sequence indicates that it belongs to the MAP kinase family and that it is distinct from the other plant MAP kinases previously implicated in stress responses, suggesting that different members of the MAP kinase family are activated by different stresses. PMID:9165755

  2. Deguelin-induced blockade of PI3K/protein kinase B/MAP kinase signaling in zebrafish and breast cancer cell lines is mediated by down-regulation of fibroblast growth factor receptor 4 activity.

    PubMed

    Wu, Wei; Hai, Yang; Chen, Lu; Liu, Rui-Jin; Han, Yu-Xiang; Li, Wen-Hao; Li, Song; Lin, Shuo; Wu, Xin-Rong

    2016-04-01

    Deguelin, a natural component derived from leguminous plants, has been used as pesticide in some regions. Accumulating evidence show that deguelin has promising chemopreventive and therapeutic activities against cancer cells. This study shows that low concentrations of deguelin can lead to significant delay in zebrafish embryonic development through growth inhibition and induction of apoptosis. Furthermore, we identified fibroblast growth factor receptor 4 (FGFR4) as the putative target of deguelin. The candidate was initially identified by a microarray approach and then validated through in vitro experiments using hormone-responsive (MCF-7) and nonresponsive (MDA-MB-231) human breast cancer cell lines. The results show that deguelin suppressed cell proliferation and induced apoptosis in both cancer cell lines, but not in Hs 578Bst cells, by blocking PI3K/AKT and mitogen-activated protein kinases (MAPK) signaling. The FGFR4 mRNA and protein level also diminished in a dose-dependent manner. Interestingly, we found that forced FGFR4 overexpression attenuated deguelin-induced proliferative suppression and apoptotic cell death in both zebrafish and MCF-7 cell lines, p-AKT and p-ERK levels were restored upon FGFR4 overexpression. Taken together, our results strongly suggest that deguelin inhibition of PI3K/AKT and MAPK signaling in zebrafish and breast cancer cell lines is partially mediated through down-regulation of FGFR4 activity. PMID:27069628

  3. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

    PubMed

    Kohansal-Nodehi, Mahdokht; Chua, John Je; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. PMID:27115346

  4. Activity-Based Protein Profiling of Microbes

    SciTech Connect

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  5. Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

    PubMed Central

    Tan, Dan; Li, Qiang; Zhang, Mei-Jun; Liu, Chao; Ma, Chengying; Zhang, Pan; Ding, Yue-He; Fan, Sheng-Bo; Tao, Li; Yang, Bing; Li, Xiangke; Ma, Shoucai; Liu, Junjie; Feng, Boya; Liu, Xiaohui; Wang, Hong-Wei; He, Si-Min; Gao, Ning; Ye, Keqiong; Dong, Meng-Qiu; Lei, Xiaoguang

    2016-01-01

    To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction. DOI: http://dx.doi.org/10.7554/eLife.12509.001 PMID:26952210

  6. An Inquiry Activity for Genetics Using Chromosome Mapping.

    ERIC Educational Resources Information Center

    Leonard, William H.; Snodgrass, George

    1982-01-01

    Concepts to be developed, objectives, and student instructions are provided for an activity useful as an introduction to or review of Mendelian genetics and sex determination. Universal codes (read by optical scanners at supermarket checkout stands) from soup can labels are used as chromosome maps during the activity. (JN)

  7. Computational methods for constructing protein structure models from 3D electron microscopy maps

    PubMed Central

    Esquivel-Rodríguez, Juan; Kihara, Daisuke

    2013-01-01

    Protein structure determination by cryo-electron microscopy (EM) has made significant progress in the past decades. Resolutions of EM maps have been improving as evidenced by recently reported structures that are solved at high resolutions close to 3 Å. Computational methods play a key role in interpreting EM data. Among many computational procedures applied to an EM map to obtain protein structure information, in this article we focus on reviewing computational methods that model protein three-dimensional (3D) structures from a 3D EM density map that is constructed from two-dimensional (2D) maps. The computational methods we discuss range from de novo methods, which identify structural elements in an EM map, to structure fitting methods, where known high resolution structures are fit into a low-resolution EM map. A list of available computational tools is also provided. PMID:23796504

  8. Dietary protein considerations to support active aging.

    PubMed

    Wall, Benjamin T; Cermak, Naomi M; van Loon, Luc J C

    2014-11-01

    Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the application of dietary protein supplementation to support exercise-induced gains in muscle mass in younger individuals. In contrast, the role of dietary protein in supporting the maintenance (or gain) of skeletal muscle mass in active older persons has received less attention. Older individuals display a blunted muscle protein synthetic response to dietary protein ingestion. However, this reduced anabolic response can largely be overcome when physical activity is performed in close temporal proximity to protein consumption. Moreover, recent evidence has helped elucidate the optimal type and amount of dietary protein that should be ingested by the older adult throughout the day in order to maximize the skeletal muscle adaptive response to physical activity. Evidence demonstrates that when these principles are adhered to, muscle maintenance or hypertrophy over prolonged periods can be further augmented in active older persons. The present review outlines the current understanding of the role that dietary protein occupies in the lifestyle of active older adults as a means to increase skeletal muscle mass, strength and function, and thus support healthier aging. PMID:25355192

  9. Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases.

    PubMed Central

    Belham, C M; Tate, R J; Scott, P H; Pemberton, A D; Miller, H R; Wadsworth, R M; Gould, G W; Plevin, R

    1996-01-01

    We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species. PMID:9003384

  10. Activity-dependent plasticity of hippocampal place maps.

    PubMed

    Schoenenberger, Philipp; O'Neill, Joseph; Csicsvari, Jozsef

    2016-01-01

    Hippocampal neurons encode a cognitive map of space. These maps are thought to be updated during learning and in response to changes in the environment through activity-dependent synaptic plasticity. Here we examine how changes in activity influence spatial coding in rats using halorhodopsin-mediated, spatially selective optogenetic silencing. Halorhoposin stimulation leads to light-induced suppression in many place cells and interneurons; some place cells increase their firing through disinhibition, whereas some show no effect. We find that place fields of the unaffected subpopulation remain stable. On the other hand, place fields of suppressed place cells were unstable, showing remapping across sessions before and after optogenetic inhibition. Disinhibited place cells had stable maps but sustained an elevated firing rate. These findings suggest that place representation in the hippocampus is constantly governed by activity-dependent processes, and that disinhibition may provide a mechanism for rate remapping. PMID:27282121

  11. Activity-dependent plasticity of hippocampal place maps

    PubMed Central

    Schoenenberger, Philipp; O'Neill, Joseph; Csicsvari, Jozsef

    2016-01-01

    Hippocampal neurons encode a cognitive map of space. These maps are thought to be updated during learning and in response to changes in the environment through activity-dependent synaptic plasticity. Here we examine how changes in activity influence spatial coding in rats using halorhodopsin-mediated, spatially selective optogenetic silencing. Halorhoposin stimulation leads to light-induced suppression in many place cells and interneurons; some place cells increase their firing through disinhibition, whereas some show no effect. We find that place fields of the unaffected subpopulation remain stable. On the other hand, place fields of suppressed place cells were unstable, showing remapping across sessions before and after optogenetic inhibition. Disinhibited place cells had stable maps but sustained an elevated firing rate. These findings suggest that place representation in the hippocampus is constantly governed by activity-dependent processes, and that disinhibition may provide a mechanism for rate remapping. PMID:27282121

  12. Asymmetric friction of nonmotor MAPs can lead to their directional motion in active microtubule networks.

    PubMed

    Forth, Scott; Hsia, Kuo-Chiang; Shimamoto, Yuta; Kapoor, Tarun M

    2014-04-10

    Diverse cellular processes require microtubules to be organized into distinct structures, such as asters or bundles. Within these dynamic motifs, microtubule-associated proteins (MAPs) are frequently under load, but how force modulates these proteins' function is poorly understood. Here, we combine optical trapping with TIRF-based microscopy to measure the force dependence of microtubule interaction for three nonmotor MAPs (NuMA, PRC1, and EB1) required for cell division. We find that frictional forces increase nonlinearly with MAP velocity across microtubules and depend on filament polarity, with NuMA's friction being lower when moving toward minus ends, EB1's lower toward plus ends, and PRC1's exhibiting no directional preference. Mathematical models predict, and experiments confirm, that MAPs with asymmetric friction can move directionally within actively moving microtubule pairs they crosslink. Our findings reveal how nonmotor MAPs can generate frictional resistance in dynamic cytoskeletal networks via micromechanical adaptations whose anisotropy may be optimized for MAP localization and function within cellular structures. PMID:24725408

  13. De novo protein structure determination from near-atomic resolution cryo-EM maps

    PubMed Central

    Wang, Ray Yu-Ruei; Kudryashev, Mikhail; Li, Xueming; Egelman, Edward H.; Basler, Marek; Cheng, Yifan; Baker, David; DiMaio, Frank

    2015-01-01

    We present a de novo model building approach that combines predicted backbone conformations with side-chain density-fit to accurately assign sequence into density maps. We show this method yields accurate models for six experimental maps at 3.3–4.8 Å resolution, and produces a nearly complete model for an unsolved map containing a 660-residue hetero-dimeric protein. This method should enable rapid and reliable protein structure determination from near-atomic resolution cryo-EM maps. PMID:25707029

  14. Mass spectrometric protein maps for biomarker discovery and clinical research

    PubMed Central

    Liu, Yansheng; Hüttenhain, Ruth; Collins, Ben; Aebersold, Ruedi

    2013-01-01

    Among the wide range of proteomic technologies, targeted mass spectrometry (MS) has shown great potential for biomarker studies. To extend the degree of multiplexing achieved by selected reaction monitoring (SRM), we recently developed SWATH MS. SWATH MS is a variant of the emerging class of data-independent acquisition (DIA) methods and essentially converts the molecules in a physical sample into perpetually re-usable digital maps. The thus generated SWATH maps are then mined using a targeted data extraction strategy, allowing us to profile disease-related proteomes at a high degree of reproducibility. The successful application of both SRM and SWATH MS requires the a priori generation of reference spectral maps that provide coordinates for quantification. Herein, we demonstrate that the application of the mass spectrometric reference maps and the acquisition of personalized SWATH maps hold a particular promise for accelerating the current process of biomarker discovery. PMID:24138574

  15. RefSOFI for Mapping Nanoscale Organization of Protein-protein Interactions in Living cells

    PubMed Central

    Hertel, Fabian; Mo, Gary C. H.; Duwé, Sam; Dedecker, Peter; Zhang, Jin

    2015-01-01

    Summary It has become increasingly clear that protein-protein interactions (PPIs) are compartmentalized in nanoscale domains that define the biochemical architecture of the cell. Despite tremendous advances in super-resolution imaging, strategies to observe PPIs at sufficient resolution to discern their organization are just emerging. Here we describe a strategy in which PPIs induce reconstitution of fluorescent proteins (FPs) that are capable of exhibiting single-molecule fluctuations suitable for Stochastic Optical Fluctuation Imaging (SOFI). Subsequently, spatial maps of these interactions can be resolved in super-resolution in living cells. Using this strategy, termed reconstituted fluorescence-based SOFI (refSOFI), we investigated the interaction between the endoplasmic reticulum Ca2+ sensor STIM1 and the pore-forming channel subunit ORAI1, a crucial process in store-operated Ca2+ entry (SOCE). Stimulating SOCE does not appear to change the size of existing STIM1/ORAI1 interaction puncta at the ER-plasma membrane junctions, but results in an apparent increase in the number of interaction puncta. PMID:26748717

  16. Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

    PubMed Central

    Corbit, Kevin C.; Foster, David A.; Rosner, Marsha Rich

    1999-01-01

    In several neuronal cell systems, fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogen. The mechanisms responsible for these different cellular fates are unclear. We report here that although FGF, NGF, and EGF all activate mitogen-activated protein (MAP) kinase (extracellular signal-related kinase [ERK]) in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells, the activation of ERK by the neurogenic agents FGF and NGF is dependent upon protein kinase Cδ (PKCδ), whereas ERK activation in response to the mitogenic EGF is independent of PKCδ. Antisense PKCδ oligonucleotides or the PKCδ-specific inhibitor rottlerin inhibited FGF- and NGF-induced, but not EGF-induced, ERK activation. In contrast, EGF-induced ERK activation was inhibited by the phosphatidylinositol-3-kinase inhibitor wortmannin, which had no effect upon FGF-induced ERK activation. Rottlerin also inhibited the activation of MAP kinase kinase (MEK) in response to activated Raf, but had no effect upon c-Raf activity or ERK activation by activated MEK. These results indicate that PKCδ functions either downstream from or in parallel with c-Raf, but upstream of MEK. Inhibition of PKCδ also blocked neurite outgrowth induced by FGF and NGF in PC12 cells and by activated Raf in H19-7 cells, indicating a role for PKCδ in the neurogenic effects of FGF, NGF, and Raf. Interestingly, the PKCδ requirement is apparently cell type specific, since FGF-induced ERK activation was independent of PKCδ in NIH 3T3 murine fibroblasts, in which FGF is a mitogen. These data demonstrate that PKCδ contributes to growth factor specificity and response in neuronal cells and may also promote cell-type-specific differences in growth factor signaling. PMID:10330161

  17. Enhancer-specific modulation of E protein activity.

    PubMed

    Markus, Maurice; Du, Zhimei; Benezra, Robert

    2002-02-22

    Homodimeric complexes of members of the E protein family of basic helix-loop-helix (bHLH) transcription factors are important for tissue-specific activation of genes in B lymphocytes (Bain, G., Gruenwald, S., and Murre, C. (1993) Mol. Cell Biol. 13, 3522-3529; Shen, C. P., and Kadesch, T. (1995) Mol. Cell Biol. 15, 4518-4524; Jacobs, Y., et al. (1994) Mol. Cell Biol. 14, 4087-4096; Wilson, R. B., et al. (1991) Mol. Cell Biol. 11, 6185-6191). These homodimers, however, have little activity on myogenic enhancers (Weintraub, H., Genetta, T., and Kadesch, T. (1994) Genes Dev. 8, 2203-2211). We report here the identification of a novel cis-acting transcriptional repression domain in the E protein family of bHLH transcription factors. This domain, the Rep domain, is present in each of the known vertebrate E proteins. Extensive mapping analysis demonstrates that this domain is an acidic region of 30 amino acids with a predicted loop structure. Fusion studies indicate that the Rep domain can repress both of the E protein transactivation domains (AD1 and AD2). Physiologically, the Rep domain plays a key role in maintaining E protein homodimers in an inactive state on myogenic enhancers. In addition, we demonstrate that Rep domain mediated repression of AD1 is a necessary for the function of MyoD-E protein heterodimeric complexes. These studies demonstrate that the Rep domain is important for modulating the transcriptional activity of E proteins and provide key insights into both the selectivity and mechanism of action of E protein containing bHLH protein complexes. PMID:11724804

  18. Methods for Mapping of Interaction Networks Involving Membrane Proteins

    SciTech Connect

    Hooker, Brian S.; Bigelow, Diana J.; Lin, Chiann Tso

    2007-11-23

    Numerous approaches have been taken to study protein interactions, such as tagged protein complex isolation followed by mass spectrometry, yeast two-hybrid methods, fluorescence resonance energy transfer, surface plasmon resonance, site-directed mutagenesis, and crystallography. Membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is not suitable for the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.

  19. Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways.

    PubMed

    Abdulnour, Raja-Elie E; Peng, Xinqi; Finigan, Jay H; Han, Eugenia J; Hasan, Emile J; Birukov, Konstantin G; Reddy, Sekhar P; Watkins, James E; Kayyali, Usamah S; Garcia, Joe G N; Tuder, Rubin M; Hassoun, Paul M

    2006-09-01

    Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined. In separate experiments, rat pulmonary microvascular endothelial cells (RPMECs) were exposed to cyclic stretch (5% and 18% elongation, 20 cycles/min) for 2 h before intracellular XOR activity measurement. Lung XOR activity was significantly increased at 2 h of MV without changes in XOR expression. There was evidence of p38 MAP kinase, ERK1/2, and ERK5 phosphorylation, but no change in JNK phosphorylation. Evans blue dye extravasation and bronchoalveolar lavage protein concentration were significantly increased in response to MV, changes that were significantly attenuated by pretreatment with allopurinol. Cyclic stretch of RPMECs also caused MAP kinase phosphorylation and a 1.7-fold increase in XOR activity, which was completely abrogated by pretreatment of the cells with specific MAP kinase inhibitors. We conclude that XOR enzymatic activity is significantly increased by mechanical stress via activation of p38 MAP kinase and ERK and plays a critical role in the pathogenesis of pulmonary edema associated with VILI. PMID:16632522

  20. Structural basis for the association of MAP6 protein with microtubules and its regulation by calmodulin.

    PubMed

    Lefèvre, Julien; Savarin, Philippe; Gans, Pierre; Hamon, Loïc; Clément, Marie-Jeanne; David, Marie-Odile; Bosc, Christophe; Andrieux, Annie; Curmi, Patrick A

    2013-08-23

    Microtubules are highly dynamic αβ-tubulin polymers. In vitro and in living cells, microtubules are most often cold- and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold- and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90-177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90-177) to microtubules with a 1:1 MAP6(90-177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90-177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca(2+)-calmodulin competes with microtubules for MAP6(90-177) binding and that the binding mode of MAP6(90-177) to microtubules and Ca(2+)-calmodulin involves a common stretch of amino acid residues on the MAP6(90-177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca(2+)-calmodulin. PMID:23831686

  1. Isolation and restriction site maps of the genes encoding five Mycobacterium tuberculosis proteins.

    PubMed

    Shinnick, T M; Krat, C; Schadow, S

    1987-07-01

    A series of recombinant phage expressing five Mycobacterium tuberculosis antigens were isolated. Restriction maps for these antigens were deduced, and the size of the expressed proteins was determined. PMID:3036711

  2. Isolation and restriction site maps of the genes encoding five Mycobacterium tuberculosis proteins.

    PubMed Central

    Shinnick, T M; Krat, C; Schadow, S

    1987-01-01

    A series of recombinant phage expressing five Mycobacterium tuberculosis antigens were isolated. Restriction maps for these antigens were deduced, and the size of the expressed proteins was determined. Images PMID:3036711

  3. A two-dimensional protein map of Pleurotus ostreatus microsomes-proteome dynamics.

    PubMed

    Petráčková, Denisa; Halada, Petr; Bezoušková, Silvia; Křesinová, Zdena; Svobodová, Kateřina

    2016-01-01

    Recent studies documented that several processes in filamentous fungi are connected with microsomal enzyme activities. In this work, microsomal subproteomes of Pleurotus ostreatus were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. To assess proteome dynamics, microsomal proteins were isolated from fungal cultures after 7 and 12 days of cultivation. Additionally, 10 mg/L of 17α-ethinylestradiol (EE2) was treated with the cultures during 2 days. Despite the EE2 degradation by the fungus reached 97 and 76.3 % in 7- and 12-day-old cultures, respectively, only a minor effect on the composition of microsomal proteins was observed. The changes in protein maps related to ageing prevailed over those induced by EE2. Epoxide hydrolase, known to metabolize EE2, was detected in 12-day-old cultures only which suggests differences in EE2 degradation pathways utilized by fungal cultures of different age. The majority (32 %) of identified microsomal proteins were parts of mitochondrial energy metabolism. PMID:26122365

  4. Prolonged activation of mitogen-activated protein kinases during NSAID-induced apoptosis in HT-29 colon cancer cells.

    PubMed

    Kim, T I; Jin, S H; Kim, W H; Kang, E H; Choi, K Y; Kim, H J; Shin, S K; Kang, J K

    2001-06-01

    The mechanisms of the antineoplastic effect of nonsteroidal anti-inflammatory drugs (NSAIDs) still are unknown, but the induction of apoptosis is one of the possible mechanisms. We attempted to demonstrate the role of mitogen-activated protein (MAP) kinases, generally considered to be important mediators of proliferative and apoptotic signals, in NSAID-induced colon cancer cell apoptosis. Apoptosis was detected by demonstration of DNA fragmentation in agarose gel electrophoresis. Cell death was assessed by trypan blue dye exclusion method. MAP kinase activation was assessed by Western blot using phosphospecific antibodies to MAP kinases. Kinase assay using activating transcription factor-2 (ATF-2) fusion protein as a substrate was also performed for measuring p38 MAP kinase activity. For the inhibition of p38 MAP kinase, pyridinylimidazole compound (SB203580) was utilized. Caspase-3 activity was measured using the tetrapeptide fluorogenic substrate Ac-DEVD-AMC. Treatment of HT-29 cells with NSAIDs results in time- and dose-dependent induction of apoptosis, accompanied by sustained activation of all three MAP kinase subfamilies. The SB203580, a p38 MAP kinase inhibitor, reduced indomethacin-induced cell death by 43%, while PD098059, a MAPK/ERK kinase (MEK)1 inhibitor, did not affect cell death. p38 MAP kinase and caspase-3 activation were not significantly interlinked in indomethacin-induced apoptosis. From these results, we conclude that NSAIDs can induce prolonged activation of MAP kinases in colon cancer cells and that, of these, p38 MAP kinase may play a partial but significant role in indomethacin-induced apoptosis. PMID:11459290

  5. Mapping flexible protein domains at subnanometer resolution with the atomic force microscope.

    PubMed

    Müller, D J; Fotiadis, D; Engel, A

    1998-06-23

    The mapping of flexible protein domains with the atomic force microscope is reviewed. Examples discussed are the bacteriorhodopsin from Halobacterium salinarum, the head-tail-connector from phage phi29, and the hexagonally packed intermediate layer from Deinococcus radiodurans which all were recorded in physiological buffer solution. All three proteins undergo reversible structural changes that are reflected in standard deviation maps calculated from aligned topographs of individual protein complexes. Depending on the lateral resolution (up to 0.8 nm) flexible surface regions can ultimately be correlated with individual polypeptide loops. In addition, multivariate statistical classification revealed the major conformations of the protein surface. PMID:9678604

  6. Molecular architecture of the active mitochondrial protein gate.

    PubMed

    Shiota, Takuya; Imai, Kenichiro; Qiu, Jian; Hewitt, Victoria L; Tan, Khershing; Shen, Hsin-Hui; Sakiyama, Noriyuki; Fukasawa, Yoshinori; Hayat, Sikander; Kamiya, Megumi; Elofsson, Arne; Tomii, Kentaro; Horton, Paul; Wiedemann, Nils; Pfanner, Nikolaus; Lithgow, Trevor; Endo, Toshiya

    2015-09-25

    Mitochondria fulfill central functions in cellular energetics, metabolism, and signaling. The outer membrane translocator complex (the TOM complex) imports most mitochondrial proteins, but its architecture is unknown. Using a cross-linking approach, we mapped the active translocator down to single amino acid residues, revealing different transport paths for preproteins through the Tom40 channel. An N-terminal segment of Tom40 passes from the cytosol through the channel to recruit chaperones from the intermembrane space that guide the transfer of hydrophobic preproteins. The translocator contains three Tom40 β-barrel channels sandwiched between a central α-helical Tom22 receptor cluster and external regulatory Tom proteins. The preprotein-translocating trimeric complex exchanges with a dimeric isoform to assemble new TOM complexes. Dynamic coupling of α-helical receptors, β-barrel channels, and chaperones generates a versatile machinery that transports about 1000 different proteins. PMID:26404837

  7. Conditional independence mapping of DIGE data reveals PDIA3 protein species as key nodes associated with muscle aerobic capacity

    PubMed Central

    Burniston, Jatin G.; Kenyani, Jenna; Gray, Donna; Guadagnin, Eleonora; Jarman, Ian H.; Cobley, James N.; Cuthbertson, Daniel J.; Chen, Yi-Wen; Wastling, Jonathan M.; Lisboa, Paulo J.; Koch, Lauren G.; Britton, Steven L.

    2014-01-01

    Profiling of protein species is important because gene polymorphisms, splice variations and post-translational modifications may combine and give rise to multiple protein species that have different effects on cellular function. Two-dimensional gel electrophoresis is one of the most robust methods for differential analysis of protein species, but bioinformatic interrogation is challenging because the consequences of changes in the abundance of individual protein species on cell function are unknown and cannot be predicted. We conducted DIGE of soleus muscle from male and female rats artificially selected as either high- or low-capacity runners (HCR and LCR, respectively). In total 696 protein species were resolved and LC–MS/MS identified proteins in 337 spots. Forty protein species were differentially (P < 0.05, FDR < 10%) expressed between HCR and LCR and conditional independence mapping found distinct networks within these data, which brought insight beyond that achieved by functional annotation. Protein disulphide isomerase A3 emerged as a key node segregating with differences in aerobic capacity and unsupervised bibliometric analysis highlighted further links to signal transducer and activator of transcription 3, which were confirmed by western blotting. Thus, conditional independence mapping is a useful technique for interrogating DIGE data that is capable of highlighting latent features. PMID:24769234

  8. Automatic classification of protein structures using low-dimensional structure space mappings

    PubMed Central

    2014-01-01

    Background Protein function is closely intertwined with protein structure. Discovery of meaningful structure-function relationships is of utmost importance in protein biochemistry and has led to creation of high-quality, manually curated classification databases, such as the gold-standard SCOP (Structural Classification of Proteins) database. The SCOP database and its counterparts such as CATH provide a detailed and comprehensive description of the structural and evolutionary relationships of the proteins of known structure and are widely employed in structural and computational biology. Since manual classification is both subjective and highly laborious, automated classification of novel structures is increasingly an active area of research. The design of methods for automated structure classification has been rendered even more important since the recent past, due to the explosion in number of solved structures arising out of various structural biology initiatives. In this paper we propose an approach to the problem of structure classification based on creating and tessellating low dimensional maps of the protein structure space (MPSS). Given a set of protein structures, an MPSS is a low dimensional embedding of structural similarity-based distances between the molecules. In an MPSS, a group of proteins (such as all the proteins in the PDB or sub-samplings thereof) under consideration are represented as point clouds and structural relatedness maps to spatial adjacency of the points. In this paper we present methods and results that show that MPSS can be used to create tessellations of the protein space comparable to the clade systems within SCOP. Though we have used SCOP as the gold standard, the proposed approach is equally applicable for other structural classifications. Methods In the proposed approach, we first construct MPSS using pairwise alignment distances obtained from four established structure alignment algorithms (CE, Dali, FATCAT and MATT). The low

  9. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  10. DNA-based control of protein activity.

    PubMed

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  11. Insurance Applications of Active Fault Maps Showing Epistemic Uncertainty

    NASA Astrophysics Data System (ADS)

    Woo, G.

    2005-12-01

    Insurance loss modeling for earthquakes utilizes available maps of active faulting produced by geoscientists. All such maps are subject to uncertainty, arising from lack of knowledge of fault geometry and rupture history. Field work to undertake geological fault investigations drains human and monetary resources, and this inevitably limits the resolution of fault parameters. Some areas are more accessible than others; some may be of greater social or economic importance than others; some areas may be investigated more rapidly or diligently than others; or funding restrictions may have curtailed the extent of the fault mapping program. In contrast with the aleatory uncertainty associated with the inherent variability in the dynamics of earthquake fault rupture, uncertainty associated with lack of knowledge of fault geometry and rupture history is epistemic. The extent of this epistemic uncertainty may vary substantially from one regional or national fault map to another. However aware the local cartographer may be, this uncertainty is generally not conveyed in detail to the international map user. For example, an area may be left blank for a variety of reasons, ranging from lack of sufficient investigation of a fault to lack of convincing evidence of activity. Epistemic uncertainty in fault parameters is of concern in any probabilistic assessment of seismic hazard, not least in insurance earthquake risk applications. A logic-tree framework is appropriate for incorporating epistemic uncertainty. Some insurance contracts cover specific high-value properties or transport infrastructure, and therefore are extremely sensitive to the geometry of active faulting. Alternative Risk Transfer (ART) to the capital markets may also be considered. In order for such insurance or ART contracts to be properly priced, uncertainty should be taken into account. Accordingly, an estimate is needed for the likelihood of surface rupture capable of causing severe damage. Especially where a

  12. Analysis of low-density lipoprotein-associated proteins using the method of digitized native protein mapping.

    PubMed

    Jin, Ya; Chen, Jin; Wang, Ahui; Zhang, Jun; Chen, Shumin; Manabe, Takashi; Tan, Wen

    2016-07-01

    The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS (in data-independent acquisition mode, or MS(E) ), was improved by using a new MS/MS mode, ion mobility separation enhanced-MS(E) (HDMS(E) ), and applied to analyze the area of human plasma low-density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid-cut into 72 square gel pieces and subjected to quantitative LC-MS/MS. Compared with MS(E) , HDMS(E) showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC-HDMS(E) and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B-100 was the most abundant protein in the grid-cut area, concentrated at pI ca. 5.4-6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39-42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B-100. Twenty-two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL. PMID:27174546

  13. Immunogenicity and Reactivity of Novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and Conserved MAP1156 Proteins with Sera from Experimentally and Naturally Infected Animals ▿ †

    PubMed Central

    Bannantine, John P.; Paulson, Avery L.; Chacon, Ofelia; Fenton, Robert J.; Zinniel, Denise K.; McVey, David S.; Smith, David R.; Czuprynski, Charles J.; Barletta, Raúl G.

    2011-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5′ and 3′ regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein (P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis-infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine

  14. Motor Cortex Microcircuit Simulation Based on Brain Activity Mapping

    PubMed Central

    Chadderdon, George L.; Mohan, Ashutosh; Suter, Benjamin A.; Neymotin, Samuel A.; Kerr, Cliff C.; Francis, Joseph T.; Shepherd, Gordon M. G.; Lytton, William W.

    2016-01-01

    The deceptively simple laminar structure of neocortex belies the complexity of intra- and interlaminar connectivity. We developed a computational model based primarily on a unified set of brain activity mapping studies of mouse M1. The simulation consisted of 775 spiking neurons of 10 cell types with detailed population-to-population connectivity. Static analysis of connectivity with graph-theoretic tools revealed that the corticostriatal population showed strong centrality, suggesting that would provide a network hub. Subsequent dynamical analysis confirmed this observation, in addition to revealing network dynamics that cannot be readily predicted through analysis of the wiring diagram alone. Activation thresholds depended on the stimulated layer. Low stimulation produced transient activation, while stronger activation produced sustained oscillations where the threshold for sustained responses varied by layer: 13% in layer 2/3, 54% in layer 5A, 25% in layer 5B, and 17% in layer 6. The frequency and phase of the resulting oscillation also depended on stimulation layer. By demonstrating the effectiveness of combined static and dynamic analysis, our results show how static brain maps can be related to the results of brain activity mapping. PMID:24708371

  15. Activation of MAP kinase signaling pathway in the mussel Mytilus galloprovincialis as biomarker of environmental pollution.

    PubMed

    Châtel, A; Hamer, B; Talarmin, H; Dorange, G; Schröder, H C; Müller, W E G

    2010-03-01

    Stimulation of MAP kinase signal transduction pathway by various stressful stimuli was investigated in the marine bivalve Mytilus galloprovincialis. Analyses were performed in animals exposed in laboratory to selected pollutants and in mussels collected in winter and summer along the eastern Adriatic coast (Croatia). Effects of oxidative stress, induced by tributyltin, hydrogen peroxide and water soluble fraction of diesel fuel on the activation/phosphorylation of the three Mitogen-Activated Protein Kinases (MAPKs) p38, JNK and ERK using a newly developed ELISA procedure were evaluated. MAP kinase activation was analyzed 1h after exposure of mussels to chemical agents, and after recovery periods of 6 and 24h. Our results clearly indicated that pollutants generated different patterns of induction of the MAPK phosphorylation. Indeed, only pp38 and pJNK were activated with 11, 33 and 100 microg/L TBT, reaching a maximum activation after 6h in seawater following treatment of mussels with 11 microg/L TBT. Treatment with 0.074 and 0.222 mM H2O2 enhanced activation of both p38 and ERK. These two kinases were activated after 1h exposure, followed by a diminution after 6h of recovery in seawater and a reactivation after 24h. The levels of phosphorylated P38 and JNK were increased after mussel exposure with 7.5, 15 and 30% of water soluble fraction of diesel oil. P38 was activated concentration dependently at 1h exposure. Additionally, field study pointed out seasonal differences in MAP kinases activation as mussels collected during summer had a higher enzyme activation state than in winter, as well as sampling site differences which could be correlated to the industrial/tourism activity and environmental stresses (salinity). All the results converge towards MAP kinase signaling pathway being induced by various pollutants in M. galloprovincialis. This signaling cascade should be considered as a possible biomarker of environmental stress and pollution. PMID:19948362

  16. Computing wheat nitrogen requirements from grain yield and protein maps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Optical protein sensors and mass-flow yield monitors provide the opportunity to continuously measure grain quality and quantity during harvesting. This chapter illustrates how yield monitor and grain protein measurements may provide useful postharvest information for evaluating water or nitrogen (N)...

  17. Computing wheat nitrogen requirements from grain yield and protein maps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Optical protein sensors and mass-flow yield monitors provide the opportunity to continuously measure grain quality and quantity during harvesting. This chapter illustrates how yield monitor and grain protein measurements may provide useful post-harvest information for evaluating water or nitrogen (...

  18. Microtubule-associated proteins: a monoclonal antibody to MAP2 binds to differentiated neurons.

    PubMed

    Izant, J G; McIntosh, J R

    1980-08-01

    Hybridomas that secret IgG reacting specifically with the brain microtubule-associated protein MAP2 have been prepared with speen cells from BALB/c mice hyperimmunized with high molecular weight neurotubule-associated proteins. Immunofluorecence microscopy using dual fluorochrome labeling of tubulin and MAP2 antigens revealed identical patterns of interphase fiber networks in cells from explants of newborn mouse brain. The anti-MAP2 antibody did not stain primary mouse kidney cells or CHO, 3T3, HeLa, or PtK1 cell lines. Immunoprecipitation and antibody gel staining techniques failed to demonstrate any crossreacting antigen in these cells. MAP2 antigen was not seen in association with the mitotic spindle in any of the cells examined. Radioimmunoassay showed species crossreactivity of the anti-MAP2 antibody with mammalian but not avian neural cell extracts. Glial cells and some neuroblastoma cell lines did not appear to contain MAP2. However, in the B104 rat neuroblastoma cell line the MAP2 antigen appeared to be associated with the cytoskeleton concomitant with differentiation induced by dibutyryl cyclic AMP. In disagreement with most previously published reports, our data suggest that MAP2 is found only in differentiated neuronal cells and raises the possibility that MAP2 is involved in neuronal differentiation or neuron-specific processes. PMID:7001466

  19. Active Nuclear Import of Membrane Proteins Revisited

    PubMed Central

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

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

  20. Active Nuclear Import of Membrane Proteins Revisited.

    PubMed

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

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker's yeast. PMID:26473931

  1. Mapping the Protein-Protein Interactome Networks Using Yeast Two-Hybrid Screens.

    PubMed

    Rajagopala, Seesandra Venkatappa

    2015-01-01

    The yeast two-hybrid system (Y2H) is a powerful method to identify binary protein-protein interactions in vivo. Here we describe Y2H screening strategies that use defined libraries of open reading frames (ORFs) and cDNA libraries. The array-based Y2H system is well suited for interactome studies of small genomes with an existing ORFeome clones preferentially in a recombination based cloning system. For large genomes, pooled library screening followed by Y2H pairwise retests may be more efficient in terms of time and resources, but multiple sampling is necessary to ensure comprehensive screening. While the Y2H false positives can be efficiently reduced by using built-in controls, retesting, and evaluation of background activation; implementing the multiple variants of the Y2H vector systems is essential to reduce the false negatives and ensure comprehensive coverage of an interactome. PMID:26621469

  2. Environment Polarity in Proteins Mapped Noninvasively by FTIR Spectroscopy

    PubMed Central

    Manor, Joshua; Feldblum, Esther S.; Zanni, Martin T.; Arkin, Isaiah T.

    2012-01-01

    The polarity pattern of a macromolecule is of utmost importance to its structure and function. For example, one of the main driving forces for protein folding is the burial of hydrophobic residues. Yet polarity remains a difficult property to measure experimentally, due in part to its non-uniformity in the protein interior. Herein, we show that FTIR linewidth analysis of noninvasive 1-13C=18O labels can be used to obtain a reliable measure of the local polarity, even in a highly multi-phasic system, such as a membrane protein. We show that in the Influenza M2 H+ channel, residues that line the pore are located in an environment that is as polar as fully solvated residues, while residues that face the lipid acyl chains are located in an apolar environment. Taken together, FTIR linewidth analysis is a powerful, yet chemically non-perturbing approach to examine one of the most important properties in proteins - polarity. PMID:22563521

  3. Topological mapping methods for α-helical bacterial membrane proteins – an update and a guide

    PubMed Central

    Islam, Salim T; Lam, Joseph S

    2013-01-01

    Integral membrane proteins with α-helical transmembrane segments (TMS) are known to play important and diverse roles in prokaryotic cell physiology. The net hydrophobicity of TMS directly corresponds to the observed difficulties in expressing and purifying these proteins, let alone producing sufficient yields for structural studies using two-/three-dimensional (2D/3D) crystallographic or nuclear magnetic resonance methods. To gain insight into the function of these integral membrane proteins, topological mapping has become an important tool to identify exposed and membrane-embedded protein domains. This approach has led to the discovery of protein tracts of functional importance and to the proposition of novel mechanistic hypotheses. In this review, we synthesize the various methods available for topological mapping of α-helical integral membrane proteins to provide investigators with a comprehensive reference for choosing techniques suited to their particular topological queries and available resources. PMID:23408725

  4. Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila

    PubMed Central

    Pindyurin, Alexey V.; Pagie, Ludo; Kozhevnikova, Elena N.; van Arensbergen, Joris; van Steensel, Bas

    2016-01-01

    Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity, it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila. Here, we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress possible toxic effects of Dam. In addition, we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. These new DamID tools will be valuable for the mapping of binding patterns of chromatin proteins in Drosophila tissues and especially in cell lineages. PMID:27001518

  5. Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila.

    PubMed

    Pindyurin, Alexey V; Pagie, Ludo; Kozhevnikova, Elena N; van Arensbergen, Joris; van Steensel, Bas

    2016-07-01

    Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity, it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila Here, we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress possible toxic effects of Dam. In addition, we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. These new DamID tools will be valuable for the mapping of binding patterns of chromatin proteins in Drosophila tissues and especially in cell lineages. PMID:27001518

  6. Mapping the expression of soluble olfactory proteins in the honeybee.

    PubMed

    Dani, Francesca Romana; Iovinella, Immacolata; Felicioli, Antonio; Niccolini, Alberto; Calvello, Maria Antonietta; Carucci, Maria Giovanna; Qiao, Huili; Pieraccini, Giuseppe; Turillazzi, Stefano; Moneti, Gloriano; Pelosi, Paolo

    2010-04-01

    Chemical communication in insects is mediated by soluble binding proteins, belonging to two large families, Odorant-binding Proteins (OBPs) and Chemosensory Proteins (CSPs). Recently, evidence has been provided that OBPs are involved in recognition of chemical stimuli. We therefore decided to investigate the expression of OBPs and CSPs in the honeybee at the protein level, using a proteomic approach. Our results are in agreement with previous reports of expression at the RNA level and show that 12 of the 21 OBPs predicted in the genome of the honeybee Apis mellifera and 2 of the 6 CSPs are present in the foragers' antennae, while the larvae express only three OBPs and a single CSP. MALDI mass spectrometry on crude antennal extracts and MALDI profiling on sections of antennae demonstrated that these techniques can be applied to investigate individual differences in the expression of abundant proteins, such as OBPs and CSPs, as well as to detect the presence of proteins in different regions of the antenna. Finally, as part of a project aimed at the characterization of all OBPs and CSPs of the honeybee, we expressed 5 OBPs and 4 CSPs in a bacterial system and measured their affinity to a number of ligands. Clear differences in their binding spectra have been observed between OBPs, as well as CSPs. PMID:20155982

  7. Reverse Phase Protein Arrays: Mapping the path towards personalized medicine

    PubMed Central

    Gallagher, Rosa I.; Espina, Virginia

    2016-01-01

    Reverse phase protein array (RPPA) technology evolved from the advent of miniaturized immunoassays and gene microarray technology. Reverse phase protein arrays provide either a low throughput or high throughput methodology for quantifying proteins and their post-translationally modified forms in both cellular and non-cellular samples. As the demand for patient tailored therapies increases so does the need for precise and sensitive technology to accurately profile the molecular circuitry driving an individual patient’s disease. RPPAs are currently utilized in clinical trials for profiling and comparing the functional state of protein signaling pathways, either temporally within tumors, between patients, or within the same patients before/after treatment. RPPAs are generally employed for quantifying large numbers of samples on one array, under identical experimental conditions. However, the goal of personalized cancer medicine is to design therapies based on the molecular portrait of a patient’s tumor, which in turn result in more efficacious treatments with less toxicity. Therefore, RPPAs are also being validated for low throughput assays of individual patient samples. This review explores reverse phase protein array technology in the cancer research field, concentrating on its role as a fundamental tool for deciphering protein signaling networks and its emerging role in personalized medicine. PMID:25358623

  8. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  9. Petunia nectar proteins have ribonuclease activity.

    PubMed

    Hillwig, Melissa S; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W; Macintosh, Gustavo C

    2010-06-01

    Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar. PMID:20460362

  10. Petunia nectar proteins have ribonuclease activity

    PubMed Central

    Hillwig, Melissa S.; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W.; MacIntosh, Gustavo C.

    2010-01-01

    Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar. PMID:20460362

  11. Mapping Ultra-weak Protein-Protein Interactions between Heme Transporters of Staphylococcus aureus

    PubMed Central

    Abe, Ryota; Caaveiro, Jose M. M.; Kozuka-Hata, Hiroko; Oyama, Masaaki; Tsumoto, Kouhei

    2012-01-01

    Iron is an essential nutrient for the proliferation of Staphylococcus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host organism. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs. PMID:22427659

  12. A simple contact mapping algorithm for identifying potential peptide mimetics in protein–protein interaction partners

    PubMed Central

    Krall, Alex; Brunn, Jonathan; Kankanala, Spandana; Peters, Michael H

    2014-01-01

    A simple, static contact mapping algorithm has been developed as a first step at identifying potential peptide biomimetics from protein interaction partner structure files. This rapid and simple mapping algorithm, “OpenContact” provides screened or parsed protein interaction files based on specified criteria for interatomic separation distances and interatomic potential interactions. The algorithm, which uses all-atom Amber03 force field models, was blindly tested on several unrelated cases from the literature where potential peptide mimetics have been experimentally developed to varying degrees of success. In all cases, the screening algorithm efficiently predicted proposed or potential peptide biomimetics, or close variations thereof, and provided complete atom-atom interaction data necessary for further detailed analysis and drug development. In addition, we used the static parsing/mapping method to develop a peptide mimetic to the cancer protein target, epidermal growth factor receptor. In this case, secondary, loop structure for the peptide was indicated from the intra-protein mapping, and the peptide was subsequently synthesized and shown to exhibit successful binding to the target protein. The case studies, which all involved experimental peptide drug advancement, illustrate many of the challenges associated with the development of peptide biomimetics, in general. Proteins 2014; 82:2253–2262. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:24756879

  13. Theoretical considerations for mapping activation in human cardiac fibrillation

    NASA Astrophysics Data System (ADS)

    Rappel, Wouter-Jan; Narayan, Sanjiv M.

    2013-06-01

    Defining mechanisms for cardiac fibrillation is challenging because, in contrast to other arrhythmias, fibrillation exhibits complex non-repeatability in spatiotemporal activation but paradoxically exhibits conserved spatial gradients in rate, dominant frequency, and electrical propagation. Unlike animal models, in which fibrillation can be mapped at high spatial and temporal resolution using optical dyes or arrays of contact electrodes, mapping of cardiac fibrillation in patients is constrained practically to lower resolutions or smaller fields-of-view. In many animal models, atrial fibrillation is maintained by localized electrical rotors and focal sources. However, until recently, few studies had revealed localized sources in human fibrillation, so that the impact of mapping constraints on the ability to identify rotors or focal sources in humans was not described. Here, we determine the minimum spatial and temporal resolutions theoretically required to detect rigidly rotating spiral waves and focal sources, then extend these requirements for spiral waves in computer simulations. Finally, we apply our results to clinical data acquired during human atrial fibrillation using a novel technique termed focal impulse and rotor mapping (FIRM). Our results provide theoretical justification and clinical demonstration that FIRM meets the spatio-temporal resolution requirements to reliably identify rotors and focal sources for human atrial fibrillation.

  14. Reverse footprinting to map sites of RNA-protein interactions.

    PubMed

    Nilsen, Timothy W

    2014-06-01

    Nuclease protection of a site-specifically labeled RNA by an RNA-binding protein is an extremely powerful method for determining the site of an RNA-protein interaction. If a protein binds to the region that contains the site-specific label, it will protect the label and adjoining sequences from nuclease digestion. The protected region, or "footprint," can then be characterized by extensive fragmentation and gel electrophoresis. As the name implies, reverse footprinting produces a mirror image of a conventional footprint; the footprint is revealed by the presence, not the absence, of bands. This method has a distinct advantage over conventional footprinting in that only a small fraction of the labeled RNA must be bound. PMID:24890211

  15. You are lost without a map: Navigating the sea of protein structures.

    PubMed

    Lamb, Audrey L; Kappock, T Joseph; Silvaggi, Nicholas R

    2015-04-01

    X-ray crystal structures propel biochemistry research like no other experimental method, since they answer many questions directly and inspire new hypotheses. Unfortunately, many users of crystallographic models mistake them for actual experimental data. Crystallographic models are interpretations, several steps removed from the experimental measurements, making it difficult for nonspecialists to assess the quality of the underlying data. Crystallographers mainly rely on "global" measures of data and model quality to build models. Robust validation procedures based on global measures now largely ensure that structures in the Protein Data Bank (PDB) are largely correct. However, global measures do not allow users of crystallographic models to judge the reliability of "local" features in a region of interest. Refinement of a model to fit into an electron density map requires interpretation of the data to produce a single "best" overall model. This process requires inclusion of most probable conformations in areas of poor density. Users who misunderstand this can be misled, especially in regions of the structure that are mobile, including active sites, surface residues, and especially ligands. This article aims to equip users of macromolecular models with tools to critically assess local model quality. Structure users should always check the agreement of the electron density map and the derived model in all areas of interest, even if the global statistics are good. We provide illustrated examples of interpreted electron density as a guide for those unaccustomed to viewing electron density. PMID:25554228

  16. Epitope mapping and functional analysis of sigma A and sigma NS proteins of avian reovirus

    SciTech Connect

    Huang, Pi H.; Li, Ying J.; Su, Yu P.; Lee, Long H.; Liu, Hung J. . E-mail: hjliu@mail.npust.edu.tw

    2005-02-20

    We have previously shown that avian reovirus (ARV) {sigma}A and {sigma}NS proteins possess dsRNA and ssRNA binding activity and suggested that there are two epitopes on {sigma}A (I and II) and three epitopes (A, B, and C) on {sigma}NS. To further define the location of epitopes on {sigma}A and {sigma}NS proteins and to further elucidate the biological functions of these epitopes by using monoclonal antibodies (MAbs) 62, 1F9, H1E1, and 4A123 against the ARV S1133 strain, the full-length and deletion fragments of S2 and S4 genes of ARV generated by polymerase chain reaction (PCR) were cloned into pET32 expression vectors and the fusion proteins were overexpressed in Escherichia coli BL21 strain. Epitope mapping using MAbs and E. coli-expressed deletion fragments of {sigma}A and {sigma}NS of the ARV S1133 strain, synthetic peptides, and the cross reactivity of MAbs to heterologous ARV strains demonstrated that epitope II on {sigma}A was located at amino acid residues {sup 340}QWVMAGLVSAA{sup 350} and epitope B on {sigma}NS at amino acid residues {sup 180}MLDMVDGRP{sup 188}. The MAbs (62, 1F9, and H1E1) directed against epitopes II and B did not require the native conformation of {sigma}A and {sigma}NS, suggesting that their binding activities were conformation-independent. On the other hand, MAb 4A123 only reacted with complete {sigma}NS but not with truncated {sigma}NS fusion proteins in Western blot, suggesting that the binding activity of MAb to epitope A on {sigma}NS was conformation-dependent. Amino acid sequence analysis and the binding assays of MAb 62 to heterologous ARV strains suggested that epitope II on {sigma}A was highly conserved among ARV strains and that this epitope is suitable as a serological marker for the detection of ARV antibodies following natural infection in chickens. On the contrary, an amino acid substitution at position 183 (M to V) in epitope B of ARV could hinder the reactivity of the {sigma}NS with MAb 1F9. The {sigma}NS of ARV with ss

  17. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J.; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2012-10-01

    Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

  18. Proteins of human semen. I. Two-dimensional mapping of human seminal fluid

    SciTech Connect

    Edwards, J.J.; Tollaksen, S.L.; Anderson, N.G.

    1981-08-01

    The proteins in human seminal plasma were mapped by high-resolution two-dimensional electrophoresis (ISO-DALT and BASO-DALT systems). When analyzed under dissociating conditions, samples from normal fertile males revealed a pattern of over 200 proteins, ranging in mass from 10,000 to 100,000 daltons. Comparison of the mapped proteins from these males and those who had undergone vasectomy allowed us to identify one series of glycoproteins as missing from the semen from vasectomized individuals. Glycoproteins isolated by affinity chromatography with use of concanavalin A were also mapped. Some of the protein spots were identified either by coelectrophoresis with purified proteins or by the electrophoretic transfer of proteins to nitrocellulose sheets and subsequent detection by immunological procedures. The proteins identified include a number of serum proteins as well as prostatic acid phosphatase and creatine kinase. Proteolytic events shown to occur during the liquefaction of semen that occurs early after collection indicate the importance of carefully controlled collection and preparation methods for clinical evaluation of seminal plasma. Ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride inhibit this proteolysis.

  19. Proteins of human semen. I. Two-dimensional mapping of human seminal fluid.

    PubMed

    Edwards, J J; Tollaksen, S L; Anderson, N G

    1981-08-01

    The proteins in human seminal plasma were mapped by high-resolution two-dimensional electrophoresis (ISO-DALT and BASO-DALT systems). When analyzed under dissociating conditions, samples from normal fertile males revealed a pattern of over 200 proteins, ranging in mass from 10 000 to 100 000 daltons. Comparison of the mapped proteins from these males and those who had undergone vasectomy allowed us to identify one series of glycoproteins as missing from the semen from vasectomized individuals. Glycoproteins isolated by affinity chromatography with use of concanavalin A were also mapped. Some of the protein spots were identified either by co-electrophoresis with purified proteins or by the electrophoretic transfer of proteins to nitrocellulose sheets and subsequent detection by immunological procedures. The proteins identified include a number of serum proteins as well as prostatic acid phosphatase and creatine kinase. Proteolytic events shown to occur during the liquefaction of semen that occurs early after collection indicate the importance of carefully controlled collection and preparation methods for clinical evaluation of seminal plasma. Ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride inhibit this proteolysis. PMID:7273394

  20. Paired natural cysteine mutation mapping: aid to constraining models of protein tertiary structure.

    PubMed Central

    Kreisberg, R.; Buchner, V.; Arad, D.

    1995-01-01

    This paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural-cysteine-mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide-bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using PileUp algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three-dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur. PMID:8563638

  1. CMOS Monolithic Active Pixel Sensors (MAPS): Developments and future outlook

    NASA Astrophysics Data System (ADS)

    Turchetta, R.; Fant, A.; Gasiorek, P.; Esbrand, C.; Griffiths, J. A.; Metaxas, M. G.; Royle, G. J.; Speller, R.; Venanzi, C.; van der Stelt, P. F.; Verheij, H.; Li, G.; Theodoridis, S.; Georgiou, H.; Cavouras, D.; Hall, G.; Noy, M.; Jones, J.; Leaver, J.; Machin, D.; Greenwood, S.; Khaleeq, M.; Schulerud, H.; Østby, J. M.; Triantis, F.; Asimidis, A.; Bolanakis, D.; Manthos, N.; Longo, R.; Bergamaschi, A.

    2007-12-01

    Re-invented in the early 1990s, on both sides of the Atlantic, Monolithic Active Pixel Sensors (MAPS) in a CMOS technology are today the most sold solid-state imaging devices, overtaking the traditional technology of Charge-Coupled Devices (CCD). The slow uptake of CMOS MAPS started with low-end applications, for example web-cams, and is slowly pervading the high-end applications, for example in prosumer digital cameras. Higher specifications are required for scientific applications: very low noise, high speed, high dynamic range, large format and radiation hardness are some of these requirements. This paper will present a brief overview of the CMOS Image Sensor technology and of the requirements for scientific applications. As an example, a sensor for X-ray imaging will be presented. This sensor was developed within a European FP6 Consortium, intelligent imaging sensors (I-ImaS).

  2. Fusion of protegrin-1 and plectasin to MAP30 shows significant inhibition activity against dengue virus replication.

    PubMed

    Rothan, Hussin A; Bahrani, Hirbod; Mohamed, Zulqarnain; Abd Rahman, Noorsaadah; Yusof, Rohana

    2014-01-01

    Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities. PMID:24722532

  3. Mapping protein binding sites on the biomolecular corona of nanoparticles

    NASA Astrophysics Data System (ADS)

    Kelly, Philip M.; Åberg, Christoffer; Polo, Ester; O'Connell, Ann; Cookman, Jennifer; Fallon, Jonathan; Krpetić, Željka; Dawson, Kenneth A.

    2015-05-01

    Nanoparticles in a biological milieu are known to form a sufficiently long-lived and well-organized ‘corona’ of biomolecules to confer a biological identity to the particle. Because this nanoparticle-biomolecule complex interacts with cells and biological barriers, potentially engaging with different biological pathways, it is important to clarify the presentation of functional biomolecular motifs at its interface. Here, we demonstrate that by using antibody-labelled gold nanoparticles, differential centrifugal sedimentation and various imaging techniques it is possible to identify the spatial location of proteins, their functional motifs and their binding sites. We show that for transferrin-coated polystyrene nanoparticles only a minority of adsorbed proteins exhibit functional motifs and the spatial organization appears random, which is consistent, overall, with a stochastic and irreversible adsorption process. Our methods are applicable to a wide array of nanoparticles and can offer a microscopic molecular description of the biological identity of nanoparticles.

  4. Determination of contact maps in proteins: A combination of structural and chemical approaches

    NASA Astrophysics Data System (ADS)

    Wołek, Karol; Gómez-Sicilia, Àngel; Cieplak, Marek

    2015-12-01

    Contact map selection is a crucial step in structure-based molecular dynamics modelling of proteins. The map can be determined in many different ways. We focus on the methods in which residues are represented as clusters of effective spheres. One contact map, denoted as overlap (OV), is based on the overlap of such spheres. Another contact map, named Contacts of Structural Units (CSU), involves the geometry in a different way and, in addition, brings chemical considerations into account. We develop a variant of the CSU approach in which we also incorporate Coulombic effects such as formation of the ionic bridges and destabilization of possible links through repulsion. In this way, the most essential and well defined contacts are identified. The resulting residue-residue contact map, dubbed repulsive CSU (rCSU), is more sound in its physico-chemical justification than CSU. It also provides a clear prescription for validity of an inter-residual contact: the number of attractive atomic contacts should be larger than the number of repulsive ones — a feature that is not present in CSU. However, both of these maps do not correlate well with the experimental data on protein stretching. Thus, we propose to use rCSU together with the OV map. We find that the combined map, denoted as OV+rCSU, performs better than OV. In most situations, OV and OV+rCSU yield comparable folding properties but for some proteins rCSU provides contacts which improve folding in a substantial way. We discuss the likely residue-specificity of the rCSU contacts. Finally, we make comparisons to the recently proposed shadow contact map, which is derived from different principles.

  5. Determination of contact maps in proteins: A combination of structural and chemical approaches.

    PubMed

    Wołek, Karol; Gómez-Sicilia, Àngel; Cieplak, Marek

    2015-12-28

    Contact map selection is a crucial step in structure-based molecular dynamics modelling of proteins. The map can be determined in many different ways. We focus on the methods in which residues are represented as clusters of effective spheres. One contact map, denoted as overlap (OV), is based on the overlap of such spheres. Another contact map, named Contacts of Structural Units (CSU), involves the geometry in a different way and, in addition, brings chemical considerations into account. We develop a variant of the CSU approach in which we also incorporate Coulombic effects such as formation of the ionic bridges and destabilization of possible links through repulsion. In this way, the most essential and well defined contacts are identified. The resulting residue-residue contact map, dubbed repulsive CSU (rCSU), is more sound in its physico-chemical justification than CSU. It also provides a clear prescription for validity of an inter-residual contact: the number of attractive atomic contacts should be larger than the number of repulsive ones - a feature that is not present in CSU. However, both of these maps do not correlate well with the experimental data on protein stretching. Thus, we propose to use rCSU together with the OV map. We find that the combined map, denoted as OV+rCSU, performs better than OV. In most situations, OV and OV+rCSU yield comparable folding properties but for some proteins rCSU provides contacts which improve folding in a substantial way. We discuss the likely residue-specificity of the rCSU contacts. Finally, we make comparisons to the recently proposed shadow contact map, which is derived from different principles. PMID:26723590

  6. Determination of contact maps in proteins: A combination of structural and chemical approaches

    SciTech Connect

    Wołek, Karol; Cieplak, Marek

    2015-12-28

    Contact map selection is a crucial step in structure-based molecular dynamics modelling of proteins. The map can be determined in many different ways. We focus on the methods in which residues are represented as clusters of effective spheres. One contact map, denoted as overlap (OV), is based on the overlap of such spheres. Another contact map, named Contacts of Structural Units (CSU), involves the geometry in a different way and, in addition, brings chemical considerations into account. We develop a variant of the CSU approach in which we also incorporate Coulombic effects such as formation of the ionic bridges and destabilization of possible links through repulsion. In this way, the most essential and well defined contacts are identified. The resulting residue-residue contact map, dubbed repulsive CSU (rCSU), is more sound in its physico-chemical justification than CSU. It also provides a clear prescription for validity of an inter-residual contact: the number of attractive atomic contacts should be larger than the number of repulsive ones — a feature that is not present in CSU. However, both of these maps do not correlate well with the experimental data on protein stretching. Thus, we propose to use rCSU together with the OV map. We find that the combined map, denoted as OV+rCSU, performs better than OV. In most situations, OV and OV+rCSU yield comparable folding properties but for some proteins rCSU provides contacts which improve folding in a substantial way. We discuss the likely residue-specificity of the rCSU contacts. Finally, we make comparisons to the recently proposed shadow contact map, which is derived from different principles.

  7. Α-MMC and MAP30, two ribosome-inactivating proteins extracted from Momordica charantia, induce cell cycle arrest and apoptosis in A549 human lung carcinoma cells.

    PubMed

    Fan, Xiang; He, Lingli; Meng, Yao; Li, Gangrui; Li, Linli; Meng, Yanfa

    2015-05-01

    α‑Momorcharin (α‑MMC) and momordica anti‑human immunodeficiency virus protein (MAP30), produced by Momordica charantia, are ribosome‑inactivating proteins, which have been reported to exert inhibitory effects on cultured tumor cells. In order to further elucidate the functions of these agents, the present study aimed to investigate the effects of α‑MMC and MAP30 on cell viability, the induction of apoptosis, cell cycle arrest, DNA integrity and superoxide dismutase (SOD) activity. α‑MMC and MAP30 were purified from bitter melon seeds using ammonium sulfate precipitation in combination with sulfopropyl (SP)‑sepharose fast flow, sephacryl S‑100 and macro‑Cap‑SP chromatography. MTT, flow cytometric and DNA fragmentation analyses were then used to determine the effects of α‑MMC and MAP30 on human lung adenocarcinoma epithelial A549 cells. The results revealed that A549 cells were sensitive to α‑MMC and MAP30 cytotoxicity assays in vitro. Cell proliferation was significantly suppressed following α‑MMC and MAP30 treatment in a dose‑ and time‑dependent manner; in addition, the results indicated that MAP30 had a more potent anti‑tumor activity compared with that of α‑MMC. Cell cycle arrest in S phase and a significantly increased apoptotic rate were observed following treatment with α‑MMC and MAP30. Furthermore, DNA integrity analysis revealed that the DNA of A549 cells was degraded following treatment with α‑MMC and MAP30 for 48 h. The pyrogallol autoxidation method and nitrotetrazolium blue chloride staining were used to determine SOD activity, the results of which indicated that α‑MMC and MAP30 did not possess SOD activity. In conclusion, the results of the present study indicated that α‑MMC and MAP30 may have potential as novel therapeutic agents for the prophylaxis and treatment of cancer. PMID:25573293

  8. Ex Vivo Signaling Protein Mapping in T Lymphocytes in the Psoriatic Arthritis Joints.

    PubMed

    Fiocco, Ugo; Martini, Veronica; Accordi, Benedetta; Caso, Francesco; Costa, Luisa; Oliviero, Francesca; Scanu, Anna; Felicetti, Mara; Frallonardo, Paola; Facco, Monica; Boso, Daniele; Molena, Beatrice; Zambello, Renato; Ramonda, Roberta; Cozzi, Franco; Scarpa, Raffaele; Basso, Giuseppe; Semenzato, Gianpietro; Dayer, Jean-Michel; Doria, Andrea; Punzi, Leonardo

    2015-11-01

    We assessed signaling protein mapping in total T cells, to analyze the proportions of T regulatory (Treg) and TCD4+ effector (Teff) cell phenotypes, and the respective interleukin 6Rα (IL-6Rα) expression in the inflammatory microenvironment of synovial fluid (SF) of patients with sustained psoriatic arthritis (PsA). Our approach was to measure the IL-6 level in SF using a multiplex bead immunoassay. Reverse-phase protein array was used to assess Janus kinase (JAK) 1 and JAK2, extra-cellular regulated kinase (ERK) 1 and 2, protein kinase Cδ (PKCδ), signal transducer and activator and transcription (STAT) 1, STAT3, and STAT5 phosphoproteins in total T cell lysates from SF of patients with PsA. Frequencies of CD4+IL-17A-F+IL-23+ CD4+ Th cells producing IL-17A and IL-17F (Th17) and CD4+CD25high intracellular forkhead box transcription factor+ (FOXP3+) phenotypes, and the percentage of Treg- and Teff- cells were quantified in SF and matched peripheral blood (PB) of patients with PsA and PB of healthy controls (HC) by flow cytometry. Our results were the following: In PsA SF samples, a coordinate increase of JAK1, ERK1/2, STAT1, STAT3, and STAT5 phosphoproteins was found in total T cells in SF of PsA; where IL-6 levels were higher than in PB from HC. Expanded CD4+IL-17A-F+IL-23+ Th17, CD4+ CD25- Teff- and CD4+CD25(high) FoxP3+Treg subsets, showing similar levels of enhanced IL-6Rδ expression, were confined to PsA joints. In our studies, the transcriptional network profile identified by ex vivo signaling protein mapping in T lymphocytes in PsA joints revealed the complex interplay between IL-1, IL-6, and IL-23 signaling and differentiation of Th17 cells and CD4+Tregs in sustained joint inflammation in PsA. PMID:26523057

  9. Spm1, a stress-activated MAP kinase that regulates morphogenesis in S.pombe.

    PubMed Central

    Zaitsevskaya-Carter, T; Cooper, J A

    1997-01-01

    A gene encoding a novel MAP kinase family member, Spm1, was isolated from the fission yeast Schizosaccharomyces pombe. Overproduction of Spm1 inhibits proliferation. Disruption of the spm1+ gene interferes with cell separation and morphogenesis. Under conditions of nutrient limitation, hypertonic stress or elevated temperature, spm1 delta cells grow as short branched filaments in which the cell walls and septa are thickened, suggesting defects in polarized growth and cell wall remodeling. At high osmolarity, spm1 delta cells fail to form colonies. The Spm1 protein is tyrosine phosphorylated and activated in response to osmotic and heat stress, consistent with a role for Spm1 in adaptation to these conditions. Two other S.pombe MAP kinases are known, Spk1, required for sexual differentiation and sporulation, and Spc1/Sty1/Phh1, which is activated in hypertonic conditions. However, the distinctive features of the spm1 delta mutant phenotype and direct biochemical assays suggest that Spm1 does not lie on other known MAP kinase pathways. Our results demonstrate the existence of a new MAP kinase pathway that regulates cell wall remodeling and cytokinesis in response to environmental stresses. PMID:9135147

  10. Mapping of plumage colour and blood protein loci on the microsatellite linkage map of the Japanese quail.

    PubMed

    Miwa, M; Inoue-Murayama, M; Kayang, B B; Vignal, A; Minvielle, F; Monvoisin, J L; Takahashi, H; Ito, S

    2005-10-01

    The objective of this work was to map classical markers (plumage colours and blood proteins) on the microsatellite linkage map of the Japanese quail (Coturnix japonica). The segregation data on two plumage colours and three blood proteins were obtained from 25 three-generation families (193 F2 birds). Linkage analysis was carried out for these five classical markers and 80 microsatellite markers. A total of 15 linkage groups that included the five classical loci and 69 of the 80 microsatellite markers were constructed. Using the BLAST homology search against the chicken genome sequence, three quail linkage groups, QL8, QL10 and QL13, were suggested to be homologous to chicken chromosomes GGA9, GGA20 and GGA24, respectively. Two plumage colour loci, black at hatch (Bh) and yellow (Y), and the three blood protein loci, transferrin (Tf), haemoglobin (Hb-1) and prealbumin-1 (Pa-1), were assigned to CJA01, QL10, QL8, CJA14 and QL13, respectively. PMID:16167982

  11. Total Cellular RNA Modulates Protein Activity.

    PubMed

    Majumder, Subhabrata; DeMott, Christopher M; Reverdatto, Sergey; Burz, David S; Shekhtman, Alexander

    2016-08-16

    RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states. PMID:27456029

  12. MAP kinase cascades: scaffolding signal specificity.

    PubMed

    van Drogen, Frank; Peter, Matthias

    2002-01-22

    Scaffold proteins organize many MAP kinase pathways by interacting with several components of these cascades. Recent studies suggest that scaffold proteins provide local activation platforms that contribute to signal specificity by insulating different MAP kinase pathways. PMID:11818078

  13. Mapping protein abundance patterns in the brain using voxelation combined with liquid chromatography and mass spectrometry

    SciTech Connect

    Petyuk, Vladislav A.; Qian, Weijun; Smith, Richard D.; Smith, Desmond J.

    2010-02-01

    Voxelation creates expression atlases by high-throughput analysis of spatially registered cubes or voxels harvested from the brain. The modality independence of voxelation allows a variety of bioanalytical techniques to be used to map abundance. Protein expression patterns in the brain can be obtained using liquid chromatography (LC) combined with mass spectrometry (MS). Here we describe the methodology of voxelation as it pertains particularly to LC-MS proteomic analysis: sample preparation, instrumental set up and analysis, peptide identification and protein relative abundance quantitation. We also briefly describe some of the advantages, limitations and insights into the brain that can be obtained using combined proteomic and transcriptomic maps

  14. Synthetic Physical Interactions Map Kinetochore-Checkpoint Activation Regions

    PubMed Central

    Ólafsson, Guðjón; Thorpe, Peter H.

    2016-01-01

    The spindle assembly checkpoint (SAC) is a key mechanism to regulate the timing of mitosis and ensure that chromosomes are correctly segregated to daughter cells. The recruitment of the Mad1 and Mad2 proteins to the kinetochore is normally necessary for SAC activation. This recruitment is coordinated by the SAC kinase Mps1, which phosphorylates residues at the kinetochore to facilitate binding of Bub1, Bub3, Mad1, and Mad2. There is evidence that the essential function of Mps1 is to direct recruitment of Mad1/2. To test this model, we have systematically recruited Mad1, Mad2, and Mps1 to most proteins in the yeast kinetochore, and find that, while Mps1 is sufficient for checkpoint activation, recruitment of either Mad1 or Mad2 is not. These data indicate an important role for Mps1 phosphorylation in SAC activation, beyond the direct recruitment of Mad1 and Mad2. PMID:27280788

  15. Synthetic Physical Interactions Map Kinetochore-Checkpoint Activation Regions.

    PubMed

    Ólafsson, Guðjón; Thorpe, Peter H

    2016-01-01

    The spindle assembly checkpoint (SAC) is a key mechanism to regulate the timing of mitosis and ensure that chromosomes are correctly segregated to daughter cells. The recruitment of the Mad1 and Mad2 proteins to the kinetochore is normally necessary for SAC activation. This recruitment is coordinated by the SAC kinase Mps1, which phosphorylates residues at the kinetochore to facilitate binding of Bub1, Bub3, Mad1, and Mad2. There is evidence that the essential function of Mps1 is to direct recruitment of Mad1/2. To test this model, we have systematically recruited Mad1, Mad2, and Mps1 to most proteins in the yeast kinetochore, and find that, while Mps1 is sufficient for checkpoint activation, recruitment of either Mad1 or Mad2 is not. These data indicate an important role for Mps1 phosphorylation in SAC activation, beyond the direct recruitment of Mad1 and Mad2. PMID:27280788

  16. PDB2Graph: A toolbox for identifying critical amino acids map in proteins based on graph theory.

    PubMed

    Niknam, Niloofar; Khakzad, Hamed; Arab, Seyed Shahriar; Naderi-Manesh, Hossein

    2016-05-01

    The integrative and cooperative nature of protein structure involves the assessment of topological and global features of constituent parts. Network concept takes complete advantage of both of these properties in the analysis concomitantly. High compatibility to structural concepts or physicochemical properties in addition to exploiting a remarkable simplification in the system has made network an ideal tool to explore biological systems. There are numerous examples in which different protein structural and functional characteristics have been clarified by the network approach. Here, we present an interactive and user-friendly Matlab-based toolbox, PDB2Graph, devoted to protein structure network construction, visualization, and analysis. Moreover, PDB2Graph is an appropriate tool for identifying critical nodes involved in protein structural robustness and function based on centrality indices. It maps critical amino acids in protein networks and can greatly aid structural biologists in selecting proper amino acid candidates for manipulating protein structures in a more reasonable and rational manner. To introduce the capability and efficiency of PDB2Graph in detail, the structural modification of Calmodulin through allosteric binding of Ca(2+) is considered. In addition, a mutational analysis for three well-identified model proteins including Phage T4 lysozyme, Barnase and Ribonuclease HI, was performed to inspect the influence of mutating important central residues on protein activity. PMID:27043857

  17. A New MAP Kinase Protein Involved in Estradiol-Stimulated Reproduction of the Helminth Parasite Taenia crassiceps

    PubMed Central

    Escobedo, Galileo; Soldevila, Gloria; Ortega-Pierres, Guadalupe; Chávez-Ríos, Jesús Ramsés; Nava, Karen; Fonseca-Liñán, Rocío; López-Griego, Lorena; Hallal-Calleros, Claudia; Ostoa-Saloma, Pedro; Morales-Montor, Jorge

    2010-01-01

    MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17β-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17β-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design. PMID:20145710

  18. Translational Control of Myelin Basic Protein Expression by ERK2 MAP Kinase Regulates Timely Remyelination in the Adult Brain

    PubMed Central

    Michel, Kelly; Zhao, Tianna; Karl, Molly; Lewis, Katherine

    2015-01-01

    Successful myelin repair in the adult CNS requires the robust and timely production of myelin proteins to generate new myelin sheaths. The underlying regulatory mechanisms and complex molecular basis of myelin regeneration, however, remain poorly understood. Here, we investigate the role of ERK MAP kinase signaling in this process. Conditional deletion of Erk2 from cells of the oligodendrocyte lineage resulted in delayed remyelination following demyelinating injury to the adult mouse corpus callosum. The delayed repair occurred as a result of a specific deficit in the translation of the major myelin protein, MBP. In the absence of ERK2, activation of the ribosomal protein S6 kinase (p70S6K) and its downstream target, ribosomal protein S6 (S6RP), was impaired at a critical time when premyelinating oligodendrocytes were transitioning to mature cells capable of generating new myelin sheaths. Thus, we have described an important link between the ERK MAP kinase signaling cascade and the translational machinery specifically in remyelinating oligodendrocytes in vivo. These results suggest an important role for ERK2 in the translational control of MBP, a myelin protein that appears critical for ensuring the timely generation of new myelin sheaths following demyelinating injury in the adult CNS. PMID:25995471

  19. Translational control of myelin basic protein expression by ERK2 MAP kinase regulates timely remyelination in the adult brain.

    PubMed

    Michel, Kelly; Zhao, Tianna; Karl, Molly; Lewis, Katherine; Fyffe-Maricich, Sharyl L

    2015-05-20

    Successful myelin repair in the adult CNS requires the robust and timely production of myelin proteins to generate new myelin sheaths. The underlying regulatory mechanisms and complex molecular basis of myelin regeneration, however, remain poorly understood. Here, we investigate the role of ERK MAP kinase signaling in this process. Conditional deletion of Erk2 from cells of the oligodendrocyte lineage resulted in delayed remyelination following demyelinating injury to the adult mouse corpus callosum. The delayed repair occurred as a result of a specific deficit in the translation of the major myelin protein, MBP. In the absence of ERK2, activation of the ribosomal protein S6 kinase (p70S6K) and its downstream target, ribosomal protein S6 (S6RP), was impaired at a critical time when premyelinating oligodendrocytes were transitioning to mature cells capable of generating new myelin sheaths. Thus, we have described an important link between the ERK MAP kinase signaling cascade and the translational machinery specifically in remyelinating oligodendrocytes in vivo. These results suggest an important role for ERK2 in the translational control of MBP, a myelin protein that appears critical for ensuring the timely generation of new myelin sheaths following demyelinating injury in the adult CNS. PMID:25995471

  20. Map and Compass Skills for the Elementary School. Instructional Activities Series IA/E-9.

    ERIC Educational Resources Information Center

    Larkin, Robert P.; Grogger, Paul K.

    Twenty activities are described that can be used to develop map and compass skills in elementary grades. The activities range from simple, beginners' projects to more complex tasks as students acquire more skills. Most can be carried out in the classroom, schoolyard, or local neighborhood. Map activities include drawing maps of the classroom,…

  1. De Novo Construction of Redox Active Proteins.

    PubMed

    Moser, C C; Sheehan, M M; Ennist, N M; Kodali, G; Bialas, C; Englander, M T; Discher, B M; Dutton, P L

    2016-01-01

    Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways. PMID:27586341

  2. M2SG: mapping human disease-related genetic variants to protein sequences and genomic loci

    PubMed Central

    Ji, Renkai; Cong, Qian; Li, Wenlin; Grishin, Nick V.

    2013-01-01

    Summary: Online Mendelian Inheritance in Man (OMIM) is a manually curated compendium of human genetic variants and the corresponding phenotypes, mostly human diseases. Instead of directly documenting the native sequences for gene entries, OMIM links its entries to protein and DNA sequences in other databases. However, because of the existence of gene isoforms and errors in OMIM records, mapping a specific OMIM mutation to its corresponding protein sequence is not trivial. Combining computer programs and extensive manual curation of OMIM full-text descriptions and original literature, we mapped 98% of OMIM amino acid substitutions (AASs) and all SwissProt Variant (SwissVar) disease-related AASs to reference sequences and confidently mapped 99.96% of all AASs to the genomic loci. Based on the results, we developed an online database and interactive web server (M2SG) to (i) retrieve the mapped OMIM and SwissVar variants for a given protein sequence; and (ii) obtain related proteins and mutations for an input disease phenotype. This database will be useful for analyzing sequences, understanding the effect of mutations, identifying important genetic variations and designing experiments on a protein of interest. Availability and implementation: The database and web server are freely available at http://prodata.swmed.edu/M2S/mut2seq.cgi. Contact: grishin@chop.swmed.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24002112

  3. Synaptic Vesicle Proteins and Active Zone Plasticity

    PubMed Central

    Kittel, Robert J.; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention. PMID:27148040

  4. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

    PubMed Central

    Kohansal-Nodehi, Mahdokht; Chua, John JE; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. DOI: http://dx.doi.org/10.7554/eLife.14530.001 PMID:27115346

  5. FTMAP: extended protein mapping with user-selected probe molecules

    PubMed Central

    Ngan, Chi Ho; Bohnuud, Tanggis; Mottarella, Scott E.; Beglov, Dmitri; Villar, Elizabeth A.; Hall, David R.; Kozakov, Dima; Vajda, Sandor

    2012-01-01

    Binding hot spots, protein sites with high-binding affinity, can be identified using X-ray crystallography or NMR by screening libraries of small organic molecules that tend to cluster at such regions. FTMAP, a direct computational analog of the experimental screening approaches, globally samples the surface of a target protein using small organic molecules as probes, finds favorable positions, clusters the conformations and ranks the clusters on the basis of the average energy. The regions that bind several probe clusters predict the binding hot spots, in good agreement with experimental results. Small molecules discovered by fragment-based approaches to drug design also bind at the hot spot regions. To identify such molecules and their most likely bound positions, we extend the functionality of FTMAP (http://ftmap.bu.edu/param) to accept any small molecule as an additional probe. In its updated form, FTMAP identifies the hot spots based on a standard set of probes, and for each additional probe shows representative structures of nearby low energy clusters. This approach helps to predict bound poses of the user-selected molecules, detects if a compound is not likely to bind in the hot spot region, and provides input for the design of larger ligands. PMID:22589414

  6. Human uroporphyrinogen III synthase: NMR-based mapping of the active site.

    PubMed

    Cunha, Luis; Kuti, Miklos; Bishop, David F; Mezei, Mihaly; Zeng, Lei; Zhou, Ming-Ming; Desnick, Robert J

    2008-05-01

    Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex. PMID:18004775

  7. Phosphorylation of MAP kinase-like proteins mediate the response of the halotolerant alga Dunaliella viridis to hypertonic shock.

    PubMed

    Jiménez, Carlos; Berl, Tomas; Rivard, Christopher J; Edelstein, Charles L; Capasso, Juan M

    2004-02-01

    The microalga Dunaliella viridis has the ability to adapt to a variety of environmental stresses including osmotic and thermal shocks, UV irradiation and nitrogen starvation. Lacking a rigid cell wall, Dunaliella provides an excellent model to study stress signaling in eukaryotic unicellular organisms. When exposed to hyperosmotic stress, UV irradiation or high temperature, a 57-kDa protein is recognized by antibodies specific to mammalian p38, to its yeast homologue Hog1, and to the phospho-p38 MAP kinase motif. This 57-kDa protein appears to be both up-regulated and phosphorylated. Three other proteins (50, 45, 43 kDa) were transiently phosphorylated under stress conditions as detected with an antibody specific to the mammalian phospho c-Jun N-terminal kinase (JNK) motif. Treatment with specific inhibitors of p38 MAP kinase (SB203580) and JNK (SP600125) activities markedly impaired the adaptation of Dunaliella to osmotic stress. From an evolutionary standpoint, these data strongly suggest that MAP kinase signaling pathways, other than ERK, were already operating in the common ancestor of plant and animal kingdoms, probably as early as 1400 million years ago. PMID:14741745

  8. Mapping fast protein folding with multiple-site fluorescent probes

    PubMed Central

    Prigozhin, Maxim B.; Chao, Shu-Han; Sukenik, Shahar; Pogorelov, Taras V.; Gruebele, Martin

    2015-01-01

    Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein λ6–85 by engineering into it three fluorescent tryptophan–tyrosine contact probes. The probes report on distances between three different helix pairs: 1–2, 1–3, and 3–2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1–3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same “slow” and “fast” distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1–2 and 3–2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test. PMID:26080403

  9. Mapping.

    ERIC Educational Resources Information Center

    Kinney, Douglas M.; McIntosh, Willard L.

    1979-01-01

    The area of geological mapping in the United States in 1978 increased greatly over that reported in 1977; state geological maps were added for California, Idaho, Nevada, and Alaska last year. (Author/BB)

  10. Small glutamine-rich protein/viral protein U–binding protein is a novel cochaperone that affects heat shock protein 70 activity

    PubMed Central

    Angeletti, Peter C.; Walker, Doriann; Panganiban, Antonito T.

    2002-01-01

    Molecular chaperone complexes containing heat shock protein (Hsp) 70 and Hsp90 are regulated by cochaperones, including a subclass of regulators, such as Hsp70 interacting protein (Hip), C-terminus of Hsp70 interacting protein (CHIP), and Hsp70-Hsp90 organizing factor (Hop), that contain tetratricopeptide repeats (TPRs), where Hsp70 refers to Hsp70 and its nearly identical constitutive counterpart, Hsc70, together. These proteins interact with the Hsp70 to regulate adenosine triphosphatase (ATPase) and folding activities or to generate the chaperone complex. Here we provide evidence that small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) is a cochaperone that negatively regulates Hsp70. By “Far-Western” and pull-down assays, SGT/UBP was shown to interact directly with Hsp70 and weakly with Hsp90. The interaction of SGT/UBP with both these protein chaperones was mapped to 3 TPRs in SGT/UBP (amino acids 95–195) that are flanked by charged residues. Moreover, SGT/UBP caused an approximately 30% reduction in both the intrinsic ATPase activity of Hsc70 and the ability of Hsc70 to refold denatured luciferase in vitro. This negative effect of SGT/UBP on Hsc70 is similar in magnitude to that observed for the cochaperone CHIP. A role for SGT/UBP in protein folding is also supported by evidence that a yeast strain containing a deletion in the yeast homolog to SGT/UBP (ΔSGT/UBP) displays a 50-fold reduction in recovery from heat shock compared with the wild type parent. Together, these results are consistent with a regulatory role for SGT/UBP in the chaperone complex. PMID:12482202

  11. Detection of protein C activation in humans.

    PubMed Central

    Bauer, K A; Kass, B L; Beeler, D L; Rosenberg, R D

    1984-01-01

    We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the

  12. Molecular Cytogenetic Mapping of Chromosomal Fragments and Immunostaining of Kinetochore Proteins in Beta

    PubMed Central

    Dechyeva, Daryna; Schmidt, Thomas

    2009-01-01

    By comparative multicolor FISH, we have physically mapped small chromosome fragments in the sugar beet addition lines PRO1 and PAT2 and analyzed the distribution of repetitive DNA families in species of the section Procumbentes of the genus Beta. Six repetitive probes were applied, including genotype-specific probes—satellites pTS4.1, pTS5, and pRp34 and a dispersed repeat pAp4, the telomere (TTTAGGG)n, and the conserved 18S-5.8S-25S rRNA genes. Pachytene-FISH analysis of the native centromere organization allowed proposing the origin of PRO1 and PAT2 fragments. Comparative analysis of the repetitive DNA distribution and organization in the wild beet and in the addition lines allowed the development of a physical model of the chromosomal fragments. Immunostaining revealed that the PRO1 chromosome fragment binds α-tubulin and the serine 10-phosphorylated histone H3 specific for the active centromere. This is the first experimental detection of the kinetochore proteins in Beta showing their active involvement in chromosome segregation in mitosis. PMID:19911065

  13. Robust Diffeomorphic Mapping via Geodesically Controlled Active Shapes

    PubMed Central

    Tward, Daniel J.; Ma, Jun; Miller, Michael I.; Younes, Laurent

    2013-01-01

    This paper presents recent advances in the use of diffeomorphic active shapes which incorporate the conservation laws of large deformation diffeomorphic metric mapping. The equations of evolution satisfying the conservation law are geodesics under the diffeomorphism metric and therefore termed geodesically controlled diffeomorphic active shapes (GDAS). Our principal application in this paper is on robust diffeomorphic mapping methods based on parameterized surface representations of subcortical template structures. Our parametrization of the GDAS evolution is via the initial momentum representation in the tangent space of the template surface. The dimension of this representation is constrained using principal component analysis generated from training samples. In this work, we seek to use template surfaces to generate segmentations of the hippocampus with three data attachment terms: surface matching, landmark matching, and inside-outside modeling from grayscale T1 MR imaging data. This is formulated as an energy minimization problem, where energy describes shape variability and data attachment accuracy, and we derive a variational solution. A gradient descent strategy is employed in the numerical optimization. For the landmark matching case, we demonstrate the robustness of this algorithm as applied to the workflow of a large neuroanatomical study by comparing to an existing diffeomorphic landmark matching algorithm. PMID:23690757

  14. A tractable genotype–phenotype map modelling the self-assembly of protein quaternary structure

    PubMed Central

    Greenbury, Sam F.; Johnston, Iain G.; Louis, Ard A.; Ahnert, Sebastian E.

    2014-01-01

    The mapping between biological genotypes and phenotypes is central to the study of biological evolution. Here, we introduce a rich, intuitive and biologically realistic genotype–phenotype (GP) map that serves as a model of self-assembling biological structures, such as protein complexes, and remains computationally and analytically tractable. Our GP map arises naturally from the self-assembly of polyomino structures on a two-dimensional lattice and exhibits a number of properties: redundancy (genotypes vastly outnumber phenotypes), phenotype bias (genotypic redundancy varies greatly between phenotypes), genotype component disconnectivity (phenotypes consist of disconnected mutational networks) and shape space covering (most phenotypes can be reached in a small number of mutations). We also show that the mutational robustness of phenotypes scales very roughly logarithmically with phenotype redundancy and is positively correlated with phenotypic evolvability. Although our GP map describes the assembly of disconnected objects, it shares many properties with other popular GP maps for connected units, such as models for RNA secondary structure or the hydrophobic-polar (HP) lattice model for protein tertiary structure. The remarkable fact that these important properties similarly emerge from such different models suggests the possibility that universal features underlie a much wider class of biologically realistic GP maps. PMID:24718456

  15. Discovering a "True" Map of the World--Learning Activities.

    ERIC Educational Resources Information Center

    Hantula, James

    "True" maps of the world, as seen from the perspective of the time in which they were produced, remain an ethnocentric visual language in modern times. Students can gain insight into such "true" maps by studying maps produced in the great traditions of the West and East. Teachers can determine a map's appropriateness by identifying its title,…

  16. Mapping of the Regions Involved in Homotypic Interactions of Tula Hantavirus N Protein

    PubMed Central

    Kaukinen, Pasi; Vaheri, Antti; Plyusnin, Alexander

    2003-01-01

    Hantavirus nucleocapsid (N) protein has been suggested to form homodimers and homotrimers that are further integrated into the nucleocapsid filaments around the viral RNA. Here we report detailed mapping of the regions involved in the homotypic N protein interactions in Tula hantavirus (TULV). Peptide scan screening was used to define the interaction regions, and the mammalian two-hybrid assay was used for the functional analysis of N protein mutants. To study linear regions responsible for N protein interaction(s), we used peptide scanning in which N peptides synthesized on membranes recognize recombinant TULV N protein. The data showed that the N protein bound to membrane-bound peptides comprising amino acids 13 to 30 and 41 to 57 in the N-terminal part and 340 to 379, 391 to 407, and 410 to 419 in the C-terminal part of the molecule. Further mapping of the interaction regions by alanine scanning indicated the importance of basic amino acids along the N protein and especially asparagine-394, histidine-395, and phenyalanine-396 in forming the binding interface. Analysis of truncated mutants in the mammalian two-hybrid assay showed that N-terminal amino acids 1 to 43 are involved in and C-terminal amino acids 393 to 398 (VNHFHL) are absolutely crucial for the homotypic interactions. Furthermore, our data suggested a tail-to-tail and head-to-head binding scheme for the N proteins. PMID:14512541

  17. Experimental Approach to Controllably Vary Protein Oxidation While Minimizing Electrode Adsorption for Boron-Doped Diamond Electrochemical Surface Mapping Applications

    SciTech Connect

    McClintock, Carlee; Hettich, Robert {Bob} L

    2013-01-01

    Oxidative protein surface mapping has become a powerful approach for measuring the solvent accessibility of folded protein structures. A variety of techniques exist for generating the key reagent hydroxyl radicals for these measurements; however, many of these approaches require use of radioactive sources or caustic oxidizing chemicals. The purpose of this research was to evaluate and optimize the use of boron-doped diamond (BDD) electrochemistry as a highly accessible tool for producing hydroxyl radicals as a means to induce a controllable level of oxidation on a range of intact proteins. These experiments utilize a relatively high flow rates to reduce protein residence time inside the electrochemical flow chamber, along with a unique cell activation approach to improve control over the intact protein oxidation yield. Studies were conducted to evaluate the level of protein adsorption onto the electrode surface. This report demonstrates a robust protocol for the use of BDD electrochemistry and high performance LC-MS/MS as a high-throughput experimental pipeline for probing higher order protein structure, and illustrates how it is complementary to predictive computational modeling efforts.

  18. LOCALIZATION, FERTILITY INHIBITION, AND EPITOPE MAPS USING ANTIBODIES TO THE SPERM PROTEIN SP22

    EPA Science Inventory

    LOCALIZATION, FERTILITY INHIBITION, AND EPITOPE MAPS USING ANTIBODIES TO THE SPERM PROTEIN SP22. GR Klinefelter1, JE Welch*1, HDM Moore*2, K Bobseine*1, J Suarez*1 ,N Roberts*1 ,R Zucker *1 1U.S. EPA, NHEERL, Reproductive Toxicology Division, RTP, NC and 2University of Sheffield...

  19. Crowding Activates Heat Shock Protein 90.

    PubMed

    Halpin, Jackson C; Huang, Bin; Sun, Ming; Street, Timothy O

    2016-03-18

    Hsp90 is a dimeric ATP-dependent chaperone involved in the folding, maturation, and activation of diverse target proteins. Extensive in vitro structural analysis has led to a working model of Hsp90's ATP-driven conformational cycle. An implicit assumption is that dilute experimental conditions do not significantly perturb Hsp90 structure and function. However, Hsp90 undergoes a dramatic open/closed conformational change, which raises the possibility that this assumption may not be valid for this chaperone. Indeed, here we show that the ATPase activity of Hsp90 is highly sensitive to molecular crowding, whereas the ATPase activities of Hsp60 and Hsp70 chaperones are insensitive to crowding conditions. Polymer crowders activate Hsp90 in a non-saturable manner, with increasing efficacy at increasing concentration. Crowders exhibit a non-linear relationship between their radius of gyration and the extent to which they activate Hsp90. This experimental relationship can be qualitatively recapitulated with simple structure-based volume calculations comparing open/closed configurations of Hsp90. Thermodynamic analysis indicates that crowding activation of Hsp90 is entropically driven, which is consistent with a model in which excluded volume provides a driving force that favors the closed active state of Hsp90. Multiple Hsp90 homologs are activated by crowders, with the endoplasmic reticulum-specific Hsp90, Grp94, exhibiting the highest sensitivity. Finally, we find that crowding activation works by a different mechanism than co-chaperone activation and that these mechanisms are independent. We hypothesize that Hsp90 has a higher intrinsic activity in the cell than in vitro. PMID:26797120

  20. Role of emergent neural activity in visual map development.

    PubMed

    Ackman, James B; Crair, Michael C

    2014-02-01

    The initial structural and functional development of visual circuits in reptiles, birds, and mammals happens independent of sensory experience. After eye opening, visual experience further refines and elaborates circuits that are critical for normal visual function. Innate genetic programs that code for gradients of molecules provide gross positional information for developing nerve cells, yet much of the cytoarchitectural complexity and synaptogenesis of neurons depends on calcium influx, neurotransmitter release, and neural activity before the onset of vision. In fact, specific spatiotemporal patterns of neural activity, or 'retinal waves', emerge amidst the development of the earliest connections made between excitable cells in the developing eye. These patterns of spontaneous activity, which have been observed in all amniote retinae examined to date, may be an evolved adaptation for species with long gestational periods before the onset of functional vision, imparting an informational robustness and redundancy to guide development of visual maps across the nervous system. Recent experiments indicate that retinal waves play a crucial role in the development of interconnections between different parts of the visual system, suggesting that these spontaneous patterns serve as a template-matching mechanism to prepare higher-order visually associative circuits for the onset of visuomotor learning and behavior. Key questions for future studies include determining the exact sources and nature of spontaneous activity during development, characterizing the interactions between neural activity and transcriptional gene regulation, and understanding the extent of circuit connectivity governed by retinal waves within and between sensory-motor systems. PMID:24492092

  1. A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells

    PubMed Central

    Wang, Xiao Min; Zhai, Ye; Ferrell, James E.

    1997-01-01

    The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole—the chromosomes decondensed and the nuclear envelope re-formed—whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells. PMID:9128253

  2. Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes.

    PubMed

    Jin, Ya; Yuan, Qi; Zhang, Jun; Manabe, Takashi; Tan, Wen

    2015-09-01

    Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an "overlap score," (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 "overlap factors," (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells. PMID:26031785

  3. Immunogenicity and reactivity of novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and conserved MAP1156 proteins with sera from experimentally and naturally infected animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s Disease (JD) in ruminants. Development of genetic tools and completion of the MAP genome sequencing project expanded opportunities for antigen discovery. In this study, we determined the seroreactivity of two proteins encoded for at th...

  4. [Peptide mapping of the major proteins in 2 strains of influenza virus].

    PubMed

    Savich, I M; Gerasimova, L M; Mertvetsov, N P

    1985-01-01

    The protein composition of two influenza A virus strains from different serological groups was studied. For preliminary separation of envelope and core proteins the virus was treated with nonionic detergent Triton X-100 solution followed by chromatography in ultragel AcA 34. After their separation by disk electrophoresis and treatment with trypsin they were labeled with tritium and peptide analysis was carried out. Differences in peptide maps between the appropriate light and heavy chains of hemagglutinins of the strains under study were demonstrated. Differences in the composition of nucleoprotein and matrix protein were insignificant. PMID:3922120

  5. Fine Mapping of the Interaction between C4b-Binding Protein and Outer Membrane Proteins LigA and LigB of Pathogenic Leptospira interrogans

    PubMed Central

    Breda, Leandro C. D.; Hsieh, Ching-Lin; Castiblanco Valencia, Mónica M.; da Silva, Ludmila B.; Barbosa, Angela S.; Blom, Anna M.; Yung-Fu, Chang; Isaac, Lourdes

    2015-01-01

    The complement system consists of more than 40 proteins that participate in the inflammatory response and in pathogen killing. Complement inhibitors are necessary to avoid the excessive consumption and activation of this system on host cells. Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Pathogenic leptospires are able to escape from complement activation by binding to host complement inhibitors Factor H [FH] and C4b-binding protein (C4BP) while non-pathogenic leptospires are rapidly killed in the presence of fresh serum. In this study, we demonstrate that complement control protein domains (CCP) 7 and 8 of C4BP α-chain interact with the outer membrane proteins LcpA, LigA and LigB from the pathogenic leptospire L. interrogans. The interaction between C4BP and LcpA, LigA and LigB is sensitive to ionic strength and inhibited by heparin. We fine mapped the LigA and LigB domains involved in its binding to C4BP and heparin and found that both interactions are mediated through the bacterial immunoglobulin-like (Big) domains 7 and 8 (LigA7-8 and LigB7-8) of both LigA and LigB and also through LigB9-10. Therefore, C4BP and heparin may share the same binding sites on Lig proteins. PMID:26517116

  6. Epitope Mapping of Avian Influenza M2e Protein: Different Species Recognise Various Epitopes

    PubMed Central

    Hasan, Noor Haliza; Ignjatovic, Jagoda; Tarigan, Simson; Peaston, Anne; Hemmatzadeh, Farhid

    2016-01-01

    A common approach for developing diagnostic tests for influenza virus detection is the use of mouse or rabbit monoclonal and/or polyclonal antibodies against a target antigen of the virus. However, comparative mapping of the target antigen using antibodies from different animal sources has not been evaluated before. This is important because identification of antigenic determinants of the target antigen in different species plays a central role to ensure the efficiency of a diagnostic test, such as competitive ELISA or immunohistochemistry-based tests. Interest in the matrix 2 ectodomain (M2e) protein of avian influenza virus (AIV) as a candidate for a universal vaccine and also as a marker for detection of virus infection in vaccinated animals (DIVA) is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs) recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb) and chicken antibodies (cAbs) recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development. PMID:27362795

  7. Heat dissipation guides activation in signaling proteins.

    PubMed

    Weber, Jeffrey K; Shukla, Diwakar; Pande, Vijay S

    2015-08-18

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein-coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  8. Comparing Two Forms of Concept Map Critique Activities to Facilitate Knowledge Integration Processes in Evolution Education

    ERIC Educational Resources Information Center

    Schwendimann, Beat A.; Linn, Marcia C.

    2016-01-01

    Concept map activities often lack a subsequent revision step that facilitates knowledge integration. This study compares two collaborative critique activities using a Knowledge Integration Map (KIM), a form of concept map. Four classes of high school biology students (n?=?81) using an online inquiry-based learning unit on evolution were assigned…

  9. Activation and activities of the p53 tumour suppressor protein

    PubMed Central

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pathways that regulate p53 and the pathways that are induced by p53, as well as their implications for cancer therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11747320

  10. Phosphorylation of the 27-kDa heat shock protein via p38 MAP kinase and MAPKAP kinase in smooth muscle.

    PubMed

    Larsen, J K; Yamboliev, I A; Weber, L A; Gerthoffer, W T

    1997-11-01

    The 27-kDa heat shock protein (HSP27) is expressed in a variety of tissues in the absence of stress and is thought to regulate actin filament dynamics, possibly by a phosphorylation/dephosphorylation mechanism. HSP27 has also been suggested to be involved in contraction of intestinal smooth muscle. We have investigated phosphorylation of HSP27 in airway smooth muscle in response to the muscarinic agonist carbachol. Carbachol increased 32P incorporation into canine tracheal HSP27 and induced a shift in the distribution of charge isoforms on two-dimensional gels to more acidic, phosphorylated forms. The canine HSP27 amino acid sequence includes three serine residues corresponding to sites in human HSP27 known to be phosphorylated by mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2. To determine whether muscarinic receptors are coupled to a "stress response" pathway in smooth muscle culminating in phosphorylation of HSP27, we assayed MAPKAP kinase-2 activity and tyrosine phosphorylation of p38 mitogen-activated protein (MAP) kinase, the enzyme thought to activate MAPKAP kinase-2. Recombinant canine HSP27 expressed in Escherichia coli was a substrate for MAPKAP kinase-2 in vitro as well as a substrate for endogenous smooth muscle HSP27 kinase, which was activated by carbachol. Carbachol also increased tyrosine phosphorylation of p38 MAP kinase. SB-203580, an inhibitor of p38 MAP kinases, reduced activation of endogenous HSP27 kinase activity and blocked the shift in HSP27 charge isoforms to acidic forms. We suggest that HSP27 in airway smooth muscle, in addition to being a stress response protein, is phosphorylated by a receptor-initiated signaling cascade involving muscarinic receptors, tyrosine phosphorylation of p38 MAP kinase, and activation of MAPKAP kinase-2. PMID:9374719

  11. Mapping the potential energy landscape of intrinsically disordered proteins at amino acid resolution.

    PubMed

    Ozenne, Valéry; Schneider, Robert; Yao, Mingxi; Huang, Jie-rong; Salmon, Loïc; Zweckstetter, Markus; Jensen, Malene Ringkjøbing; Blackledge, Martin

    2012-09-12

    Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling. PMID:22901047

  12. Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

    NASA Astrophysics Data System (ADS)

    Yan, Yuetian; Chen, Guodong; Wei, Hui; Huang, Richard Y.-C.; Mo, Jingjie; Rempel, Don L.; Tymiak, Adrienne A.; Gross, Michael L.

    2014-12-01

    Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin (10Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein-protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.

  13. Protein-protein interaction and gene co-expression maps of ARFs and Aux/IAAs in Arabidopsis

    PubMed Central

    Piya, Sarbottam; Shrestha, Sandesh K.; Binder, Brad; Stewart, C. Neal; Hewezi, Tarek

    2014-01-01

    The phytohormone auxin regulates nearly all aspects of plant growth and development. Based on the current model in Arabidopsis thaliana, Auxin/indole-3-acetic acid (Aux/IAA) proteins repress auxin-inducible genes by inhibiting auxin response transcription factors (ARFs). Experimental evidence suggests that heterodimerization between Aux/IAA and ARF proteins are related to their unique biological functions. The objective of this study was to generate the Aux/IAA-ARF protein-protein interaction map using full length sequences and locate the interacting protein pairs to specific gene co-expression networks in order to define tissue-specific responses of the Aux/IAA-ARF interactome. Pairwise interactions between 19 ARFs and 29 Aux/IAAs resulted in the identification of 213 specific interactions of which 79 interactions were previously unknown. The incorporation of co-expression profiles with protein-protein interaction data revealed a strong correlation of gene co-expression for 70% of the ARF-Aux/IAA interacting pairs in at least one tissue/organ, indicative of the biological significance of these interactions. Importantly, ARF4-8 and 19, which were found to interact with almost all Aux-Aux/IAA showed broad co-expression relationships with Aux/IAA genes, thus, formed the central hubs of the co-expression network. Our analyses provide new insights into the biological significance of ARF-Aux/IAA associations in the morphogenesis and development of various plant tissues and organs. PMID:25566309

  14. Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries.

    SciTech Connect

    Kay, B. K.; Castagnoli, L.; Biosciences Division; Univ. of Rome

    2003-01-01

    This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead-based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.

  15. Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries and alanine scanning.

    PubMed

    Kokoszka, Malgorzata E; Kay, Brian K

    2015-01-01

    One avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. Among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry such peptide sequences interact with the target protein in the cell. With the protocols described in this chapter, a laboratory with skills in microbiology, molecular biology, and protein biochemistry can readily identify peptides in the library that bind selectively, and with micromolar affinity, to a given target protein on the time scale of 2 months. To illustrate this approach, we use a library of bacteriophage M13 particles, which display 12-mer combinatorial peptides, to affinity select different peptide ligands for two different targets, the SH3 domain of the human Lyn protein tyrosine kinase and a segment of the yeast serine/threonine protein kinase Cbk1. The binding properties of the selected peptide ligands are then dissected by sequence alignment, Kunkel mutagenesis, and alanine scanning. Finally, the peptide ligands can be used to predict cellular interacting proteins and serve as the starting point for drug discovery. PMID:25616333

  16. Mapping the DNA-binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI.

    PubMed

    Sheldon, Paul J; Busarow, Sara B; Hutchinson, C Richard

    2002-04-01

    Streptomyces antibiotic regulatory proteins (SARPs) constitute a novel family of transcriptional activators that control the expression of several diverse anti-biotic biosynthetic gene clusters. The Streptomyces peucetius DnrI protein, one of only a handful of these proteins yet discovered, controls the biosynthesis of the polyketide antitumour antibiotics daunorubicin and doxorubicin. Recently, comparative analyses have revealed significant similarities among the predicted DNA-binding domains of the SARPs and the C-terminal DNA-binding domain of the OmpR family of regulatory proteins. Using the crystal structure of the OmpR-binding domain as a template, DnrI was mapped by truncation and site-directed mutagenesis. Several highly conserved residues within the N-terminus are crucial for DNA binding and protein function. Tandemly arranged heptameric imperfect repeat sequences are found within the -35 promoter regions of target genes. Substitutions for each nucleotide within the repeats of the dnrG-dpsABCD promoter were performed by site-directed mutagenesis. The mutant promoter fragments were found to have modified binding characteristics in gel mobility shift assays. The spacing between the repeat target sequences is also critical for successful occupation by DnrI and, therefore, competent transcriptional activation of the dnrG-dpsABCD operon. PMID:11972782

  17. In-depth mapping of human testicular and epididymal proteins and their functional association with spermatozoa

    PubMed Central

    LIU, XUEXIA; LIU, FUJUN

    2015-01-01

    The mammalian testis and epididymis are responsible for spermatozoa production and maturation, which contributes to male fertility. Predominantly expressed proteins in the testis and epididymis were suggested to be involved in the key functions or pathways in spermatogenesis and sperm maturation. To further investigate these proteins and their associations with sperm, large protein profiles of human testis and epididymis were mapped. Predominantly-expressed testicular (173) and epididymal (244) secreted proteins were further screened and functionally characterized. Differential expression levels of solute carrier family 2 (facilitated glucose transporter), member 3, solute carrier family 25 (carnitine/acylcarnitine translocase), member 20, WAP-type four-disulfide core domain protein 8 and prostate and testis expressed 1 were validated using western blot and immunohistochemical analyses. The results may provide novel insight into the understanding of testicular and epididymal physiology and function, and facilitate sperm maturation research. PMID:25760095

  18. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  19. Targeted killing of rhabdomyosarcoma cells by a MAP-based human cytolytic fusion protein.

    PubMed

    Brehm, Hannes; Hristodorov, Dmitrij; Pardo, Alessa; Mladenov, Radoslav; Niesen, Judith; Fischer, Rainer; Tur, Mehmet K; Barth, Stefan

    2015-09-01

    The treatment of rhabdomyosarcoma (RMS) is challenging, and the prognosis remains especially poor for high-grade RMS with metastasis. The conventional treatment of RMS is based on multi-agent chemotherapy combined with resection and radiotherapy, which are often marked by low success rate. Alternative therapeutic options include the combination of standard treatments with immunotherapy. We generated a microtubule-associated protein (MAP)-based fully human cytolytic fusion protein (hCFP) targeting the fetal acetylcholine receptor, which is expressed on RMS cells. We were able to express and purify functional scFv35-MAP from Escherichia coli cells. Moreover, we found that scFv35-MAP is rapidly internalized by target cells after binding its receptor, and exhibits specific cytotoxicity toward FL-OH1 and RD cells in vitro. We also confirmed that scFv35-MAP induces apoptosis in FL-OH1 and RD cells. The in vivo potential of scFv35-MAP will need to be considered in further studies. PMID:25888452

  20. AlphaSpace: Fragment-Centric Topographical Mapping To Target Protein–Protein Interaction Interfaces

    PubMed Central

    2016-01-01

    Inhibition of protein–protein interactions (PPIs) is emerging as a promising therapeutic strategy despite the difficulty in targeting such interfaces with drug-like small molecules. PPIs generally feature large and flat binding surfaces as compared to typical drug targets. These features pose a challenge for structural characterization of the surface using geometry-based pocket-detection methods. An attractive mapping strategy—that builds on the principles of fragment-based drug discovery (FBDD)—is to detect the fragment-centric modularity at the protein surface and then characterize the large PPI interface as a set of localized, fragment-targetable interaction regions. Here, we introduce AlphaSpace, a computational analysis tool designed for fragment-centric topographical mapping (FCTM) of PPI interfaces. Our approach uses the alpha sphere construct, a geometric feature of a protein’s Voronoi diagram, to map out concave interaction space at the protein surface. We introduce two new features—alpha-atom and alpha-space—and the concept of the alpha-atom/alpha-space pair to rank pockets for fragment-targetability and to facilitate the evaluation of pocket/fragment complementarity. The resulting high-resolution interfacial map of targetable pocket space can be used to guide the rational design and optimization of small molecule or biomimetic PPI inhibitors. PMID:26225450

  1. Phospholipases as GTPase activity accelerating proteins (GAPs) in plants.

    PubMed

    Pandey, Sona

    2016-05-01

    GTPase activity accelerating proteins (GAPs) are key regulators of the G-protein signaling cycle. By facilitating effective hydrolysis of the GTP bound on Gα proteins, GAPs control the timing and amplitude of the signaling cycle and ascertain the availability of the inactive heterotrimer for the next round of activation. Until very recently, the studies of GAPs in plants were focused exclusively on the regulator of G-protein signaling (RGS) protein. We now show that phospholipase Dα1 (PLDα1) is also a bona fide GAP in plants and together with the RGS protein controls the level of activeprotein. PMID:27124090

  2. Monooxygenase activity of type 3 copper proteins.

    PubMed

    Itoh, Shinobu; Fukuzumi, Shunichi

    2007-07-01

    The molecular mechanism of the monooxygenase (phenolase) activity of type 3 copper proteins has been examined in detail both in the model systems and in the enzymatic systems. The reaction of a side-on peroxo dicopper(II) model compound ( A) and neutral phenols proceeds via a proton-coupled electron-transfer (PCET) mechanism to generate phenoxyl radical species, which collapse each other to give the corresponding C-C coupling dimer products. In this reaction, a bis(mu-oxo)dicopper(III) complex ( B) generated by O-O bond homolysis of A is suggested to be a real active species. On the other hand, the reaction of lithium phenolates (deprotonated form of phenols) with the same side-on peroxo dicopper(II) complex proceeds via an electrophilic aromatic substitution mechanism to give the oxygenated products (catechols). The mechanistic difference between these two systems has been discussed on the basis of the Marcus theory of electron transfer and Hammett analysis. Mechanistic details of the monooxygenase activity of tyrosinase have also been examined using a simplified enzymatic reaction system to demonstrate that the enzymatic reaction mechanism is virtually the same as that of the model reaction, that is, an electrophilic aromatic substitution mechanism. In addition, the monooxygenase activity of the oxygen carrier protein hemocyanin has been explored for the first time by employing urea as an additive in the reaction system. In this case as well, the ortho-hydroxylation of phenols to catechols has been demonstrated to involve the same ionic mechanism. PMID:17461541

  3. Tctex2: a sperm tail surface protein mapping to the t-complex.

    PubMed

    Huw, L Y; Goldsborough, A S; Willison, K; Artzt, K

    1995-07-01

    Transmission ratio distortion (TRD) in mouse t-haplotypes remains the most significant example of meiotic drive in vertebrates. While the underlying mechanism that fuels it is still mysterious, TRD is clearly a complex multigene phenomenon. The characterization of Tctex2 (t-complex testis expressed 2) shows it to be one of several candidates for involvement in TRD. Tctex2 maps to the t-complex and encodes a membrane-associated protein found exclusively on the sperm tail. The t-haplotype form of Tctex2 is aberrant in both the level of its expression and its primary amino acid sequence, but is nonetheless translated and transported to its normal location. The multiple amino acid changes in the t-form make it extremely unlikely that it can function normally and, since it is found on sperm tails, suggest that it may actively interfere with the development of normal gamete function in males. The possible role of Tctex2 in t-complex transmission ratio distortion and sterility is discussed. PMID:7601308

  4. Improving Protocols for Protein Mapping through Proper Comparison to Crystallography Data

    PubMed Central

    Lexa, Katrina W.; Carlson, Heather A.

    2013-01-01

    Computational approaches to fragment-based drug design (FBDD) can complement experiments and facilitate the identification of potential hot spots along the protein surface. However, the evaluation of computational methods for mapping binding sites frequently focuses upon the ability to reproduce crystallographic coordinates to within a low RMSD threshold. This dependency on the deposited coordinate data overlooks the original electron density from the experiment, thus techniques may be developed based upon subjective - or even erroneous - atomic coordinates. This can become a significant drawback in applications to systems where the location of hot spots is unknown. Based on comparison to crystallographic density, we previously showed that mixed-solvent molecular dynamics (MixMD) accurately identifies the active site for HEWL, with acetonitrile as an organic solvent. Here, we concentrated on the influence of protic solvent on simulation and refined the optimal MixMD approach for extrapolation of the method to systems without established sites. Our results establish an accurate approach for comparing simulations to experiment. We have outlined the most efficient strategy for MixMD, based on simulation length and number of runs. The development outlined here makes MixMD a robust method which should prove useful across a broad range of target structures. Lastly, our results with MixMD match experimental data so well that consistency between simulations and density may be a useful way to aid the identification of probes vs waters during the refinement of future MSCS crystallographic structures. PMID:23327200

  5. Improving protocols for protein mapping through proper comparison to crystallography data.

    PubMed

    Lexa, Katrina W; Carlson, Heather A

    2013-02-25

    Computational approaches to fragment-based drug design (FBDD) can complement experiments and facilitate the identification of potential hot spots along the protein surface. However, the evaluation of computational methods for mapping binding sites frequently focuses upon the ability to reproduce crystallographic coordinates to within a low RMSD threshold. This dependency on the deposited coordinate data overlooks the original electron density from the experiment, thus techniques may be developed based upon subjective-or even erroneous-atomic coordinates. This can become a significant drawback in applications to systems where the location of hot spots is unknown. On the basis of comparison to crystallographic density, we previously showed that mixed-solvent molecular dynamics (MixMD) accurately identifies the active site for HEWL, with acetonitrile as an organic solvent. Here, we concentrated on the influence of protic solvent on simulation and refined the optimal MixMD approach for extrapolation of the method to systems without established sites. Our results establish an accurate approach for comparing simulations to experiment. We have outlined the most efficient strategy for MixMD, based on simulation length and number of runs. The development outlined here makes MixMD a robust method which should prove useful across a broad range of target structures. Lastly, our results with MixMD match experimental data so well that consistency between simulations and density may be a useful way to aid the identification of probes vs waters during the refinement of future multiple solvent crystallographic structures. PMID:23327200

  6. Heat dissipation guides activation in signaling proteins

    PubMed Central

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein–coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  7. Protein domain mapping by internal labeling and single particle electron microscopy.

    PubMed

    Ciferri, Claudio; Lander, Gabriel C; Nogales, Eva

    2015-11-01

    In recent years, electron microscopy (EM) and single particle analysis have emerged as essential tools for investigating the architecture of large biological complexes. When high resolution is achievable, crystal structure docking and de-novo modeling allows for precise assignment of individual protein domain sequences. However, the achievable resolution may limit the ability to do so, especially when small or flexible complexes are under study. In such cases, protein labeling has emerged as an important complementary tool to characterize domain architecture and elucidate functional mechanistic details. All labeling strategies proposed to date are either focused on the identification of the position of protein termini or require multi-step labeling strategies, potentially interfering with the final labeling efficiency. Here we describe a strategy for determining the position of internal protein domains within EM maps using a recombinant one-step labeling approach named Efficient Mapping by Internal Labeling (EMIL). EMIL takes advantage of the close spatial proximity of the GFP's N- and C-termini to generate protein chimeras containing an internal GFP at desired locations along the main protein chain. We apply this method to characterize the subunit domain localization of the human Polycomb Repressive Complex 2. PMID:26431894

  8. The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis

    PubMed Central

    Nadarajan, Saravanapriah; Mohideen, Firaz; Tzur, Yonatan B; Ferrandiz, Nuria; Crawley, Oliver; Montoya, Alex; Faull, Peter; Snijders, Ambrosius P; Cutillas, Pedro R; Jambhekar, Ashwini; Blower, Michael D; Martinez-Perez, Enrique; Harper, J Wade; Colaiacovo, Monica P

    2016-01-01

    Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.12039.001 PMID:26920220

  9. Inhibition of Fast Axonal Transport by Pathogenic SOD1 Involves Activation of p38 MAP Kinase

    PubMed Central

    Morfini, Gerardo A.; Bosco, Daryl A.; Brown, Hannah; Gatto, Rodolfo; Kaminska, Agnieszka; Song, Yuyu; Molla, Linda; Baker, Lisa; Marangoni, M. Natalia; Berth, Sarah; Tavassoli, Ehsan; Bagnato, Carolina; Tiwari, Ashutosh; Hayward, Lawrence J.; Pigino, Gustavo F.; Watterson, D. Martin; Huang, Chun-Fang; Banker, Gary; Brown, Robert H.; Brady, Scott T.

    2013-01-01

    Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS. PMID:23776455

  10. Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation.

    PubMed

    Yamano, Koji; Queliconi, Bruno B; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki

    2015-10-16

    Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser(65) by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

  11. Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

    PubMed

    Hay, Debbie L; Walker, Christopher S; Gingell, Joseph J; Ladds, Graham; Reynolds, Christopher A; Poyner, David R

    2016-04-15

    Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine. PMID:27068971

  12. Protein-protein interactions in plant mitogen-activated protein kinase cascades.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2016-02-01

    Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense. However, the molecular mechanisms underlying MAPK cascades are still elusive, due largely to our poor understanding of how they relay the signals. Extensive effort has been devoted to characterization of MAPK-substrate interactions to illustrate phosphorylation-based signaling. The diverse MAPK substrates identified also shed light on how spatiotemporal-specific protein-protein interactions function in distinct MAPK cascade-mediated biological processes. This review surveys various technologies used for characterizing MAPK-substrate interactions and presents case studies of MPK4 and MPK6, highlighting the multiple functions of MAPKs. Mass spectrometry-based approaches in identifying MAPK-interacting proteins are emphasized due to their increasing utility and effectiveness. The potential for using MAPKs and their substrates in enhancing plant stress tolerance is also discussed. PMID:26646897

  13. Monolithic active pixel sensors (MAPS) in a VLSI CMOS technology

    NASA Astrophysics Data System (ADS)

    Turchetta, R.; French, M.; Manolopoulos, S.; Tyndel, M.; Allport, P.; Bates, R.; O'Shea, V.; Hall, G.; Raymond, M.

    2003-03-01

    Monolithic Active Pixel Sensors (MAPS) designed in a standard VLSI CMOS technology have recently been proposed as a compact pixel detector for the detection of high-energy charged particle in vertex/tracking applications. MAPS, also named CMOS sensors, are already extensively used in visible light applications. With respect to other competing imaging technologies, CMOS sensors have several potential advantages in terms of low cost, low power, lower noise at higher speed, random access of pixels which allows windowing of region of interest, ability to integrate several functions on the same chip. This brings altogether to the concept of 'camera-on-a-chip'. In this paper, we review the use of CMOS sensors for particle physics and we analyse their performances in term of the efficiency (fill factor), signal generation, noise, readout speed and sensor area. In most of high-energy physics applications, data reduction is needed in the sensor at an early stage of the data processing before transfer of the data to tape. Because of the large number of pixels, data reduction is needed on the sensor itself or just outside. This brings in stringent requirements on the temporal noise as well as to the sensor uniformity, expressed as a Fixed Pattern Noise (FPN). A pixel architecture with an additional transistor is proposed. This architecture, coupled to correlated double sampling of the signal will allow cancellation of the two dominant noise sources, namely the reset or kTC noise and the FPN. A prototype has been designed in a standard 0.25 μm CMOS technology. It has also a structure for electrical calibration of the sensor. The prototype is functional and detailed tests are under way.

  14. Mitogen Activated Protein Kinase Family Proteins and c-jun Signaling in Injury-induced Schwann Cell Plasticity.

    PubMed

    Lee, Hye Jeong; Shin, Yoon Kyung; Park, Hwan Tae

    2014-06-01

    Schwann cells (SCs) in the peripheral nerves myelinate axons during postnatal development to allow saltatory conduction of nerve impulses. Well-organized structures of myelin sheathes are maintained throughout life unless nerves are insulted. After peripheral nerve injury, unidentified signals from injured nerves drive SC dedifferentiation into an immature state. Dedifferentiated SCs participate in axonal regeneration by producing neurotrophic factors and removing degenerating nerve debris. In this review, we focus on the role of mitogen activated protein kinase family proteins (MAP kinases) in SC dedifferentiation. In addition, we will highlight neuregulin 1 and the transcription factor c-jun as upstream and downstream signals for MAP kinases in SC responses to nerve injury. PMID:24963277

  15. The mapping of yeast's G-protein coupled receptor with an atomic force microscope

    NASA Astrophysics Data System (ADS)

    Takenaka, Musashi; Miyachi, Yusuke; Ishii, Jun; Ogino, Chiaki; Kondo, Akihiko

    2015-03-01

    An atomic force microscope (AFM) can measure the adhesion force between a sample and a cantilever while simultaneously applying a rupture force during the imaging of a sample. An AFM should be useful in targeting specific proteins on a cell surface. The present study proposes the use of an AFM to measure the adhesion force between targeting receptors and their ligands, and to map the targeting receptors. In this study, Ste2p, one of the G protein-coupled receptors (GPCRs), was chosen as the target receptor. The specific force between Ste2p on a yeast cell surface and a cantilever modified with its ligand, α-factor, was measured and found to be approximately 250 pN. In addition, through continuous measuring of the cell surface, a mapping of the receptors on the cell surface could be performed, which indicated the differences in the Ste2p expression levels. Therefore, the proposed AFM system is accurate for cell diagnosis.

  16. Microtubule-associated Proteins 1 (MAP1) Promote Human Immunodeficiency Virus Type I (HIV-1) Intracytoplasmic Routing to the Nucleus

    PubMed Central

    Fernandez, Juliette; Portilho, Débora M.; Danckaert, Anne; Munier, Sandie; Becker, Andreas; Roux, Pascal; Zambo, Anaba; Shorte, Spencer; Jacob, Yves; Vidalain, Pierre-Olivier; Charneau, Pierre; Clavel, François; Arhel, Nathalie J.

    2015-01-01

    After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules. PMID:25505242

  17. Structural analysis and mapping of individual protein complexes by infrared nanospectroscopy

    PubMed Central

    Amenabar, Iban; Poly, Simon; Nuansing, Wiwat; Hubrich, Elmar H.; Govyadinov, Alexander A.; Huth, Florian; Krutokhvostov, Roman; Zhang, Lianbing; Knez, Mato; Heberle, Joachim; Bittner, Alexander M.; Hillenbrand, Rainer

    2013-01-01

    Mid-infrared spectroscopy is a widely used tool for material identification and secondary structure analysis in chemistry, biology and biochemistry. However, the diffraction limit prevents nanoscale protein studies. Here we introduce mapping of protein structure with 30 nm lateral resolution and sensitivity to individual protein complexes by Fourier transform infrared nanospectroscopy (nano-FTIR). We present local broadband spectra of one virus, ferritin complexes, purple membranes and insulin aggregates, which can be interpreted in terms of their α-helical and/or β-sheet structure. Applying nano-FTIR for studying insulin fibrils—a model system widely used in neurodegenerative disease research—we find clear evidence that 3-nm-thin amyloid-like fibrils contain a large amount of α-helical structure. This reveals the surprisingly high level of protein organization in the fibril’s periphery, which might explain why fibrils associate. We envision a wide application potential of nano-FTIR, including cellular receptor in vitro mapping and analysis of proteins within quaternary structures. PMID:24301518

  18. Velocity Mapping of Proteins via Space-Time Image Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Hebert, Benedict; Wiseman, Paul W.

    2004-03-01

    The imaging analogs of fluorescence correlation spectroscopy (FCS), image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS), have proven successful in assessing the degree of aggregation and the membrane density of fluorescently labeled macromolecule clusters, as well as measuring diffusion coefficients and flow speeds within the plasma membrane in living cells. We present a new extension of ICS and ICCS that relies on complete analysis of the temporal and all spatial correlation lags for intensity fluctuations from laser scanning microscopy image series. This new approach allows measurement of diffusion coefficients and velocity vectors (magnitude and direction) for labeled proteins in vivo. By filtering in Fourier space, we can measure the protein transport even in the presence of a large fraction of immobile proteins. We will present computer simulations and fluorescent microsphere sample analysis to demonstrate proof of principle, capabilities and limitations of the technique. We will also show correlation velocity mapping of proteins on living cells. This new method permits us to generate complete transport maps for sub-regions of the basal membrane even if the protein concentration is too high to perform single particle tracking studies.

  19. Mutations in the Microtubule-Associated Protein 1A (Map1a) Gene Cause Purkinje Cell Degeneration

    PubMed Central

    Liu, Ye; Lee, Jeong Woong

    2015-01-01

    The structural microtubule-associated proteins (MAPs) are critical for the organization of neuronal microtubules (MTs). Microtubule-associated protein 1A (MAP1A) is one of the most abundantly expressed MAPs in the mammalian brain. However, its in vivo function remains largely unknown. Here we describe a spontaneous mouse mutation, nm2719, which causes tremors, ataxia, and loss of cerebellar Purkinje neurons in aged homozygous mice. The nm2719 mutation disrupts the Map1a gene. We show that targeted deletion of mouse Map1a gene leads to similar neurodegenerative defects. Before neuron death, Map1a mutant Purkinje cells exhibited abnormal focal swellings of dendritic shafts and disruptions in axon initial segment (AIS) morphology. Furthermore, the MT network was reduced in the somatodendritic and AIS compartments, and both the heavy and light chains of MAP1B, another brain-enriched MAP, was aberrantly distributed in the soma and dendrites of mutant Purkinje cells. MAP1A has been reported to bind to the membrane-associated guanylate kinase (MAGUK) scaffolding proteins, as well as to MTs. Indeed, PSD-93, the MAGUK specifically enriched in Purkinje cells, was reduced in Map1a−/− Purkinje cells. These results demonstrate that MAP1A functions to maintain both the neuronal MT network and the level of PSD-93 in neurons of the mammalian brain. PMID:25788676

  20. Mutagenesis Mapping of the Protein-Protein Interaction Underlying FusB-Type Fusidic Acid Resistance

    PubMed Central

    Cox, Georgina; Edwards, Thomas A.

    2013-01-01

    FusB-type proteins represent the predominant mechanism of resistance to fusidic acid in staphylococci and act by binding to and modulating the function of the drug target (elongation factor G [EF-G]). To gain further insight into this antibiotic resistance mechanism, we sought to identify residues important for the interaction of FusB with EF-G and thereby delineate the binding interface within the FusB–EF-G complex. Replacement with alanine of any one of four conserved residues within the C-terminal domain of FusB (F156, K184, Y187, and F208) abrogated the ability of the protein to confer resistance to fusidic acid; the purified mutant proteins also lost the ability to bind S. aureus EF-G in vitro. E. coli EF-G, which is not ordinarily able to bind FusB-type proteins, was rendered competent for binding to FusB following deletion of a 3-residue tract (529SNP531) from domain IV of the protein. This study has identified key regions of both FusB and EF-G that are important for the interaction between the proteins, findings which corroborate our previous in silico prediction for the architecture of the complex formed between the resistance protein and the drug target (G. Cox, G. S. Thompson, H. T. Jenkins, F. Peske, A. Savelsbergh, M. V. Rodnina, W. Wintermeyer, S. W. Homans, T. A. Edwards, and A. J. O'Neill, Proc. Natl. Acad. Sci. U. S. A. 109:2102-2107, 2012). PMID:23836182

  1. Mutagenesis mapping of the protein-protein interaction underlying FusB-type fusidic acid resistance.

    PubMed

    Cox, Georgina; Edwards, Thomas A; O'Neill, Alex J

    2013-10-01

    FusB-type proteins represent the predominant mechanism of resistance to fusidic acid in staphylococci and act by binding to and modulating the function of the drug target (elongation factor G [EF-G]). To gain further insight into this antibiotic resistance mechanism, we sought to identify residues important for the interaction of FusB with EF-G and thereby delineate the binding interface within the FusB-EF-G complex. Replacement with alanine of any one of four conserved residues within the C-terminal domain of FusB (F156, K184, Y187, and F208) abrogated the ability of the protein to confer resistance to fusidic acid; the purified mutant proteins also lost the ability to bind S. aureus EF-G in vitro. E. coli EF-G, which is not ordinarily able to bind FusB-type proteins, was rendered competent for binding to FusB following deletion of a 3-residue tract (529SNP531) from domain IV of the protein. This study has identified key regions of both FusB and EF-G that are important for the interaction between the proteins, findings which corroborate our previous in silico prediction for the architecture of the complex formed between the resistance protein and the drug target (G. Cox, G. S. Thompson, H. T. Jenkins, F. Peske, A. Savelsbergh, M. V. Rodnina, W. Wintermeyer, S. W. Homans, T. A. Edwards, and A. J. O'Neill, Proc. Natl. Acad. Sci. U. S. A. 109:2102-2107, 2012). PMID:23836182

  2. Inhibition of the mitogen-activated protein kinase pathway triggers B16 melanoma cell differentiation.

    PubMed

    Englaro, W; Bertolotto, C; Buscà, R; Brunet, A; Pagès, G; Ortonne, J P; Ballotti, R

    1998-04-17

    In B16 melanoma cells, mitogen-activated protein (MAP) kinases are activated during cAMP-induced melanogenesis (Englaro, W., Rezzonico, R., Durand-Clément, M., Lallemand, D., Ortonne, J. P., and Ballotti, R. (1995) J. Biol. Chem. 270, 24315-24320). To establish the role of the MAP kinases in melanogenesis, we studied the effects of a specific MAP kinase kinase (MEK) inhibitor PD 98059 on different melanogenic parameters. We showed that PD 98059 inhibits the activation of MAP kinase extracellular signal-regulated kinase 1 by cAMP, but does not impair the effects of cAMP either on the morphological differentiation, characterized by an increase in dendrite outgrowth, or on the up-regulation of tyrosinase that is the key enzyme in melanogenesis. On the contrary, PD 98059 promotes by itself cell dendricity and increases the tyrosinase amount and activity. Moreover, down-regulation of the MAP kinase pathway by PD 98059, or with dominant negative mutants of p21(ras) and MEK, triggers a stimulation of the tyrosinase promoter activity and enhances the effect of cAMP on this parameter. Conversely, activation of the MAP kinase pathway, using constitutive active mutants of p21(ras) and MEK, leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription. These results demonstrate that the MAP kinase pathway activation is not required for cAMP-induced melanogenesis. Furthermore, the inhibition of this pathway induces B16 melanoma cell differentiation, while a sustained activation impairs the melanogenic effect of cAMP-elevating agents. PMID:9545341

  3. Development of Multigene Expression Signature Maps at the Protein Level from Digitized Immunohistochemistry Slides

    PubMed Central

    Metzger, Gregory J.; Dankbar, Stephen C.; Henriksen, Jonathan; Rizzardi, Anthony E.; Rosener, Nikolaus K.; Schmechel, Stephen C.

    2012-01-01

    Molecular classification of diseases based on multigene expression signatures is increasingly used for diagnosis, prognosis, and prediction of response to therapy. Immunohistochemistry (IHC) is an optimal method for validating expression signatures obtained using high-throughput genomics techniques since IHC allows a pathologist to examine gene expression at the protein level within the context of histologically interpretable tissue sections. Additionally, validated IHC assays may be readily implemented as clinical tests since IHC is performed on routinely processed clinical tissue samples. However, methods have not been available for automated n-gene expression profiling at the protein level using IHC data. We have developed methods to compute expression level maps (signature maps) of multiple genes from IHC data digitized on a commercial whole slide imaging system. Areas of cancer for these expression level maps are defined by a pathologist on adjacent, co-registered H&E slides, allowing assessment of IHC statistics and heterogeneity within the diseased tissue. This novel way of representing multiple IHC assays as signature maps will allow the development of n-gene expression profiling databases in three dimensions throughout virtual whole organ reconstructions. PMID:22438942

  4. Mapping the Protein Interaction Network for TFIIB-Related Factor Brf1 in the RNA Polymerase III Preinitiation Complex

    PubMed Central

    Khoo, Seok-Kooi; Wu, Chih-Chien; Lin, Yu-Chun; Lee, Jin-Cheng

    2014-01-01

    TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III recruitment and open-promoter formation in transcription initiation. We site specifically incorporated a nonnatural amino acid cross-linker into Brf1 to map its protein interaction targets in the preinitiation complex (PIC). Our cross-linking analysis in the N-terminal domain of Brf1 indicated a pattern of multiple protein interactions reminiscent of TFIIB in the Pol active-site cleft. In addition to the TFIIB-like protein interactions, the Brf1 cyclin repeat subdomain is in contact with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, we further revealed the binding between Brf1 cyclin repeats and the highly conserved region connecting C34 winged-helix domains 2 and 3. In contrast to the N-terminal domain of Brf1, the C-terminal domain contains extensive binding sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. Overall, the domain architecture of the PIC derived from our cross-linking data explains how individual structural subdomains of Brf1 integrate the protein network from the Pol III active center to the promoter for transcription initiation. PMID:24277937

  5. Mapping the protein interaction network for TFIIB-related factor Brf1 in the RNA polymerase III preinitiation complex.

    PubMed

    Khoo, Seok-Kooi; Wu, Chih-Chien; Lin, Yu-Chun; Lee, Jin-Cheng; Chen, Hung-Ta

    2014-02-01

    TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III recruitment and open-promoter formation in transcription initiation. We site specifically incorporated a nonnatural amino acid cross-linker into Brf1 to map its protein interaction targets in the preinitiation complex (PIC). Our cross-linking analysis in the N-terminal domain of Brf1 indicated a pattern of multiple protein interactions reminiscent of TFIIB in the Pol active-site cleft. In addition to the TFIIB-like protein interactions, the Brf1 cyclin repeat subdomain is in contact with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, we further revealed the binding between Brf1 cyclin repeats and the highly conserved region connecting C34 winged-helix domains 2 and 3. In contrast to the N-terminal domain of Brf1, the C-terminal domain contains extensive binding sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. Overall, the domain architecture of the PIC derived from our cross-linking data explains how individual structural subdomains of Brf1 integrate the protein network from the Pol III active center to the promoter for transcription initiation. PMID:24277937

  6. Protein interaction mapping with ribosome-displayed using PLATO ORF libraries

    PubMed Central

    Zhu, Jian; Larman, H. Benjamin; Gao, Geng; Somwar, Romel; Zhang, Zijuan; Laserson, Uri; Ciccia, Alberto; Pavlova, Natalya; Church, George; Zhang, Wei; Kesari, Santosh; Elledge, Stephen J.

    2013-01-01

    Identifying physical interactions between proteins and other molecules is a critical aspect of biological analysis. Here we describe PLATO, an in vitro method for mapping such interactions by affinity enrichment of a library of full-length open reading frames displayed on ribosomes, followed by massively parallel analysis using DNA sequencing. We demonstrate the broad utility of the method by identifying known and new interacting partners of LYN kinase, patient autoantibodies and the small molecules gefitinib and dasatinib. PMID:24336473

  7. Mapping of B-cell epitopes in the nucleocapsid protein of Puumala hantavirus.

    PubMed

    Lundkvist, A; Meisel, H; Koletzki, D; Lankinen, H; Cifire, F; Geldmacher, A; Sibold, C; Gött, P; Vaheri, A; Krüger, D H; Ulrich, R

    2002-01-01

    Hantavirus nucleocapsid protein (N) has been proven to induce highly protective immune responses in animal models. The knowledge on the mechanisms behind N-induced protection is still limited, although recent data suggest that both cellular and humoral immune responses are of importance. For a detailed B-cell epitope mapping of Puumala hantavirus (PUUV) N, we used recombinant N derivatives of the Russian strain CG18-20 and the Swedish strain Vranica/Hällnäs, as well as overlapping synthetic peptides corresponding to the Finnish prototype strain Sotkamo. The majority of a panel of monoclonal antibodies (mAbs) reacted with proteins derived from all included PUUV strains demonstrating the antigenic similarity of these proteins. In line with previous results, the epitopes of most mAbs were mapped within the 80 N-terminal amino acids of N. The present study further revealed that the epitopes of four mAbs raised against native viral N were located within amino acids 14-45, whereas one mAb raised against recombinant N was mapped to amino acids 14-39. Differences between the reactivity of the PUUV strains Vranica/Hällnäs and CG18-20 N suggested the importance of amino acid position 35 for the integrity of the epitopes. In line with the patterns obtained by the truncated recombinant proteins, mapping by overlapping peptides (PEPSCAN) confirmed a complex recognition pattern for most analyzed mAbs. Together, the results revealed the existence of several, partially overlapping, and discontinuous B-cell epitopes. In addition, based on differences within the same competition group, novel epitopes were defined. PMID:11952140

  8. A fluorescent reporter for mapping cellular protein-protein interactions in time and space

    PubMed Central

    Moreno, Daniel; Neller, Joachim; Kestler, Hans A; Kraus, Johann; Dünkler, Alexander; Johnsson, Nils

    2013-01-01

    We introduce a fluorescent reporter for monitoring protein–protein interactions in living cells. The method is based on the Split-Ubiquitin method and uses the ratio of two auto-fluorescent reporter proteins as signal for interaction (SPLIFF). The mating of two haploid yeast cells initiates the analysis and the interactions are followed online by two-channel time-lapse microscopy of the diploid cells during their first cell cycle. Using this approach we could with high spatio-temporal resolution visualize the differences between the interactions of the microtubule binding protein Stu2p with two of its binding partners, monitor the transient association of a Ran-GTPase with its receptors at the nuclear pore, and distinguish between protein interactions at the polar cortical domain at different phases of polar growth. These examples further demonstrate that protein–protein interactions identified from large-scale screens can be effectively followed up by high-resolution single-cell analysis. PMID:23511205

  9. Boosting AthaMap Database Content with Data from Protein Binding Microarrays.

    PubMed

    Hehl, Reinhard; Norval, Leo; Romanov, Artyom; Bülow, Lorenz

    2016-01-01

    The AthaMap database generates a map of predicted transcription factor binding sites (TFBS) and small RNA target sites for the whole Arabidopsis thaliana genome. With the advent of protein binding microarrays (PBM), the number of known TFBS for A. thaliana transcription factors (TFs) has increased dramatically. Using 113 positional weight matrices (PWMs) generated from a single PBM study and representing a total number of 68 different TFs, the number of predicted TFBS in AthaMap was now increased by about 3.8 × 10(7) to 4.9 × 10(7). The number of TFs with PWM-predicted TFBS annotated in AthaMap has increased to 126, representing a total of 29 TF families and newly including ARF, AT-Hook, YABBY, LOB/AS2 and SRS. Furthermore, links from all Arabidopsis TFs and genes to the newly established Arabidopsis Information Portal (AIP) have been implemented. With this qualitative and quantitative update, the improved AthaMap increases its value as a resource for the analysis of A. thaliana gene expression regulation at www.athamap.de. PMID:26542109

  10. Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Huang, Y.; Kieber, J.; Luan, S.; Evans, M. L. (Principal Investigator)

    1998-01-01

    Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.

  11. Global mapping of herpesvirus-host protein complexes reveals a novel transcription strategy for late genes

    PubMed Central

    Davis, Zoe H.; Verschueren, Erik; Jang, Gwendolyn M.; Kleffman, Kevin; Johnson, Jeffrey R.; Park, Jimin; Von Dollen, John; Maher, M. Cyrus; Johnson, Tasha; Newton, William; Jäger, Stefanie; Shales, Michael; Horner, Julie; Hernandez, Ryan D.; Krogan, Nevan J.; Glaunsinger, Britt A.

    2014-01-01

    SUMMARY Mapping host-pathogen interactions has proven instrumental for understanding how viruses manipulate host machinery and how numerous cellular processes are regulated. DNA viruses such as herpesviruses have relatively large coding capacity and thus can target an extensive network of cellular proteins. To identify the host proteins hijacked by this pathogen, we systematically affinity tagged and purified all 89 proteins of Kaposi’s sarcoma-associated herpesvirus (KSHV) from human cells. Mass spectrometry of this material identified over 500 virus-host interactions. KSHV causes AIDS-associated cancers and its interaction network is enriched for proteins linked to cancer and overlaps with proteins that are also targeted by HIV-1. We found that the conserved KSHV protein ORF24 binds to RNA polymerase II and brings it to viral late promoters by mimicking and replacing cellular TATA-box-binding protein (TBP). This is required for herpesviral late gene expression, a complex and poorly understood phase of the viral lifecycle. PMID:25544563

  12. ConSole: using modularity of Contact maps to locate Solenoid domains in protein structures

    PubMed Central

    2014-01-01

    Background Periodic proteins, characterized by the presence of multiple repeats of short motifs, form an interesting and seldom-studied group. Due to often extreme divergence in sequence, detection and analysis of such motifs is performed more reliably on the structural level. Yet, few algorithms have been developed for the detection and analysis of structures of periodic proteins. Results ConSole recognizes modularity in protein contact maps, allowing for precise identification of repeats in solenoid protein structures, an important subgroup of periodic proteins. Tests on benchmarks show that ConSole has higher recognition accuracy as compared to Raphael, the only other publicly available solenoid structure detection tool. As a next step of ConSole analysis, we show how detection of solenoid repeats in structures can be used to improve sequence recognition of these motifs and to detect subtle irregularities of repeat lengths in three solenoid protein families. Conclusions The ConSole algorithm provides a fast and accurate tool to recognize solenoid protein structures as a whole and to identify individual solenoid repeat units from a structure. ConSole is available as a web-based, interactive server and is available for download at http://console.sanfordburnham.org. PMID:24766872

  13. Size-selective fractionation and visual mapping of allergen protein chemistry in Arachis hypogaea.

    PubMed

    Hebling, Christine M; Ross, Mark M; Callahan, John H; McFarland, Melinda A

    2012-11-01

    Peanuts (Arachis hypogaea) in addition to milk, eggs, fish, crustaceans, wheat, tree nuts, and soybean are commonly referred to as the "big eight" foods that contribute to the majority of food allergies worldwide. Despite the severity of allergic reactions and growing prevalence in children and adults, there is no cure for peanut allergy, leaving avoidance as the primary mode of treatment. To improve analytical methods for peanut allergen detection, researchers must overcome obstacles involved in handling complex food matrices while attempting to decipher the chemistry that underlies allergen protein interactions. To address such challenges, we conducted a global proteome characterization of raw peanuts using a sophisticated GELFrEE-PAGE-LC-MS/MS platform consisting of gel-based protein fractionation followed by mass spectrometric identification. The in-solution mass-selective protein fractionation: (1) enhances the number of unique peptide identifications, (2) provides a visual map of protein isoforms, and (3) aids in the identification of disulfide-linked protein complexes. GELFrEE-PAGE-LC-MS/MS not only overcomes many of the challenges involved in the study of plant proteomics, but enriches the understanding of peanut protein chemistry, which is typically unattainable in a traditional bottom-up proteomic analysis. A global understanding of protein chemistry in Arachis hypogaea ultimately will aid the development of improved methods for allergen detection in food. PMID:23020697

  14. Areas activated during naturalistic reading comprehension overlap topological visual, auditory, and somatotomotor maps.

    PubMed

    Sood, Mariam R; Sereno, Martin I

    2016-08-01

    Cortical mapping techniques using fMRI have been instrumental in identifying the boundaries of topological (neighbor-preserving) maps in early sensory areas. The presence of topological maps beyond early sensory areas raises the possibility that they might play a significant role in other cognitive systems, and that topological mapping might help to delineate areas involved in higher cognitive processes. In this study, we combine surface-based visual, auditory, and somatomotor mapping methods with a naturalistic reading comprehension task in the same group of subjects to provide a qualitative and quantitative assessment of the cortical overlap between sensory-motor maps in all major sensory modalities, and reading processing regions. Our results suggest that cortical activation during naturalistic reading comprehension overlaps more extensively with topological sensory-motor maps than has been heretofore appreciated. Reading activation in regions adjacent to occipital lobe and inferior parietal lobe almost completely overlaps visual maps, whereas a significant portion of frontal activation for reading in dorsolateral and ventral prefrontal cortex overlaps both visual and auditory maps. Even classical language regions in superior temporal cortex are partially overlapped by topological visual and auditory maps. By contrast, the main overlap with somatomotor maps is restricted to a small region on the anterior bank of the central sulcus near the border between the face and hand representations of M-I. Hum Brain Mapp 37:2784-2810, 2016. © 2016 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc. PMID:27061771

  15. Areas activated during naturalistic reading comprehension overlap topological visual, auditory, and somatotomotor maps

    PubMed Central

    2016-01-01

    Abstract Cortical mapping techniques using fMRI have been instrumental in identifying the boundaries of topological (neighbor‐preserving) maps in early sensory areas. The presence of topological maps beyond early sensory areas raises the possibility that they might play a significant role in other cognitive systems, and that topological mapping might help to delineate areas involved in higher cognitive processes. In this study, we combine surface‐based visual, auditory, and somatomotor mapping methods with a naturalistic reading comprehension task in the same group of subjects to provide a qualitative and quantitative assessment of the cortical overlap between sensory‐motor maps in all major sensory modalities, and reading processing regions. Our results suggest that cortical activation during naturalistic reading comprehension overlaps more extensively with topological sensory‐motor maps than has been heretofore appreciated. Reading activation in regions adjacent to occipital lobe and inferior parietal lobe almost completely overlaps visual maps, whereas a significant portion of frontal activation for reading in dorsolateral and ventral prefrontal cortex overlaps both visual and auditory maps. Even classical language regions in superior temporal cortex are partially overlapped by topological visual and auditory maps. By contrast, the main overlap with somatomotor maps is restricted to a small region on the anterior bank of the central sulcus near the border between the face and hand representations of M‐I. Hum Brain Mapp 37:2784–2810, 2016. © 2016 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc. PMID:27061771

  16. The TMS Map Scales with Increased Stimulation Intensity and Muscle Activation.

    PubMed

    van de Ruit, Mark; Grey, Michael J

    2016-01-01

    One way to study cortical organisation, or its reorganisation, is to use transcranial magnetic stimulation (TMS) to construct a map of corticospinal excitability. TMS maps are reported to be acquired with a wide variety of stimulation intensities and levels of muscle activation. Whilst MEPs are known to increase both with stimulation intensity and muscle activation, it remains to be established what the effect of these factors is on the map's centre of gravity (COG), area, volume and shape. Therefore, the objective of this study was to systematically examine the effect of stimulation intensity and muscle activation on these four key map outcome measures. In a first experiment, maps were acquired with a stimulation intensity of 110, 120 and 130% of resting threshold. In a second experiment, maps were acquired at rest and at 5, 10, 20 and 40% of maximum voluntary contraction. Map area and map volume increased with both stimulation intensity (P < 0.01) and muscle activation (P < 0.01). Neither the COG nor the map shape changed with either stimulation intensity or muscle activation (P > 0.09 in all cases). This result indicates the map simply scales with stimulation intensity and muscle activation. PMID:26337508

  17. A Framework of Active Learning by Concept Mapping

    ERIC Educational Resources Information Center

    Chen, Wang-Kun; Wang, Ping

    2012-01-01

    This study presents a student-centered teaching model based on concept mapping and problem-solving. The concept map is used as a tool to develop curriculum and evaluate teaching performance. Case-based teaching was implemented on the course of building energy conservation. The results of this study, which include teaching plans, evaluation tools,…

  18. Nucleolar protein B23 has molecular chaperone activities.

    PubMed Central

    Szebeni, A.; Olson, M. O.

    1999-01-01

    Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis. PMID:10211837

  19. Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities

    PubMed Central

    Shen, Hsin-Hui; Lithgow, Trevor; Martin, Lisandra L.

    2013-01-01

    The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. We also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. Understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. It is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro. PMID:23344058

  20. Human adenosine A1 receptor and P2Y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells

    PubMed Central

    Dickenson, John M; Blank, Jonathan L; Hill, Stephen J

    1998-01-01

    The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric Gi/Go protein-coupled and Gq/G11 protein-coupled receptors. The aims of the current study were: (i) to investigate whether the Gi/Go protein-coupled adenosine A1 receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor.The selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1±0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 μM; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block Gi/Go-dependent pathways).Adenosine A1 receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 μM; 6±10% of control). In contrast, daidzein (100 μM), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55±8% inhibition) and LY 294002 (30 μM; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 μM).Activation of the endogenous P2Y2 purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50=1.6±0.3 μM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 μM; 45±5% inhibition), indicating the possible involvement of both Gi/Go protein and Gq protein-dependent pathways in the overall response to UTP.CPA and UTP stimulated concentration

  1. Stimulation by endothelin-1 of mitogen-activated protein kinases and DNA synthesis in bovine tracheal smooth muscle cells.

    PubMed Central

    Malarkey, K.; Chilvers, E. R.; Lawson, M. F.; Plevin, R.

    1995-01-01

    1. In cultures of bovine tracheal smooth muscle cells, platelet-derived growth factor-BB (PDGF), bradykinin (BK) and endothelin-1 (ET-1) stimulated the tyrosine phosphorylation and activation of both pp42 and pp44 kDa forms of mitogen-activated protein (MAP) kinase. 2. Both ET-1 and PDGF stimulated a sustained activation of MAP kinase whilst the response to BK was transient. 3. Activation of MAP kinase occurred in a concentration-dependent manner (EC50 values: ET-1, 2.3 +/- 1.3 nM; BK, 8.7 +/- 4.1 nM, PDGF, 9.7 +/- 3.2 ng ml-1). 4. Pretreatment with the protein kinase C (PKC) inhibitor Ro-318220, significantly reduced ET-1 activation of MAP kinase at 2 and 5 min but enhanced MAP kinase activation at 60 min. 5. Following chronic phorbol ester pretreatment, BK-stimulated activation of MAP kinase was abolished whilst the responses to PDGF and ET-1 were only partly reduced (80 and 45% inhibition respectively). 6. Pretreatment with pertussis toxin reduced ET-1 stimulated activation of MAP kinase particularly at later times (60 min), but left the responses to both PDGF and BK unaffected. 7. ET-1 also stimulated a 3 fold increase in [3H]-thymidine incorporation which was abolished by pertussis toxin pretreatment. In contrast, PDGF stimulated a 131 fold increase in [3H]-thymidine incorporation which was not affected by pertussis toxin. 8. These results suggest that a pertussis toxin-sensitive activation of MAP kinase may play an important role in ET-1-stimulated DNA synthesis but that activation of MAP kinase alone is not sufficient to induce the magnitude of DNA synthesis observed in response to PDGF. Images Figure 1 Figure 2 Figure 5 Figure 6 Figure 7 PMID:8564258

  2. Designing Mimics of Membrane Active Proteins

    PubMed Central

    Sgolastra, Federica; deRonde, Brittany M.; Sarapas, Joel M.; Som, Abhigyan; Tew, Gregory N.

    2014-01-01

    CONSPECTUS As a semi-permeable barrier that controls the flux of biomolecules in and out the cell, the plasma membrane is critical in cell function and survival. Many proteins interact with the plasma membrane and modulate its physiology. Within this large landscape of membrane-active molecules, researchers have focused significant attention on two specific classes of peptides, antimicrobial peptides (AMPs) and cell penetrating peptides (CPPs) because of their unique properties. In this account, we describe our efforts over the last decade to build and understand synthetic mimics of antimicrobial peptides (SMAMPs). These endeavors represent one specific example of a much larger effort to understand how synthetic molecules interact with and manipulate the plasma membrane. Using both defined molecular weight oligomers and easier to produce, but heterogeneous, polymers, it has been possible to generate scaffolds with biological potency superior to the natural analogs. In one case, a compound has progressed through a phase II clinical trial for pan)staph infections. Modern biophysical assays highlighted the interplay between the synthetic scaffold and lipid composition leading to negative Gaussian curvature, a requirement for both pore formation and endosomal escape. The complexity of this interplay between lipids, bilayer components, and the scaffolds remains to be better resolved, but significant new insight has been provided. It is worthwhile to consider the various aspects of permeation and how these are related to ‘pore formation.’ More recently, our efforts have expanded toward protein transduction domains, or cell penetrating peptide, mimics. The combination of unique molecular scaffolds and guanidinium) rich side chains has produced an array of polymers with robust transduction (and delivery) activity. Being a new area, the fundamental interactions between these new scaffolds and the plasma membrane are just beginning to be understood. Negative Gaussian

  3. Discovery and fine mapping of serum protein loci through transethnic meta-analysis.

    PubMed

    Franceschini, Nora; van Rooij, Frank J A; Prins, Bram P; Feitosa, Mary F; Karakas, Mahir; Eckfeldt, John H; Folsom, Aaron R; Kopp, Jeffrey; Vaez, Ahmad; Andrews, Jeanette S; Baumert, Jens; Boraska, Vesna; Broer, Linda; Hayward, Caroline; Ngwa, Julius S; Okada, Yukinori; Polasek, Ozren; Westra, Harm-Jan; Wang, Ying A; Del Greco M, Fabiola; Glazer, Nicole L; Kapur, Karen; Kema, Ido P; Lopez, Lorna M; Schillert, Arne; Smith, Albert V; Winkler, Cheryl A; Zgaga, Lina; Bandinelli, Stefania; Bergmann, Sven; Boban, Mladen; Bochud, Murielle; Chen, Y D; Davies, Gail; Dehghan, Abbas; Ding, Jingzhong; Doering, Angela; Durda, J Peter; Ferrucci, Luigi; Franco, Oscar H; Franke, Lude; Gunjaca, Grog; Hofman, Albert; Hsu, Fang-Chi; Kolcic, Ivana; Kraja, Aldi; Kubo, Michiaki; Lackner, Karl J; Launer, Lenore; Loehr, Laura R; Li, Guo; Meisinger, Christa; Nakamura, Yusuke; Schwienbacher, Christine; Starr, John M; Takahashi, Atsushi; Torlak, Vesela; Uitterlinden, André G; Vitart, Veronique; Waldenberger, Melanie; Wild, Philipp S; Kirin, Mirna; Zeller, Tanja; Zemunik, Tatijana; Zhang, Qunyuan; Ziegler, Andreas; Blankenberg, Stefan; Boerwinkle, Eric; Borecki, Ingrid B; Campbell, Harry; Deary, Ian J; Frayling, Timothy M; Gieger, Christian; Harris, Tamara B; Hicks, Andrew A; Koenig, Wolfgang; O' Donnell, Christopher J; Fox, Caroline S; Pramstaller, Peter P; Psaty, Bruce M; Reiner, Alex P; Rotter, Jerome I; Rudan, Igor; Snieder, Harold; Tanaka, Toshihiro; van Duijn, Cornelia M; Vollenweider, Peter; Waeber, Gerard; Wilson, James F; Witteman, Jacqueline C M; Wolffenbuttel, Bruce H R; Wright, Alan F; Wu, Qingyu; Liu, Yongmei; Jenny, Nancy S; North, Kari E; Felix, Janine F; Alizadeh, Behrooz Z; Cupples, L Adrienne; Perry, John R B; Morris, Andrew P

    2012-10-01

    Many disorders are associated with altered serum protein concentrations, including malnutrition, cancer, and cardiovascular, kidney, and inflammatory diseases. Although these protein concentrations are highly heritable, relatively little is known about their underlying genetic determinants. Through transethnic meta-analysis of European-ancestry and Japanese genome-wide association studies, we identified six loci at genome-wide significance (p < 5 × 10(-8)) for serum albumin (HPN-SCN1B, GCKR-FNDC4, SERPINF2-WDR81, TNFRSF11A-ZCCHC2, FRMD5-WDR76, and RPS11-FCGRT, in up to 53,190 European-ancestry and 9,380 Japanese individuals) and three loci for total protein (TNFRS13B, 6q21.3, and ELL2, in up to 25,539 European-ancestry and 10,168 Japanese individuals). We observed little evidence of heterogeneity in allelic effects at these loci between groups of European and Japanese ancestry but obtained substantial improvements in the resolution of fine mapping of potential causal variants by leveraging transethnic differences in the distribution of linkage disequilibrium. We demonstrated a functional role for the most strongly associated serum albumin locus, HPN, for which Hpn knockout mice manifest low plasma albumin concentrations. Other loci associated with serum albumin harbor genes related to ribosome function, protein translation, and proteasomal degradation, whereas those associated with serum total protein include genes related to immune function. Our results highlight the advantages of transethnic meta-analysis for the discovery and fine mapping of complex trait loci and have provided initial insights into the underlying genetic architecture of serum protein concentrations and their association with human disease. PMID:23022100

  4. Discovery and Fine Mapping of Serum Protein Loci through Transethnic Meta-analysis

    PubMed Central

    Franceschini, Nora; van Rooij, Frank J.A.; Prins, Bram P.; Feitosa, Mary F.; Karakas, Mahir; Eckfeldt, John H.; Folsom, Aaron R.; Kopp, Jeffrey; Vaez, Ahmad; Andrews, Jeanette S.; Baumert, Jens; Boraska, Vesna; Broer, Linda; Hayward, Caroline; Ngwa, Julius S.; Okada, Yukinori; Polasek, Ozren; Westra, Harm-Jan; Wang, Ying A.; Del Greco M., Fabiola; Glazer, Nicole L.; Kapur, Karen; Kema, Ido P.; Lopez, Lorna M.; Schillert, Arne; Smith, Albert V.; Winkler, Cheryl A.; Zgaga, Lina; Bandinelli, Stefania; Bergmann, Sven; Boban, Mladen; Bochud, Murielle; Chen, Y.D.; Davies, Gail; Dehghan, Abbas; Ding, Jingzhong; Doering, Angela; Durda, J. Peter; Ferrucci, Luigi; Franco, Oscar H.; Franke, Lude; Gunjaca, Grog; Hofman, Albert; Hsu, Fang-Chi; Kolcic, Ivana; Kraja, Aldi; Kubo, Michiaki; Lackner, Karl J.; Launer, Lenore; Loehr, Laura R.; Li, Guo; Meisinger, Christa; Nakamura, Yusuke; Schwienbacher, Christine; Starr, John M.; Takahashi, Atsushi; Torlak, Vesela; Uitterlinden, André G.; Vitart, Veronique; Waldenberger, Melanie; Wild, Philipp S.; Kirin, Mirna; Zeller, Tanja; Zemunik, Tatijana; Zhang, Qunyuan; Ziegler, Andreas; Blankenberg, Stefan; Boerwinkle, Eric; Borecki, Ingrid B.; Campbell, Harry; Deary, Ian J.; Frayling, Timothy M.; Gieger, Christian; Harris, Tamara B.; Hicks, Andrew A.; Koenig, Wolfgang; O’Donnell, Christopher J.; Fox, Caroline S.; Pramstaller, Peter P.; Psaty, Bruce M.; Reiner, Alex P.; Rotter, Jerome I.; Rudan, Igor; Snieder, Harold; Tanaka, Toshihiro; van Duijn, Cornelia M.; Vollenweider, Peter; Waeber, Gerard; Wilson, James F.; Witteman, Jacqueline C.M.; Wolffenbuttel, Bruce H.R.; Wright, Alan F.; Wu, Qingyu; Liu, Yongmei; Jenny, Nancy S.; North, Kari E.; Felix, Janine F.; Alizadeh, Behrooz Z.; Cupples, L. Adrienne; Perry, John R.B.; Morris, Andrew P.

    2012-01-01

    Many disorders are associated with altered serum protein concentrations, including malnutrition, cancer, and cardiovascular, kidney, and inflammatory diseases. Although these protein concentrations are highly heritable, relatively little is known about their underlying genetic determinants. Through transethnic meta-analysis of European-ancestry and Japanese genome-wide association studies, we identified six loci at genome-wide significance (p < 5 × 10−8) for serum albumin (HPN-SCN1B, GCKR-FNDC4, SERPINF2-WDR81, TNFRSF11A-ZCCHC2, FRMD5-WDR76, and RPS11-FCGRT, in up to 53,190 European-ancestry and 9,380 Japanese individuals) and three loci for total protein (TNFRS13B, 6q21.3, and ELL2, in up to 25,539 European-ancestry and 10,168 Japanese individuals). We observed little evidence of heterogeneity in allelic effects at these loci between groups of European and Japanese ancestry but obtained substantial improvements in the resolution of fine mapping of potential causal variants by leveraging transethnic differences in the distribution of linkage disequilibrium. We demonstrated a functional role for the most strongly associated serum albumin locus, HPN, for which Hpn knockout mice manifest low plasma albumin concentrations. Other loci associated with serum albumin harbor genes related to ribosome function, protein translation, and proteasomal degradation, whereas those associated with serum total protein include genes related to immune function. Our results highlight the advantages of transethnic meta-analysis for the discovery and fine mapping of complex trait loci and have provided initial insights into the underlying genetic architecture of serum protein concentrations and their association with human disease. PMID:23022100

  5. Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis.

    PubMed

    Takiar, Vinita; Nishio, Saori; Seo-Mayer, Patricia; King, J Darwin; Li, Hui; Zhang, Li; Karihaloo, Anil; Hallows, Kenneth R; Somlo, Stefan; Caplan, Michael J

    2011-02-01

    Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD. PMID:21262823

  6. Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins

    SciTech Connect

    Tsukamotot, A.S.; Ponticelli, A.; Berk, A.J.; Gaynor, R.B.

    1986-07-01

    Adenovirus early region 1A (E1A) encodes two acidic phosphoproteins which are required for transactivation of viral transcription, efficient viral DNA replication in phase G/sub 0/-arrested human cells, and oncogenic transformation of rodent cells. Biochemical analysis of in vivo /sup 32/P-labeled adenovirus type 2 E1A proteins purified with monoclonal antibodies demonstrated that these proteins were phosphorylated at multiple serine residues. Two-dimensional phosphotryptic peptide maps of wild-type and mutant E1A proteins were used to locate a major site of E1A protein phosphorylation at serine-219 of the large E1A protein. Although this serine fell within a consensus sequence for phosphorylation by the cyclic AMP-dependent protein kinases, experiments with mutant CHO cells defective in these enzymes indicated that it was not. Oligonucleotide-directed mutagenesis was used to substitute an alanine for serine-219. This mutation prevented phosphorylation at this site. Nonetheless, the mutant was indistinguishable from the wild type for early gene transactivation, replication on G/sub 0/-arrested WI-38 cells, and transformation of cloned rat embryo fibroblast cells.

  7. Activity landscape modeling of PPAR ligands with dual-activity difference maps.

    PubMed

    Méndez-Lucio, Oscar; Pérez-Villanueva, Jaime; Castillo, Rafael; Medina-Franco, José L

    2012-06-01

    Activation of peroxisome proliferator-activated receptor (PPAR) subtypes offers a promising strategy for the treatment of diabetes mellitus and metabolic diseases. Selective and dual PPAR agonists have been developed and the systematic characterization of their structure-activity relationships (SAR) is of major significance. Herein, we report a systematic description of the SAR of 168 compounds screened against the three PPAR subtypes using the principles of activity landscape modeling. As part of our effort to develop and apply chemoinformatic tools to navigate through activity landscapes, we employed consensus dual-activity difference maps recently reported. The analysis is based on pairwise relationships of potency difference and structure-similarity which were calculated from the combination of four different 2D and 3D structure representations. Dual-activity difference maps uncovered regions in the landscape with similar SAR for two or three receptor subtypes as well as regions with inverse SAR, that is, changes in structure that increase activity for one subtype but decrease activity for the other subtype. Analysis of pairs of compounds with high structure similarity revealed the presence of single-, dual-, and 'pan-receptor' activity cliffs, that is, small changes in structure with high changes in potency for one, two, or three receptor subtypes, respectively. Single-, dual-, and pan-receptor scaffold hops are also discussed. The analysis of the chemical structures of selected data points reported in this paper points to specific structural features that are helpful for the design of new PPAR agonists. The approach presented in this work is general and can be extended to analyze larger data sets. PMID:22564380

  8. Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation

    PubMed Central

    LEE, JIN-HAENG; JEONG, JAEHO; JEONG, EUI MAN; CHO, SUNG-YUP; KANG, JEONG WOOK; LIM, JISUN; HEO, JINBEOM; KANG, HYUNSOOK; KIM, IN-GYU; SHIN, DONG-MYUNG

    2014-01-01

    Aberrant activation of transglutaminase 2 (TGase2) contributes to a variety of protein conformational disorders such as neurodegenerative diseases and age-related cataracts. The accumulation of improperly folded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), which promotes either repair or degradation of the damaged proteins. Inadequate UPR results in protein aggregation that may contribute to the development of age-related degenerative diseases. TGase2 is a calcium-dependent enzyme that irreversibly modifies proteins by forming cross-linked protein aggregates. Intracellular TGase2 is activated by oxidative stress which generates large quantities of unfolded proteins. However, the relationship between TGase2 activity and UPR has not yet been established. In the present study, we demonstrated that ER stress activated TGase2 in various cell types. TGase2 activation was dependent on the ER stress-induced increase in the intracellular calcium ion concentration but not on the TGase2 protein expression level. Enzyme substrate analysis revealed that TGase2-mediated protein modification promoted protein aggregation concurrently with decreasing water solubility. Moreover, treatment with KCC009, a TGase2 inhibitor, abrogated ER stress-induced TGase2 activation and subsequent protein aggregation. However, TGase2 activation had no effect on ER stress-induced cell death. These results demonstrate that the accumulation of misfolded proteins activates TGase2, which further accelerates the formation of protein aggregates. Therefore, we suggest that inhibition of TGase2 may be a novel strategy by which to prevent the protein aggregation in age-related degenerative diseases. PMID:24481335

  9. Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes.

    PubMed Central

    Abrieu, A; Dorée, M; Picard, A

    1997-01-01

    The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation. Images PMID:9190205

  10. Studies of Xenopus laevis mitochondrial DNA: D-loop mapping and characterization of DNA-binding proteins

    SciTech Connect

    Cairns, S.S.

    1987-01-01

    In X. laevis oocytes, mitochondrial DNA accumulates to 10/sup 5/ times the somatic cell complement, and is characterized by a high frequency of a triple-stranded displacement hoop structure at the origin of replication. To map the termini of the single strands, it was necessary to correct the nucleotide sequence of the D-loop region. The revised sequence of 2458 nucleotides contains 54 discrepancies in comparison to a previously published sequence. Radiolabeling of the nascent strands of the D-loop structure either at the 5' end or at the 3' end identifies a major species with a length of 1670 nucleotides. Cleavage of the 5' labeled strands reveals two families of ends located near several matches to an element, designated CSB-1, that is conserved in this location in several vertebrate genomes. Cleavage of 3' labeled strands produced one fragment. The unique 3' end maps to about 15 nucleotides preceding the tRNA/sup Pro/ gene. A search for proteins which may bind to mtDNA in this region to regulate nucleic acid synthesis has identified three activities in lysates of X. laevis mitochondria. The DNA-binding proteins were assayed by monitoring their ability to retard the migration of labeled double- or single-stranded DNA fragments in polyacrylamide gels. The DNA binding preference was determined by competition with an excess of either ds- or ssDNA.

  11. Combining recombinant ribonuclease U2 and protein phosphatase for RNA modification mapping by liquid chromatography-mass spectrometry.

    PubMed

    Houser, Whitney M; Butterer, Annika; Addepalli, Balasubrahmanym; Limbach, Patrick A

    2015-06-01

    Ribonuclease (RNase) mapping of modified nucleosides onto RNA sequences is limited by RNase availability. A codon-optimized gene for RNase U2, a purine selective RNase with preference for adenosine, has been designed for overexpression using Escherichia coli as the host. Optimal expression conditions were identified enabling generation of milligram-scale quantities of active RNase U2. RNase U2 digestion products were found to terminate in both 2',3'-cyclic phosphates and 3'-linear phosphates. To generate a homogeneous 3'-linear phosphate set of products, an enzymatic approach was investigated. Bacteriophage lambda protein phosphatase was identified as the optimal enzyme for hydrolyzing cyclic phosphates from RNase U2 products. The compatibility of this enzymatic approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) RNA modification mapping was then demonstrated. RNase U2 digestion followed by subsequent phosphatase treatment generated nearly 100% 3'-phosphate-containing products that could be characterized by LC-MS/MS. In addition, bacteriophage lambda protein phosphatase can be used to introduce (18)O labels within the 3'-phosphate of digestion products when incubated in the presence of H2(18)O, allowing prior isotope labeling methods for mass spectrometry to include digestion products from RNase U2. PMID:25797349

  12. Asymmetric friction of non-motor MAPs can lead to their directional motion in active microtubule networks

    PubMed Central

    Forth, Scott; Hsia, Kuo-Chiang; Shimamoto, Yuta; Kapoor, Tarun M.

    2014-01-01

    Summary Diverse cellular processes require microtubules to be organized into distinct structures, such as asters or bundles. Within these dynamic motifs, microtubule-associated proteins (MAPs) are frequently under load, but how force modulates these proteins’ function is poorly understood. Here, we combine optical-trapping with TIRF-based microscopy to measure the force-dependence of microtubule interaction for three non-motor MAPs (NuMA, PRC1, and EB1) required for cell division. We find that frictional forces increase non-linearly with MAP velocity across microtubules and depend on filament polarity, with NuMA’s friction being lower when moving towards minus-ends, EB1’s lower towards plus-ends, and PRC1 exhibiting no directional preference. Mathematical models predict, and experiments confirm, that MAPs with asymmetric friction can move directionally within active microtubule pairs they crosslink. Our findings reveal how non-motor MAPs can generate frictional resistance in dynamic cytoskeletal networks via micromechanical adaptations whose anisotropy may be optimized for MAP localization and function within cellular structures. PMID:24725408

  13. Spa2p functions as a scaffold-like protein to recruit the Mpk1p MAP kinase module to sites of polarized growth.

    PubMed

    van Drogen, Frank; Peter, Matthias

    2002-10-01

    Scaffold proteins play a major role in regulating MAP kinase pathways. In yeast, the Mpk1p-MAP kinase pathway functions to maintain the integrity of the cytoskeleton and the cell wall. In this module, the MEKK Bck1p functions upstream of the MEKs Mkk1p and Mkk2p, which in turn activate the MAP kinase Mpk1p. Mpk1p regulates several nuclear targets, including the transcription factors Rlm1p and SBF, and the two HMG1-like proteins NHP6A and NHP6B. Here we show that Mpk1p constitutively shuttles between the nucleus and the cytoplasm, and both Mpk1p and Mkk1p localize to sites of polarized growth in a Spa2p-dependent manner. Spa2p belongs to a group of proteins that includes Bni1p, Bud6p, and Pea2p, which are involved in the dynamic organization of the actin cytoskeleton during polarized growth. FRAP analysis shows that Spa2p-GFP is stably anchored at bud tips, whereas Mpk1p binds transiently. Spa2p interacts with Mkk1p and Mpk1p, and membrane bound Spa2p is sufficient to recruit Mkk1p and Mpk1p but not other MAP kinases to the cell cortex. Taken together, these results suggest that Spa2p functions as a scaffold-like protein for the cell wall integrity pathway during polarized growth. PMID:12361575

  14. Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase

    PubMed Central

    Mandal, Goutam; Sharma, Mansi; Kruse, Martin; Sander-Juelch, Claudia; Munro, Laura Anne; Wang, Yong; Vilg, Jenny Veide; Tamás, Markus J; Bhattacharjee, Hiranmoy; Wiese, Martin; Mukhopadhyay, Rita

    2012-01-01

    Summary Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defense against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen-activated protein kinase, LmjMPK2. Leishmania parasites co-expressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr197 and this phosphorylation requires LmjMPK2 activity. Lys42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. L. mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a MAP kinase. PMID:22779703

  15. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways.

    PubMed

    Qiao, Xiaoyong; Yong Qiao, Xiao; Nie, Ying; Ma, Yaxian; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yin, Weiyao; Yao Yinrg, Wei; Hu, Ying; Xu, Wenming; Ming Xu, Wen; Xu, Liangzhi; Zhi Xu, Liang

    2016-01-01

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued. PMID:26738434

  16. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways

    PubMed Central

    Yong Qiao, Xiao; Nie, Ying; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yao Yinrg, Wei; Hu, Ying; Ming Xu, Wen; Zhi Xu, Liang

    2016-01-01

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued. PMID:26738434

  17. MapA, an iron-regulated, cytoplasmic membrane protein in the cyanobacterium Synechococcus sp. strain PCC7942.

    PubMed Central

    Webb, R; Troyan, T; Sherman, D; Sherman, L A

    1994-01-01

    Growth of Synechococcus sp. strain PCC 7942 in iron-deficient media leads to the accumulation of an approximately 34-kDa protein. The gene encoding this protein, mapA (membrane-associated protein A), has been cloned and sequenced (GenBank accession number, L01621). The mapA transcript is not detectable in normally grown cultures but is stably accumulated by cells grown in iron-deficient media. However, the promoter sequence for this gene does not resemble other bacterial iron-regulated promoters described to date. The carboxyl-terminal region of the derived amino acid sequence of MapA resembles bacterial proteins involved in iron acquisition, whereas the amino-terminal end of MapA has a high degree of amino acid identity with the abundant, chloroplast envelope protein E37. An approach employing improved cellular fractionation techniques as well as electron microscopy and immunocytochemistry was essential in localizing MapA protein to the cytoplasmic membrane of Synechococcus sp. strain PCC 7942. When these cells were grown under iron-deficient conditions, a significant fraction of MapA could also be localized to the thylakoid membranes. Images PMID:8051004

  18. LRR Conservation Mapping to Predict Functional Sites within Protein Leucine-Rich Repeat Domains

    PubMed Central

    Helft, Laura; Reddy, Vignyan; Chen, Xiyang; Koller, Teresa; Federici, Luca; Fernández-Recio, Juan; Gupta, Rishabh; Bent, Andrew

    2011-01-01

    Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs) are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM), a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP) receptors, EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2). In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area), Consurf, and PAML (positive selection) analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/RCM, and the approach should be modifiable for use with other types of repeat protein domains. PMID:21789174

  19. AlgPred: prediction of allergenic proteins and mapping of IgE epitopes

    PubMed Central

    Saha, Sudipto; Raghava, G. P. S.

    2006-01-01

    In this study a systematic attempt has been made to integrate various approaches in order to predict allergenic proteins with high accuracy. The dataset used for testing and training consists of 578 allergens and 700 non-allergens obtained from A. K. Bjorklund, D. Soeria-Atmadja, A. Zorzet, U. Hammerling and M. G. Gustafsson (2005) Bioinformatics, 21, 39–50. First, we developed methods based on support vector machine using amino acid and dipeptide composition and achieved an accuracy of 85.02 and 84.00%, respectively. Second, a motif-based method has been developed using MEME/MAST software that achieved sensitivity of 93.94 with 33.34% specificity. Third, a database of known IgE epitopes was searched and this predicted allergenic proteins with 17.47% sensitivity at specificity of 98.14%. Fourth, we predicted allergenic proteins by performing BLAST search against allergen representative peptides. Finally hybrid approaches have been developed, which combine two or more than two approaches. The performance of all these algorithms has been evaluated on an independent dataset of 323 allergens and on 101 725 non-allergens obtained from Swiss-Prot. A web server AlgPred has been developed for the predicting allergenic proteins and for mapping IgE epitopes on allergenic proteins (). AlgPred is available at . PMID:16844994

  20. Mapping the Ligand-Binding Region of Borrelia hermsii Fibronectin-Binding Protein

    PubMed Central

    Brenner, Christiane; Bomans, Katharina; Habicht, Jüri; Simon, Markus M.; Wallich, Reinhard

    2013-01-01

    Many pathogenic microorganisms express fibronectin-binding molecules that facilitate their adherence to the extracellular matrix and/or entry into mammalian cells. We have previously described a Borrelia recurrentis gene, cihC that encodes a 40-kDa surface receptor for both, fibronectin and the complement inhibitors C4bp and C1-Inh. We now provide evidence for the expression of a group of highly homologues surface proteins, termed FbpA, in three B. hermsii isolates and two tick-borne relapsing fever spirochetes, B. parkeri and B. turicatae. When expressed in Escherichia coli or B. burgdorferi, four out of five proteins were shown to selectively bind fibronectin, whereas none of five proteins were able to bind the human complement regulators, C4bp and C1-Inh. By applying deletion mutants of the B. hermsii fibronectin-binding proteins a putative high-affinity binding site for fibronectin was mapped to its central region. In addition, the fibronectin-binding proteins of B. hermsii were found to share sequence homology with BBK32 of the Lyme disease spirochete B. burgdorferi with similar function suggesting its involvement in persistence and/or virulence of relapsing fever spirochetes. PMID:23658828

  1. Label-free Raman mapping of surface distribution of protein a and IgG biomolecules.

    PubMed

    Combs, Zachary A; Chang, Sehoon; Clark, Tolecia; Singamaneni, Srikanth; Anderson, Kyle D; Tsukruk, Vladimir V

    2011-03-15

    We have demonstrated a nanoengineered substrate composed of micropatterned silver nanoparticles to be used for the label-free mapping of adsorbed biomolecules. We utilized surface-enhanced Raman scattering (SERS) phenomenon to monitor the known bioanalytes, protein A and human immunoglobulin G (IgG). The SERS substrate was composed of a poly(alylamine hydrochloride) (PAH)/poly(styrenesulfonate) (PSS) layer-by-layer (LbL) nanocoating micropatterned with silver nanoparticles confined to microscopic stripes. Selective adsorption of biomacromolecules is facilitated by the amine-terminated LbL nanocoating, which prevents the surface adsorption of positively charged protein A across the surface except on the patterned regions containing negatively charged silver nanoparticles. Furthermore, adsorption of IgG on predetermined regions is facilitated by the selective binding of the Fc region of IgG to protein A. This label-free SERS approach provides accurate, selective, and fast detection of protein A and IgG solutions with a nanomolar concentration, down to below 1 nM for IgG in solution. This method could also be utilized for the facile detection of proteins in field conditions as well as in clinical, forensic, industrial, and environmental laboratories. PMID:21294559

  2. Modeling Protein Folding and Applying It to a Relevant Activity

    ERIC Educational Resources Information Center

    Nelson, Allan; Goetze, Jim

    2004-01-01

    The different levels of protein structure that can be easily understood by creating a model that simulates protein folding, which can then be evaluated by applying it to a relevant activity, is presented. The materials required and the procedure for constructing a protein folding model are mentioned.

  3. The U.S. Geological Survey mapping and cartographic database activities, 2006-2010

    USGS Publications Warehouse

    Craun, Kari J.; Donnelly, John P.; Allord, Gregory J.

    2011-01-01

    The U.S. Geological Survey (USGS) began systematic topographic mapping of the United States in the 1880s, beginning with scales of 1:250,000 and 1:125,000 in support of geological mapping. Responding to the need for higher resolution and more detail, the 1:62,500-scale, 15-minute, topographic map series was begun in the beginning of the 20th century. Finally, in the 1950s the USGS adopted the 1:24,000-scale, 7.5-minute topographic map series to portray even more detail, completing the coverage of the conterminous 48 states of the United States with this series in 1992. In 2001, the USGS developed the vision and concept of The National Map, a topographic database for the 21st century and the source for a new generation of topographic maps (http://nationalmap.gov/). In 2008, the initial production of those maps began with a 1:24,000-scale digital product. In a separate, but related project, the USGS began scanning the existing inventory of historical topographic maps at all scales to accompany the new topographic maps. The USGS also had developed a digital database of The National Atlas of the United States. The digital version of Atlas is now Web-available and supports a mapping engine for small scale maps of the United States and North America. These three efforts define topographic mapping activities of the USGS during the last few years and are discussed below.

  4. Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase.

    PubMed Central

    Villalba, M; Pajares, M A; Renart, M F; Mato, J M

    1987-01-01

    When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed. Images Fig. 1. Fig. 4. PMID:3593229

  5. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  6. An Experimental Approach to Controllably Vary Protein Oxidation While Minimizing Electrode Adsorption for Boron-Doped Diamond Electrochemical Surface Mapping Applications

    PubMed Central

    McClintock, Carlee S; Hettich, Robert L.

    2012-01-01

    Oxidative protein surface mapping has become a powerful approach for measuring the solvent accessibility of folded protein structures. A variety of techniques exist for generating the key reagent – hydroxyl radicals – for these measurements; however, these approaches range significantly in their complexity and expense of operation. This research expands upon earlier work to enhance the controllability of boron-doped diamond (BDD) electrochemistry as an easily accessible tool for producing hydroxyl radicals in order to oxidize a range of intact proteins. Efforts to modulate oxidation level while minimizing the adsorption of protein to the electrode involved the use of relatively high flow rates to reduce protein residence time inside the electrochemical flow chamber. Additionally, a different cell activation approach using variable voltage to supply a controlled current allowed us to precisely tune the extent of oxidation in a protein-dependent manner. In order to gain perspective on the level of protein adsorption onto the electrode surface, studies were conducted to monitor protein concentration during electrolysis and gauge changes in the electrode surface between cell activation events. This report demonstrates the successful use of BDD electrochemistry for greater precision in generating a target number of oxidation events upon intact proteins. PMID:23210708

  7. Global Analysis of Protein Activities Using Proteome Chips

    NASA Astrophysics Data System (ADS)

    Zhu, Heng; Bilgin, Metin; Bangham, Rhonda; Hall, David; Casamayor, Antonio; Bertone, Paul; Lan, Ning; Jansen, Ronald; Bidlingmaier, Scott; Houfek, Thomas; Mitchell, Tom; Miller, Perry; Dean, Ralph A.; Gerstein, Mark; Snyder, Michael

    2001-09-01

    To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

  8. Mapping of the regions involved in self-interaction of rice stripe virus P3 protein.

    PubMed

    Zhao, S L; Hao, J H; Xue, Y N; Liang, C Y

    2016-03-01

    Rice stripe virus (RSV) protein P3 is a suppressor of RNA silencing in plants. P3 has been shown by biomolecular fluorescence complementation assay to self-interact in planta but the regions responsible for homotypic interaction have not been determined. Here we analyzed the domains for the self-interaction of P3 by using yeast two-hybrid, co-immunoprecipitation and fluorescence experiments. The results showed that P3 was also able to interact with itself in yeast and insect cells. The domain responsible for P3-P3 interaction was mapped to amino acids 15-30 at the N-terminal region of P3. Furthermore, subcellular localization suggested that the homo-oligomerization was the prerequisite for P3 to form larger protein aggregates in the nucleus of insect cell. PMID:26982473

  9. Genomic organization of the human VP16 accessory protein, a housekeeping gene (HCFC1) mapping to Xq28

    SciTech Connect

    Frattini, A.; Faranda, S.; Redolfi, E.

    1994-09-01

    The region between DXS52 and Factor VIII gene in the human Xq28 chromosomal band contains a G+C-rich isochore to which many genes have been mapped. We report here the isolation and characterization of a transcript mapping about 50 kb telomeric from the vasopressin type 2 receptor gene in a 180-kb YAC/cosmid contig containing the L1CAM gene at its centromeric end. The determined transcribed sequence from a human fetal brain library is identical to that of the recently identified accessory protein HCFC1 (host cell factor, also called C1) that activates herpes simplex virus VP16 ({alpha}TIF) transactivator protein for association with the octamer motif-binding protein Oct-1. The gene is expressed in a ubiquitous pattern and a larger transcript of approximately 10 kb is present in all the tissues tested, while an alternatively spliced RNA of approximately 8.0 kb is present in muscle and heart tissues. Genomic sequencing allowed us to determine that the sequenced transcript is assembled from 26 exons spread over a relatively small genomic region of approximately 24 kb. This allowed us to determine that a previously reported cDNA clone arises from the splicing out of an internal portion of exon 8 which does not change the reading frame. All together these results raise the possibility that alternative mRNA processing could partly contribute to the diversity of the polypeptide HCFC1 family in a subset of tissues. 31 refs., 3 figs., 1 tab.

  10. Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition

    PubMed Central

    Goodier, John L.; Cheung, Ling E.; Kazazian, Haig H.

    2013-01-01

    LINE1s occupy 17% of the human genome and are its only active autonomous mobile DNA. L1s are also responsible for genomic insertion of processed pseudogenes and >1 million non-autonomous retrotransposons (Alus and SVAs). These elements have significant effects on gene organization and expression. Despite the importance of retrotransposons for genome evolution, much about their biology remains unknown, including cellular factors involved in the complex processes of retrotransposition and forming and transporting L1 ribonucleoprotein particles. By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein. These include RNA transport proteins, gene expression regulators, post-translational modifiers, helicases and splicing factors. Many cellular proteins co-localize with L1 ORF1 protein in cytoplasmic granules. We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses. These data suggest candidate cofactors that interact with the L1 to modulate its activity and increase our understanding of the means by which the cell coexists with these genomic ‘parasites’. PMID:23749060

  11. Mycobacterium avium subsp. paratuberculosis PPE Protein MAP1152 and Conserved Protein MAP1156 are Antigenic in Experimentally and Naturally Infected Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s Disease (JD) in ruminants resulting in significant production losses. An insertion mutation upstream from the MAP1152-MAP1156 region causes a change in colony morphotype and results in an attenuated phenotype in bovine monocyte derive...

  12. Building Disease-Specific Drug-Protein Connectivity Maps from Molecular Interaction Networks and PubMed Abstracts

    PubMed Central

    Li, Jiao; Zhu, Xiaoyan; Chen, Jake Yue

    2009-01-01

    The recently proposed concept of molecular connectivity maps enables researchers to integrate experimental measurements of genes, proteins, metabolites, and drug compounds under similar biological conditions. The study of these maps provides opportunities for future toxicogenomics and drug discovery applications. We developed a computational framework to build disease-specific drug-protein connectivity maps. We integrated gene/protein and drug connectivity information based on protein interaction networks and literature mining, without requiring gene expression profile information derived from drug perturbation experiments on disease samples. We described the development and application of this computational framework using Alzheimer's Disease (AD) as a primary example in three steps. First, molecular interaction networks were incorporated to reduce bias and improve relevance of AD seed proteins. Second, PubMed abstracts were used to retrieve enriched drug terms that are indirectly associated with AD through molecular mechanistic studies. Third and lastly, a comprehensive AD connectivity map was created by relating enriched drugs and related proteins in literature. We showed that this molecular connectivity map development approach outperformed both curated drug target databases and conventional information retrieval systems. Our initial explorations of the AD connectivity map yielded a new hypothesis that diltiazem and quinidine may be investigated as candidate drugs for AD treatment. Molecular connectivity maps derived computationally can help study molecular signature differences between different classes of drugs in specific disease contexts. To achieve overall good data coverage and quality, a series of statistical methods have been developed to overcome high levels of data noise in biological networks and literature mining results. Further development of computational molecular connectivity maps to cover major disease areas will likely set up a new model for

  13. Building disease-specific drug-protein connectivity maps from molecular interaction networks and PubMed abstracts.

    PubMed

    Li, Jiao; Zhu, Xiaoyan; Chen, Jake Yue

    2009-07-01

    The recently proposed concept of molecular connectivity maps enables researchers to integrate experimental measurements of genes, proteins, metabolites, and drug compounds under similar biological conditions. The study of these maps provides opportunities for future toxicogenomics and drug discovery applications. We developed a computational framework to build disease-specific drug-protein connectivity maps. We integrated gene/protein and drug connectivity information based on protein interaction networks and literature mining, without requiring gene expression profile information derived from drug perturbation experiments on disease samples. We described the development and application of this computational framework using Alzheimer's Disease (AD) as a primary example in three steps. First, molecular interaction networks were incorporated to reduce bias and improve relevance of AD seed proteins. Second, PubMed abstracts were used to retrieve enriched drug terms that are indirectly associated with AD through molecular mechanistic studies. Third and lastly, a comprehensive AD connectivity map was created by relating enriched drugs and related proteins in literature. We showed that this molecular connectivity map development approach outperformed both curated drug target databases and conventional information retrieval systems. Our initial explorations of the AD connectivity map yielded a new hypothesis that diltiazem and quinidine may be investigated as candidate drugs for AD treatment. Molecular connectivity maps derived computationally can help study molecular signature differences between different classes of drugs in specific disease contexts. To achieve overall good data coverage and quality, a series of statistical methods have been developed to overcome high levels of data noise in biological networks and literature mining results. Further development of computational molecular connectivity maps to cover major disease areas will likely set up a new model for

  14. Stochastic Interaction between Neural Activity and Molecular Cues in the Formation of Topographic Maps.

    PubMed

    Owens, Melinda T; Feldheim, David A; Stryker, Michael P; Triplett, Jason W

    2015-09-23

    Topographic maps in visual processing areas maintain the spatial order of the visual world. Molecular cues and neuronal activity both play critical roles in map formation, but their interaction remains unclear. Here, we demonstrate that when molecular- and activity-dependent cues are rendered nearly equal in force, they drive topographic mapping stochastically. The functional and anatomical representation of azimuth in the superior colliculus of heterozygous Islet2-EphA3 knockin (Isl2(EphA3/+)) mice is variable: maps may be single, duplicated, or a combination of the two. This heterogeneity is not due to genetic differences, since map organizations in individual mutant animals often differ between colliculi. Disruption of spontaneous waves of retinal activity resulted in uniform map organization in Isl2(EphA3/+) mice, demonstrating that correlated spontaneous activity is required for map heterogeneity. Computational modeling replicates this heterogeneity, revealing that molecular- and activity-dependent forces interact simultaneously and stochastically during topographic map formation. PMID:26402608

  15. DIETARY PROTEIN AND LACTOSE INCREASE TRANSLATION INITIATION FACTOR ACTIVATION AND TISSUE PROTEIN SYNTHESIS IN NEONATAL PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in muscle and liver of pigs parenterally infused with amino acids and insulin. To examine the effects of enteral protein and carbohydrate on protein synthesis, pigs (n = 42, 1.7 kg body wt) were fed isocaloric milk die...

  16. Retrieving the intracellular topology from multi-scale protein mobility mapping in living cells

    PubMed Central

    Baum, Michael; Erdel, Fabian; Wachsmuth, Malte; Rippe, Karsten

    2014-01-01

    In living cells, most proteins diffuse over distances of micrometres within seconds. Protein translocation is constrained due to the cellular organization into subcompartments that impose diffusion barriers and guide enzymatic activities to their targets. Here, we introduce an approach to retrieve structural features from the scale-dependent mobility of green fluorescent protein monomer and multimers in human cells. We measure protein transport simultaneously between hundreds of positions by multi-scale fluorescence cross-correlation spectroscopy using a line-illuminating confocal microscope. From these data we derive a quantitative model of the intracellular architecture that resembles a random obstacle network for diffusing proteins. This topology partitions the cellular content and increases the dwell time of proteins in their local environment. The accessibility of obstacle surfaces depends on protein size. Our method links multi-scale mobility measurements with a quantitative description of intracellular structure that can be applied to evaluate how drug-induced perturbations affect protein transport and interactions. PMID:25058002

  17. Instantaneous, parallel mapping of protein electronic function with angle-resolved coherent wave-mixing

    NASA Astrophysics Data System (ADS)

    Mercer, Ian

    2010-03-01

    We present a novel laser method, angle-resolved coherent (ARC) wave-mixing, that separates out coherent electronic couplings from energy transfers in an instantaneous two-dimensional mapping (Ian P. Mercer et.al., Phys. Rev. Lett. 102, 57402, 2009). For this we use an ultra-broadband hollow fibre laser source. The power of the new method is demonstrated with the light harvesting complex II (LH2) of purple bacteria at ambient temperature. We observe signaturs of a coherent quantum electronic beating, a correlation between excitation and emission energies in the protein and a coherent component to the energy transfer between molecular rings. We are interested in exploring avenues for high throughput fingerprinting of molecular structure and function. Massively parallel maps, rich in detail, can be taken from solutions, surface films or solids of between 1 and 1000 microL. Each ARC map is generated instantaneously, with high throughput (currently up to 1kHz frame rate) and is noninvasive.

  18. Analyzing Large-Scale Structural Change in Proteins: Comparison of Principal Component Projection and Sammon Mapping

    SciTech Connect

    Mesentean, Sidonia; Fischer, S.; Smith, Jeremy C

    2006-04-01

    Effective analysis of large-scale conformational transitions in macromolecules requires transforming them into a lower dimensional representation that captures the dominant motions. Herein, we apply and compare two different dimensionality reduction techniques, namely, principal component analysis (PCA), a linear method, and Sammon mapping, which is nonlinear. The two methods are used to analyze four different protein transition pathways of varying complexity, obtained by using either the conjugate peak refinement method or constrained molecular dynamics. For the return-stroke in myosin, both Sammon mapping and PCA show that the conformational change is dominated by a simple rotation of a rigid body. Also, in the case of the T{yields}R transition in hemoglobin, both methods are able to identify the two main quaternary transition events. In contrast, in the cases of the unfolding transition of staphylococcal nuclease or the signaling switch of Ras p21, which are both more complex conformational transitions, only Sammon mapping is able to identify the distinct phases of motion.

  19. Bone morphogenetic proteins, eye patterning, and retinocollicular map formation in the mouse

    PubMed Central

    Plas, Daniel T.; Dhande, Onkar; Lopez, Joshua E.; Murali, Deepa; Thaller, Christina; Henkemeyer, Mark; Furuta, Yasuhide; Overbeek, Paul; Crair, Michael C.

    2009-01-01

    Patterning events during early eye formation determine retinal cell fate and can dictate the behavior of retinal ganglion cell (RGC) axons as they navigate toward central brain targets. The temporally and spatially regulated expression of bone morphogenetic proteins (BMPs) and their receptors in the retina are thought to play a key role in this process, initiating gene expression cascades that distinguish different regions of the retina, particularly along the dorsoventral axis. Here, we examine the role of BMP and a potential downstream effector, EphB, in retinotopic map formation in the lateral geniculate nucleus (LGN) and superior colliculus (SC). RGC axon behaviors during retinotopic map formation in wild type mice are compared with those in several strains of mice with engineered defects of BMP and EphB signaling. Normal RGC axon sorting produces axon order in the optic tract that reflects the dorsoventral position of the parent RGCs in the eye. A dramatic consequence of disrupting BMP signaling is a missorting of RGC axons as they exit the optic chiasm. This sorting is not dependent on EphB. When BMP signaling in the developing eye is genetically modified, RGC order in the optic tract and targeting in the LGN and SC are correspondingly disrupted. These experiments show that BMP signaling regulates dorsoventral RGC cell fate, RGC axon behavior in the ascending optic tract and retinotopic map formation in the LGN and SC through mechanisms that are in part distinct from EphB signaling in the LGN and SC. PMID:18614674

  20. Mass mapping of a protein complex with the scanning transmission electron microscope.

    PubMed Central

    Engel, A; Baumeister, W; Saxton, W O

    1982-01-01

    A mass map of the hexagonally packed intermediate layer (HPI-layer), a regular protein monolayer from the cell envelope of Micrococcus radiodurans, has been obtained by scanning transmission electron microscopy. Samples were freeze-dried within the microscope, and low-dose images were recorded in the dark-field mode directly in digital form and processed by correlation averaging. The averaged projection of the unstained structure--i.e., the mass map--thus calculated shows a resolution to 3-nm period and reveals morphological features consistent with those obtained by negative staining. The mass of individual morphological domains was extracted by using variously the mass map itself or an average from a negatively stained HPI layer to define the domain boundaries. Protrusions as small as 1,300 daltons could be measured reproducibly within the unit cell of 655,000 daltons. The method developed opens an avenue to identify molecular species in situ and to correlate topographic information with biochemical data. Images PMID:6955791

  1. A combined binary interaction and phenotypic map of C. elegans cell polarity proteins

    PubMed Central

    Koorman, Thijs; Lemmens, Irma; Ramalho, João J.; Nieuwenhuize, Susan; van den Heuvel, Sander; Tavernier, Jan; Nance, Jeremy; Boxem, Mike

    2015-01-01

    The establishment of cell polarity is an essential process for the development of multicellular organisms and the functioning of cells and tissues. Here, we combine large-scale protein interaction mapping with systematic phenotypic profiling to study the network of physical interactions that underlies polarity establishment and maintenance in the nematode Caenorhabditis elegans. Using a fragment-based yeast two-hybrid strategy, we identified 439 interactions between 296 proteins, as well as the protein regions that mediate these interactions. Phenotypic profiling of the network resulted in the identification of 100 physically interacting protein pairs for which RNAi-mediated depletion caused a defect in the same polarity-related process. We demonstrate the predictive capabilities of the network by showing that the physical interaction between the RhoGAP PAC-1 and PAR-6 is required for radial polarization of the C. elegans embryo. Our network represents a valuable resource of candidate interactions that can be used to further our insight into cell polarization. PMID:26780296

  2. Site-specific mapping and quantification of protein S-sulphenylation in cells.

    PubMed

    Yang, Jing; Gupta, Vinayak; Carroll, Kate S; Liebler, Daniel C

    2014-01-01

    Cysteine S-sulphenylation provides redox regulation of protein functions, but the global cellular impact of this transient post-translational modification remains unexplored. We describe a chemoproteomic workflow to map and quantify over 1,000 S-sulphenylation sites on more than 700 proteins in intact cells. Quantitative analysis of human cells stimulated with hydrogen peroxide or epidermal growth factor measured hundreds of site selective redox changes. Different cysteines in the same proteins displayed dramatic differences in susceptibility to S-sulphenylation. Newly discovered S-sulphenylations provided mechanistic support for proposed cysteine redox reactions and suggested novel redox mechanisms, including S-sulphenyl-mediated redox regulation of the transcription factor HIF1A by SIRT6. S-sulphenylation is favored at solvent-exposed protein surfaces and is associated with sequence motifs that are distinct from those for other thiol modifications. S-sulphenylations affect regulators of phosphorylation, acetylation and ubiquitylation, which suggest regulatory crosstalk between redox control and signalling pathways. PMID:25175731

  3. Imaging metals in proteins by combining electrophoresis with rapid x-ray fluorescence mapping.

    SciTech Connect

    Finney, L.; Chishti, Y.; Khare, T.; Giometti, C.; Levina, A.; Lay, P. A.; Vogt, S.; Univ. of Sydney; Northwestern Univ.

    2010-01-01

    Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal?protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis.

  4. Imaging metals in proteins by combining electrophoresis with rapid x-ray fluorescence mapping.

    PubMed

    Finney, Lydia; Chishti, Yasmin; Khare, Tripti; Giometti, Carol; Levina, Aviva; Lay, Peter A; Vogt, Stefan

    2010-06-18

    Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal-protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis. PMID:20392082

  5. Four paralogous protein 4.1 genes map to distinct chromosomes in mouse and human.

    PubMed

    Peters, L L; Weier, H U; Walensky, L D; Snyder, S H; Parra, M; Mohandas, N; Conboy, J G

    1998-12-01

    Four highly conserved members of the skeletal protein 4.1 gene family encode a diverse array of protein isoforms via tissue-specific transcription and developmentally regulated alternative pre-mRNA splicing. In addition to the prototypical red blood cell 4.1R (human gene symbol EPB41,) these include two homologues that are strongly expressed in the brain (4.1N, EPB41L1; and 4.1B, EPB41L3) and another that is widely expressed in many tissues (4.1G, EPB41L2). As part of a study on the structure and evolution of the 4.1 genes in human and mouse, we have now completed the chromosomal mapping of their respective loci by reporting the localization of mouse 4.1N, 4.1G, and 4.1B, as well as human 4.1B. For the mouse 4.1 genes, Southern blot analysis of RFLPs in The Jackson Laboratory BSS interspecific backcross yielded the following assignments: 4.1N (Epb4.1l1,) chromosome 2; 4.1G (Epb4.1l2,) chromosome 10; and 4.1B (Epb4.1l3,) mouse chromosome 17. Human 4.1B was physically mapped to chromosome 18p11 using fluorescence in situ hybridization. All of the mouse genes mapped within or adjacent to regions of conserved synteny with corresponding human chromosomes. We conclude that a set of four paralogous 4.1 genes has been evolutionarily conserved in rodents and primates. PMID:9828140

  6. Effects of spatial variation of skull and cerebrospinal fluid layers on optical mapping of brain activities

    NASA Astrophysics Data System (ADS)

    Wang, Shuping; Shibahara, Nanae; Kuramashi, Daishi; Okawa, Shinpei; Kakuta, Naoto; Okada, Eiji; Maki, Atsushi; Yamada, Yukio

    2010-07-01

    In order to investigate the effects of anatomical variation in human heads on the optical mapping of brain activity, we perform simulations of optical mapping by solving the photon diffusion equation for layered-models simulating human heads using the finite element method (FEM). Particularly, the effects of the spatial variations in the thicknesses of the skull and cerebrospinal fluid (CSF) layers on mapping images are investigated. Mapping images of single active regions in the gray matter layer are affected by the spatial variations in the skull and CSF layer thicknesses, although the effects are smaller than those of the positions of the active region relative to the data points. The increase in the skull thickness decreases the sensitivity of the images to active regions, while the increase in the CSF layer thickness increases the sensitivity in general. The images of multiple active regions are also influenced by their positions relative to the data points and by their depths from the skin surface.

  7. Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases.

    PubMed

    Zeke, András; Bastys, Tomas; Alexa, Anita; Garai, Ágnes; Mészáros, Bálint; Kirsch, Klára; Dosztányi, Zsuzsanna; Kalinina, Olga V; Reményi, Attila

    2015-11-01

    Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles. PMID:26538579

  8. Poliovirus protein 2C has ATPase and GTPase activities.

    PubMed

    Rodríguez, P L; Carrasco, L

    1993-04-15

    Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence. We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA. Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP). The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region. Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C. Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted. The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity. Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein. Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255. None of the fusion proteins had detectable helicase activity. We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes. These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids. PMID:8385138

  9. MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne's Disease

    PubMed Central

    Lingle, Cari K.; Stabel, Judith R.; Ramyar, Kasra X.; Garcia, Brandon L.; Raeber, Alex J.; Schacher, Pascal; Kapur, Vivek; Geisbrecht, Brian V.

    2012-01-01

    The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis. PMID:22593240

  10. Protein determination in seeds by proton activation

    NASA Astrophysics Data System (ADS)

    Morales, J. R.; Dinator, M. I.; Cerda, P.

    1989-04-01

    A proton beam of 6.6 MeV has been used to produce 11C and 13N in Araucaria Araucana seeds. Their positron decay allows determination of the N/C ratio. In seeds the nitrogen content is associated to proteins while carbon is spread in the organic material. Samples were irradiated for about 10 min with a beam intensity of 5 nA on areas of 1 mm 2. Slices of the seed were radially explored, showing a larger concentration of protein in the center.

  11. Real-time, time-frequency mapping of event-related cortical activation

    NASA Astrophysics Data System (ADS)

    Cheung, Connie; Chang, Edward F.

    2012-08-01

    Functional mapping of eloquent cortex is a common and necessary component of neurosurgical operative planning. Current electrical stimulation-based techniques are inefficient, can evoke seizures and are prone to false-negative results. Here, we present a novel cortical mapping system that extracts event-related neural activity from passive electrocorticographic recordings to quickly and accurately localize sensory and motor cortices using the precise temporal properties of spectral alteration. This procedure generates a robust functional motor and sensory cortical map in seconds, and usually with less than five to ten trial events. Our algorithm demonstrates high concordance with results derived using independent electrical cortical stimulation mapping.

  12. Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies.

    PubMed

    Hua, Rong-Hong; Liu, Li-Ke; Chen, Zhen-Shi; Li, Ye-Nan; Bu, Zhi-Gao

    2013-01-01

    Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues (5)AIDITRK(11), (72)RDELNVL(78), (251)KSKHNRREGY(260), (269)DENGIVLD(276), and (341)DETTLVRS(348). Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays. PMID:23825668

  13. Role of protein phosphatase 2A in the regulation of mitogen-activated protein kinase activity in ventricular cardiomyocytes.

    PubMed

    Braconi Quintaje, S; Church, D J; Rebsamen, M; Valloton, M B; Hemmings, B A; Lang, U

    1996-04-25

    Incubation of cultured, neonatal rat ventricular cardiomyocytes with 100 nM phorbol 12-myristate-13-acetate (PMA) induced a transient suppression of PP2A activity at 5 min, an effect that was reversed after 15 min of exposure to PMA. This inactivation was correlated with a transient increase in the phosphorylation level of the catalytic subunit of PP2A (193 +/- 38% of control levels at 5 min). Simultaneously to the transient inactivation of PP2A, we observed a rapid and reversible phosphorylation of 42-kDa MAP kinase (474 +/- 65% of control levels at 5 min, and 316 +/- 44% at 15 min) in cardiomyocytes treated with PMA. This transient phosphorylation was accompanied by a transient increase in cytosolic MAP kinase activity (209 +/- 17% of control values at 5 min and 125 +/- 7% at 15 min). Okadaic acid (1 microM ) completely blocked the decrease in the phosphorylation level and activity of MAP kinase occurring after 5 min of exposure to PMA. These data demonstrate that PP2A inactivation and MAP kinase activation are very strongly correlated in cardiomyocytes, indicating that PP2A plays a negative modulatory role in the regulation of MAP kinase activity. PMID:8629997

  14. Monitoring G protein activation in cells with BRET

    PubMed Central

    Masuho, Ikuo; Martemyanov, Kirill A.; Lambert, Nevin A.

    2016-01-01

    Summary Live-cell assays based on fluorescence and luminescence are now indispensable tools for the study of G protein signaling. Assays based on fluorescence and bioluminescence resonance energy transfer (FRET and BRET) have been particularly valuable for monitoring changes in second messengers, protein-protein interactions, and protein conformation. Here we describe a BRET assay that monitors the release of free Gβγ dimers after activation of heterotrimers containing Gα subunits from all four G protein subfamilies. This assay provides useful kinetic and pharmacological information with reasonably high throughput using standard laboratory equipment. PMID:26260597

  15. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    NASA Technical Reports Server (NTRS)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  16. Mapping of cavitational activity in a pilot plant dyeing equipment.

    PubMed

    Actis Grande, G; Giansetti, M; Pezzin, A; Rovero, G; Sicardi, S

    2015-11-01

    A large number of papers of the literature quote dyeing intensification based on the application of ultrasound (US) in the dyeing liquor. Mass transfer mechanisms are described and quantified, nevertheless these experimental results in general refer to small laboratory apparatuses with a capacity of a few hundred millilitres and extremely high volumetric energy intensity. With the strategy of overcoming the scale-up inaccuracy consequent to the technological application of ultrasounds, a dyeing pilot-plant prototype of suitable liquor capacity (about 40 L) and properly simulating several liquor to textile hydraulic relationships was designed by including US transducers with different geometries. Optimal dyeing may be obtained by optimising the distance between transducer and textile material, the liquid height being a non-negligible operating parameter. Hence, mapping the cavitation energy in the machinery is expected to provide basic data on the intensity and distribution of the ultrasonic field in the aqueous liquor. A flat ultrasonic transducer (absorbed electrical power of 600 W), equipped with eight devices emitting at 25 kHz, was mounted horizontally at the equipment bottom. Considering industrial scale dyeing, liquor and textile substrate are reciprocally displaced to achieve a uniform colouration. In this technology a non uniform US field could affect the dyeing evenness to a large extent; hence, mapping the cavitation energy distribution in the machinery is expected to provide fundamental data and define optimal operating conditions. Local values of the cavitation intensity were recorded by using a carefully calibrated Ultrasonic Energy Meter, which is able to measure the power per unit surface generated by the cavitation implosion of bubbles. More than 200 measurements were recorded to define the map at each horizontal plane positioned at a different distance from the US transducer; tap water was heated at the same temperature used for dyeing tests (60

  17. Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene.

    PubMed Central

    Guarino, L A; Summers, M D

    1986-01-01

    The temporal regulation of an early gene of the baculovirus Autographa californica nuclear polyhedrosis virus was examined. We constructed a plasmid (plasmid 39CAT) in which the bacterial gene for chloramphenicol acetyltransferase was placed under the control of the promoter for the gene for a A. californica nuclear polyhedrosis virus 39,000-dalton protein (39K). A transient expression assay of plasmid 39CAT revealed that the 39K gene was expressed in infected cells but not in uninfected cells, indicating that the 39K gene should be classified as a delayed-early gene. The 39K promoter also efficiently directed the synthesis of chloramphenicol acetyltransferase when the plasmid was cotransfected with viral DNA which had been restricted with several restriction enzymes. To map the location of the gene(s) required for the synthesis of 39K, plasmid 39CAT was cotransfected with purified restriction fragments of A. californica nuclear polyhedrosis virus DNA. Fragments which mapped between 90.7 and 100.8 map units induced plasmid 39CAT. Plasmid pEcoRI-B, containing EcoRI fragment B (90 to 100 map units), activated plasmid 39CAT. Functional mapping of plasmid pEcoRI-B indicated that the essential region was located between 95.0 and 97.5 map units. The 5' end of this gene was mapped, and the chloramphenicol acetyltransferase gene was inserted under the control of its promoter. Transient assay experiments indicated that the trans-acting regulatory gene was expressed in uninfected cells and is therefore an immediate-early gene. This gene was named IE-1. Images PMID:3944847

  18. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    PubMed Central

    Ockenga, Wymke; Kühne, Sina; Bocksberger, Simone; Banning, Antje; Tikkanen, Ritva

    2014-01-01

    Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation. PMID:25421240

  19. New constitutive latex osmotin-like proteins lacking antifungal activity.

    PubMed

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events. PMID:26231325

  20. Distribution of circular proteins in plants: large-scale mapping of cyclotides in the Violaceae

    PubMed Central

    Burman, Robert; Yeshak, Mariamawit Y.; Larsson, Sonny; Craik, David J.; Rosengren, K. Johan; Göransson, Ulf

    2015-01-01

    During the last decade there has been increasing interest in small circular proteins found in plants of the violet family (Violaceae). These so-called cyclotides consist of a circular chain of approximately 30 amino acids, including six cysteines forming three disulfide bonds, arranged in a cyclic cystine knot (CCK) motif. In this study we map the occurrence and distribution of cyclotides throughout the Violaceae. Plant material was obtained from herbarium sheets containing samples up to 200 years of age. Even the oldest specimens contained cyclotides in the preserved leaves, with no degradation products observable, confirming their place as one of the most stable proteins in nature. Over 200 samples covering 17 of the 23–31 genera in Violaceae were analyzed, and cyclotides were positively identified in 150 species. Each species contained a unique set of between one and 25 cyclotides, with many exclusive to individual plant species. We estimate the number of different cyclotides in the Violaceae to be 5000–25,000, and propose that cyclotides are ubiquitous among all Violaceae species. Twelve new cyclotides from six phylogenetically dispersed genera were sequenced. Furthermore, the first glycosylated derivatives of cyclotides were identified and characterized, further increasing the diversity and complexity of this unique protein family. PMID:26579135

  1. Two-dimensional protein map of an "ale"-brewing yeast strain: proteome dynamics during fermentation.

    PubMed

    Kobi, Dominique; Zugmeyer, Sandra; Potier, Serge; Jaquet-Gutfreund, Laurence

    2004-12-01

    The first protein map of an ale-fermenting yeast is presented in this paper: 205 spots corresponding to 133 different proteins were identified. Comparison of the proteome of this ale strain with a lager brewing yeast and the Saccharomyces cerevisiae strain S288c confirmed that this ale strain is much closer to S288c than the lager strain at the proteome level. The dynamics of the ale-brewing yeast proteome during production-scale fermentation was analysed at the beginning and end of the first and the third usage of the yeast (called generation in the brewing industry). During the first generation, most changes were related to the switch from aerobic propagation to anaerobic fermentation. Fewer changes were observed during the third generation but certain stress-response proteins such as Hsp26p, Ssa4p and Pnc1p exhibited constitutive expression in subsequent generations. The ale brewing yeast strain appears to be quite well adapted to fermentation conditions and stresses. PMID:15556083

  2. Mapping the leaf proteome of Miscanthus sinensis and its application to the identification of heat-responsive proteins.

    PubMed

    Sharmin, Shamima Akhtar; Alam, Iftekhar; Rahman, Md Atikur; Kim, Kyung-Hee; Kim, Yong-Goo; Lee, Byung-Hyun

    2013-09-01

    Miscanthus sinensis is a promising bioenergy crop; however, its genome is poorly represented in sequence databases. As an initial step in the comprehensive analysis of the M. sinensis proteome, we report a reference 2-DE protein map of the leaf. A total of 316 protein spots were excised from the gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or MALDI-TOF/TOF MS. Two hundred and thirty-two protein spots were identified, which are involved in a variety of cellular functions through distinct metabolic pathways. Functional annotation of the proteins revealed a nearly complete C3 and C4 cycle, starch and sugar synthesis pathway, glycolysis pathway, a significant portion of the pentose phosphate pathway, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, kaurene, chalcone, sesquiterpene and lignin biosynthesis. Other proteins belong to primary metabolism, transcription, protein synthesis, protein destination/storage, disease/defense, cell growth/division, transportation and signal transduction. To test the applicability of the constructed map, we studied the effect of heat stress on M. sinensis leaf proteome. Twenty-five protein spots were upregulated, five were newly induced and twenty-five spots were downregulated by heat treatment. The differentially accumulated proteins were involved in photosynthesis, energy metabolism, gene transcription, protein kinases and phosphatases, signal transduction, protein synthesis and heat shock responses. C4-specific pyruvate orthophosphate dikinase, Rubisco large subunit, Rubisco activase and some associated proteins were upregulated during heat stress and tend to restore upon recovery. Identification of these proteins provides some important clues regarding the way M. sinensis copes with hot climate. This work represents the first extensive proteomic description of M. sinensis and provides a reference map and heat

  3. Spatial Mapping of Protein Abundances in the Mouse Brain by Voxelation Integrated with High-Throughput Liquid Chromatography - Mass Spectrometry

    SciTech Connect

    Petyuk, Vladislav A; Qian, Weijun; Chin, Mark H; Wang, Haixing H; Livesay, Eric A; Monroe, Matthew E; Adkins, Joshua N; Jaitly, Navdeep; Anderson, David J; Camp, David G; Smith, Desmond J; Smith, Richard D

    2007-01-25

    Temporally and spatially resolved mapping of protein abundance patterns within the mammalian brain is of significant interest for understanding brain function and molecular etiologies of neurodegenerative diseases; however, such imaging efforts have been greatly challenged by complexity of the proteome, throughput and sensitivity of applied analytical methodologies, and accurate quantitation of protein abundances across the brain. Here, we describe a methodology for comprehensive spatial proteome mapping that addresses these challenges by employing voxelation integrated with automated microscale sample processing, high-throughput LC system coupled with high resolution Fourier transform ion cyclotron mass spectrometer and a “universal” stable isotope labeled reference sample approach for robust quantitation. We applied this methodology as a proof-of-concept trial for the analysis of protein distribution within a single coronal slice of a C57BL/6J mouse brain. For relative quantitation of the protein abundances across the slice, an 18O-isotopically labeled reference sample, derived from a whole control coronal slice from another mouse, was spiked into each voxel sample and stable isotopic intensity ratios were used to obtain measures of relative protein abundances. In total, we generated maps of protein abundance patterns for 1,028 proteins. The significant agreement of the protein distributions with previously reported data supports the validity of this methodology, which opens new opportunities for studying the spatial brain proteome and its dynamics during the course of disease progression and other important biological and associated health aspects in a discovery-driven fashion.

  4. Antibody epitopes on g protein-coupled receptors mapped with genetically encoded photoactivatable cross-linkers.

    PubMed

    Ray-Saha, Sarmistha; Huber, Thomas; Sakmar, Thomas P

    2014-03-01

    We developed a strategy for creating epitope maps of monoclonal antibodies (mAbs) that bind to G protein-coupled receptors (GPCRs) containing photo-cross-linkers. Using human CXC chemokine receptor 4 (CXCR4) as a model system, we genetically incorporated the photolabile unnatural amino acid p-azido-l-phenylalanine (azF) at various positions within extracellular loop 2 (EC2). We then mapped the interactions of the azF-CXCR4 variants with mAb 12G5 using targeted loss-of-function studies and photo-cross-linking in whole cells in a microplate-based format. We used a novel variation of a whole cell enzyme-linked immunosorbent assay to quantitate cross-linking efficiency. 12G5 cross-linked primarily to residues 184, 178, and 189 in EC2 of CXCR4. Mapping of the data to the crystal structure of CXCR4 showed a distinct mAb epitope footprint with the photo-cross-linked residues clustered around the loss-of-function sites. We also used the targeted photo-cross-linking approach to study the interaction of human CC chemokine receptor 5 (CCR5) with PRO 140, a humanized mAb that inhibits human immunodeficiency virus-1 cellular entry, and 2D7. The mAbs produced distinct cross-linking patterns on EC2 of CCR5. PRO 140 cross-linked primarily to residues 174 and 175 at the amino-terminal end of EC2, and 2D7 cross-linked mainly to residues 170, 176, and 184. These results were mapped to the recent crystal structure of CCR5 in complex with maraviroc, showing cross-linked residues at the tip of the maraviroc binding crevice formed by EC2. As a strategy for mapping mAb epitopes on GPCRs, our targeted photo-cross-linking method is complementary to loss-of-function mutagenesis results and should be especially useful for studying mAbs with discontinuous epitopes. PMID:24490954

  5. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-05-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. PMID:4004796

  6. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed Central

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-01-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. Images Fig. 1. Fig. 2. Fig. 7. Fig. 8. PMID:4004796

  7. Perceptual stability during dramatic changes in olfactory bulb activation maps and dramatic declines in activation amplitudes

    PubMed Central

    Homma, R.; Cohen, L. B.; Kosmidis, E. K.; Youngentob, S. L.

    2009-01-01

    We compared the concentration dependence of the ability of rats to identify odorants with the calcium signals in the nerve terminals of the olfactory receptor neurons. Although identification performance decreased with concentrations both above and below the training stimuli it remained well above random at all concentrations tested (between 0.0006% and 35% of saturated vapor). In contrast, the calcium signals in the same awake animals were much smaller than their maximum values at odorant concentrations less than 1% of saturated vapor. In addition, maps of activated glomeruli changed dramatically as odorant concentration was reduced. Thus perceptual stability exists in the face of dramatic changes in both the amplitude and the maps of the input to the olfactory bulb. The data for the concentration dependence of the response of the most sensitive glomeruli for each of five odorants was fitted with a Michaelis-Menten (Hill) equation. The fitted curves were extrapolated to odorant concentrations several orders of magnitude lower the smallest observed signals and suggest that the calcium response at low odorant concentrations is more than 1000 times smaller than the response at saturating odorant concentrations. We speculate that only a few spikes in olfactory sensory neurons may be sufficient for correct odorant identification. PMID:19291227

  8. The kinetics of ER fusion protein activation in vivo

    PubMed Central

    Wilson, Catherine H.; Gamper, Ivonne; Perfetto, Alessandra; Auw, Jeremy; Littlewood, Trevor D.; Evan, Gerard I.

    2014-01-01

    Reversibly switchable proteins are powerful tools with which to explore protein function in vitro and in vivo. For example, the activity of many proteins fused to the hormone-binding domain of the modified estrogen receptor (ERTAM) can be regulated by provision or removal of 4-hydroxytamoxifen (4-OHT). Despite the widespread use of ERTAM fusions in vivo, inadequate data are available as to the most efficacious routes for systemic tamoxifen delivery. In this study, we have used two well-characterised ERTAM fusion proteins, both reversibly activated by 4-OHT, to compare the effectiveness and kinetics of 4-OHT delivery in mice in vivo by either tamoxifen in food or by intraperitoneal injection. Our data indicate that dietary tamoxifen offers an effective, facile and ethically preferable means for long term activation of ERTAM fusion proteins in vivo. PMID:24662815

  9. Using MapMyFitness to Place Physical Activity into Neighborhood Context

    PubMed Central

    Hirsch, Jana A.; James, Peter; Robinson, Jamaica R. M.; Eastman, Kyler M.; Conley, Kevin D.; Evenson, Kelly R.; Laden, Francine

    2014-01-01

    It is difficult to obtain detailed information on the context of physical activity at large geographic scales, such as the entire United States, as well as over long periods of time, such as over years. MapMyFitness is a suite of interactive tools for individuals to track their workouts online or using global positioning system in their phones or other wireless trackers. This method article discusses the use of physical activity data tracked using MapMyFitness to examine patterns over space and time. An overview of MapMyFitness, including data tracked, user information, and geographic scope, is explored. We illustrate the utility of MapMyFitness data using tracked physical activity by users in Winston-Salem, NC, USA between 2006 and 2013. Types of physical activities tracked are described, as well as the percent of activities occurring in parks. Strengths of MapMyFitness data include objective data collection, low participant burden, extensive geographic scale, and longitudinal series. Limitations include generalizability, behavioral change as the result of technology use, and potential ethical considerations. MapMyFitness is a powerful tool to investigate patterns of physical activity across large geographic and temporal scales. PMID:24653982

  10. Autophagy Defects Suggested by Low Levels of Autophagy Activator MAP1S and High Levels of Autophagy Inhibitor LRPPRC Predict Poor Prognosis of Prostate Cancer Patients

    PubMed Central

    Jiang, Xianhan; Zhong, Weide; Huang, Hai; He, Huichan; Jiang, Funeng; Chen, Yanru; Yue, Fei; Zou, Jing; Li, Xun; He, Yongzhong; You, Pan; Yang, Weiqiang; Lai, Yiming; Wang, Fen; Liu, Leyuan

    2016-01-01

    MAP1S (originally named C19ORF5) is a widely distributed homolog of neuronal-specific MAP1A and MAP1B, and bridges autophagic components with microtubules and mitochondria to affect autophagosomal biogenesis and degradation. Mitochondrion-associated protein LRPPRC functions as an inhibitor for autophagy initiation to protect mitochondria from autophagy degradation. MAP1S and LRPPRC interact with each other and may collaboratively regulate autophagy although the underlying mechanism is yet unknown. Previously, we have reported that LRPPRC levels serve as a prognosis marker of patients with prostate adenocarcinomas (PCA), and that patients with high LRPPRC levels survive a shorter period after surgery than those with low levels of LRPPRC. MAP1S levels are elevated in diethylnitrosamine-induced hepatocelular carcinomas in wildtype mice and the exposed MAP1S-deficient mice develop more malignant hepatocellular carcinomas. We performed immunochemical analysis to evaluate the co-relationship among the levels of MAP1S, LRPPRC, P62, and γ-H2AX. Samples were collected from wildtype and prostate-specific PTEN-deficient mice, 111 patients with PCA who had been followed up for 10 years and 38 patients with benign prostate hyperplasia enrolled in hospitals in Guangzhou, China. The levels of MAP1S were generally elevated so the MAP1S-mediated autophagy was activated in PCA developed in either PTEN-deficient mice or patients than their respective benign tumors. The MAP1S levels among patients with PCA vary dramatically, and patients with low MAP1S levels survive a shorter period than those with high MAP1S levels. Levels of MAP1S in collaboration with levels of LRPPRC can serve as markers for prognosis of prostate cancer patients. PMID:25043940

  11. Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

    PubMed Central

    Migliaccio, A; Di Domenico, M; Castoria, G; de Falco, A; Bontempo, P; Nola, E; Auricchio, F

    1996-01-01

    The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor. Images PMID:8635462

  12. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    PubMed Central

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2013-01-01

    Membrane proteins (MPs) mediate a variety of cellular responses to extracellular signals. While MPs are intensely studied for their values as disease biomarkers and therapeutic targets, in situ investigation of binding kinetics of MPs with their ligands has been a challenge. Traditional approaches isolate MPs and then study them ex situ, which does not accurately reflect their native structures and functions. We present here a label-free plasmonic microscopy method to map the local binding kinetics of MPs in their native environment. This new analytical method can perform simultaneous plasmonic and fluorescence imaging, thus making it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we have determined the distribution of MPs on the surface of single cells, and the local binding kinetic constants of different MPs. Furthermore, we have studied the polarization of the MPs on the cell surface during chemotaxis. PMID:23000999

  13. The gene for human erythrocyte protein 4. 2 maps to chromosome 15q15

    SciTech Connect

    Najfeld, V. ); Ballard, S.G.; Menninger, J.; Ward, D.C. ); Bouhassira, E.E.; Schwartz, R.S.; Nagel, R.L.; Rybicki, A.C. )

    1992-01-01

    Protein 4.2 (P4.2), one of the major components of the red-blood-cell membrane, is located on the interior surface, where it binds with high affinity to the cytoplasmic domain of band 3. Individuals whose red blood cells are deficient in P4.2 have osmotically fragile, abnormally shaped cells and moderate hemolytic anemia. cDNA clones from both the 5{prime} and the 3{prime} coding regions of the P4.2 gene were used to map its chromosomal location by fluorescence in situ hybridization. The probes, individually or in combination, gave specific hybridization signals on chromosome 15. The hybridization locus was identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by Alu-PCR (R-like) probe and by DAPI staining (G-like). The authors results demonstrate that the locus of the P4.2 gene is located within 15q15.

  14. Modeling the SHG activities of diverse protein crystals

    SciTech Connect

    Haupert, Levi M.; DeWalt, Emma L.; Simpson, Garth J.

    2012-11-01

    The origins of the diversity in the SHG signal from protein crystals are investigated and potential protein-crystal coverage by SHG microscopy is assessed. A symmetry-additive ab initio model for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much of the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within the crystal lattice. Two or more orders-of-magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ∼84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices.

  15. Hemagglutinating activity of proteins from Parkia speciosa seeds.

    PubMed

    Chankhamjon, Kanokwan; Petsom, Amorn; Sawasdipuksa, Narumon; Sangvanich, Polkit

    2010-01-01

    Proteins from Parkia speciosa Hassk. (Fabaceae) seeds were extracted and stepwise precipitated using ammonium sulfate. Proteins precipitated with 25% ammonium sulfate were separated by affinity chromatography on Affi-Gel Blue gel followed by protein liquid chromatography on Superdex 200. The protein Gj, which was identified as a protein similar to putative aristolochene synthase, 3'-partial from Oryza sativa L. (Poaceae), had hemagglutinating activity of 0.39 mug/muL. Moreover, fraction C2 from the proteins precipitated with 60% ammonium sulfate, separated by lectin-specific adsorption chromatography using Con A Sepharose, had hemagglutinating activity of 1.17 mug/muL. Using gel electrophoresis, two proteins C2a and C2b were separated, having molecular weights of 45 kDa and 23 kDa, respectively. From protein identification, C2a was found to be similar to the hypothetical protein B1342F01.11 from Oryza sativa, and C2b was similar to the hypothetical protein At1g51560 from Arabidopsis thaliana (L.) Heynh. (Brassicaceae). PMID:20645760

  16. Protein surface topology-probing by selective chemical modification and mass spectrometric peptide mapping.

    PubMed Central

    Suckau, D; Mak, M; Przybylski, M

    1992-01-01

    Aminoacetylation of lysine residues and the modification of arginine by 1,2-cyclohexanedione to N7,N8-(dihydroxy-1,2-cyclohexylidene)arginine were used for probing the surface topology of hen-eggwhite lysozyme as a model protein. The molecular identification of lysine and arginine modification sites was provided by molecular weight determinations of modified and unmodified tryptic peptide mixtures (peptide mapping) using 252Cf plasma desorption mass spectrometry. At conditions of limited chemical modification, mass-spectrometric peptide-mapping analyses of lysozyme derivatives enabled the direct assignment of relative reactivities of lysine and arginine residues at different reaction times and reagent concentrations. The relative reactivities of lysine residues showed a direct correlation with their surface accessibilities from x-ray structure data. For the reaction with 1,2-cyclohexanedione, a selective modification at Arg-5, -125, -112, and -73 was identified, and an inverse correlation of relative reactivities with the surface accessibility ratios of the N7- and the N8-guanidino functions was obtained. By examination of the x-ray structural data of lysozyme, this selective modification was attributed to intramolecular catalysis because of the presence of neighboring proton acceptor groups, such as the Asp-119 carboxylate group for Arg-125 and the Trp-123 and Arg-125 carbonyl groups for Arg-5. PMID:1608973

  17. Evidence for a receptor protein of activated carcinogen

    PubMed Central

    Mainigi, Kumar D.; Sorof, Sam

    1977-01-01

    During carcinogenesis in rat liver by 3′-methyl-4-dimethylaminoazobenzene, the two moieties of the principal liver carcinogen-protein complex have considerably different turnover rates. With continued ingestion of the azocarcinogen by rats, the bound azo dye in the complex has a half-life of 2.5 ± 0.25 (SD) days, while the protein moiety has a half-life of 8.7 ± 1.6 days (probability of identity <0.001). In addition, the interaction of the azocarcinogen with the principal target protein in vivo appears to extend the half-life of the protein itself from 3.3 ± 0.2 days in normal liver to 8.7 ± 1.6 days (P < 0.001). The slowing of the turnover of the protein by the carcinogen appears to be readily reversible, since soon after the cessation of azocarcinogen feeding, the half-life of the protein returns to that of the target protein in normal liver. The considerable difference in turnover rates of the two moieties of the complex and the reversible effects of the carcinogen in slowing the turnover of the protein moiety suggest that the two moieties of the native azoprotein are noncovalently linked and that they have different biological activities. The native complex appears to contain azo dye in an activated state that is capable of yielding a reactive electrophile, because after protein denaturation the bound azo dye was previously found to have properties that are indicative of covalent linkage to the protein. The retardation in the biological turnover rate of the protein moiety, apparently resulting from interaction with azocarcinogen, is in agreement with the known ligand-induced stabilization in vitro and reduced rate of proteolytic degradation in vivo of other proteins that result from conformational change to a more compact configuration. Our evidence is consistent with the hypothesis that the principal liver carcinogen-protein complex contains hydrophobically bound activated azocarcinogen, whose specificity of reaction with critical macromolecule(s) in

  18. Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases

    PubMed Central

    Giordano, G.; Klintworth, H.M.; Kavanagh, T.J.; Costa, L.G.

    2008-01-01

    In mouse cerebellar granule neurons (CGN) the marine neurotoxin domoic acid (DomA) induces neuronal cell death, either by apoptosis or by necrosis, depending on its concentration, with apoptotic damage predominating in response to low concentrations (100 nM). DomA-induced apoptosis is due to selective activation of AMPA/kainate receptors, and is mediated by DomA-induced oxidative stress, leading to mitochondrial dysfunction and activation of caspase-3. The p38 MAP kinase and the c-Jun NH2-terminal protein kinase (JNK) have been shown to be preferentially activated by oxidative stress. Here we report that DomA increases p38 MAP kinase and JNK phosphorylation, and that this effect is more pronounced in CGNs from Gclm (−/−) mice, which lack the modifier subunit of glutamate-cysteine ligase, have very low glutathione (GSH) levels, and are more sensitive to DomA-induced apoptosis than CGNs from wild-type mice. The increased phosphorylation of JNK and p38 kinase was paralleled by a decreased phosphorylation of Erk 1/2. The AMPA/kainate receptor antagonist NBQX, but not the NMDA receptor antagonist MK-801, prevents DomA-induced activation of p38 and JNK kinases. Several antioxidants (GSH ethyl ester, catalase, phenylbutylnitrone) also prevent DomA-induced phosphorylation of JNK and p38 MAP kinases. Inhibitors of p38 (SB203580) and of JNK (SP600125) antagonize DomA-induced apoptosis. These results indicate the importance of oxidative stress-activated JNK and p38 MAP kinase pathways in DomA-induced apoptosis in CGNs. PMID:18164102

  19. The role of mitogen-activated protein kinase in oxytocin-induced contraction of uterine smooth muscle in pregnant rat.

    PubMed

    Nohara, A; Ohmichi, M; Koike, K; Masumoto, N; Kobayashi, M; Akahane, M; Ikegami, H; Hirota, K; Miyake, A; Murata, Y

    1996-12-24

    Oxytocin causes the rapid tyrosine phosphorylation of mitogen-activated protein (MAP) kinase in both human and rat puerperal uterine myometrial cultured cells. The potential role of the MAP kinase pathway in oxytocin action was investigated with the specific MAP kinase kinase (MEK) inhibitor, PD98059. Oxytocin stimulation of the tyrosine phosphorylation of MAP kinase in both human and rat cultured puerperal uterine cells was abolished by pretreatment of the cells with MEK inhibitor in a dose-dependent manner. Although MEK inhibitor had no effect on oxytocin-induced intracellular Ca2+ mobilization in either pregnant human or pregnant rat uterine cells, it partly inhibited oxytocin-induced pregnant rat uterine contraction in a dose-dependent manner. These results suggest that MAP kinase pathway may have some important roles in oxytocin-induced uterine contraction. PMID:8954997

  20. Roles of mitogen activated protein kinases and EGF receptor in arsenite-stimulated matrix metalloproteinase-9 production

    SciTech Connect

    Cooper, Karen L.; Myers, Terrance Alix; Rosenberg, Martina; Chavez, Miquella; Hudson, Laurie G. . E-mail: lghudson@unm.edu

    2004-11-01

    The dermatotoxicity of arsenic is well established and epidemiological studies identify an increased incidence of keratinocytic tumors (basal cell and squamous cell carcinoma) associated with arsenic exposure. Little is known about the underlying mechanisms of arsenic-mediated skin carcinogenesis, but activation of mitogen-activated protein (MAP) kinases and subsequent regulation of downstream target genes may contribute to tumor promotion and progression. In this study, we investigated activation of the extracellular signal regulated kinase (ERK) and the stress-associated kinase p38 by arsenite in HaCat cells, a spontaneously immortalized human keratinocyte cell line. Arsenite concentrations {>=}100 {mu}M stimulate rapid activation of p38 and ERK MAP kinases. However, upon extended exposure (24 h), persistent stimulation of p38 and ERK MAP kinases was detected at low micromolar concentrations of arsenite. Although ERK and p38 were activated with similar time and concentration dependence, the mechanism of activation differed for these two MAP kinases. ERK activation by arsenite was fully dependent on the catalytic activity of the epidermal growth factor (EGF) receptor and partially dependent on Src-family kinase activity. In contrast, p38 activation was independent of EGF receptor or Src-family kinase activity. Arsenite-stimulated MAP kinase signal transduction resulted in increased production of matrix metalloproteinase (MMP)-9, an AP-1 regulated gene product. MMP-9 induction by arsenite was prevented when EGF receptor or MAP kinase signaling was inhibited. These studies indicate that EGF receptor activation is a component of arsenite-mediated signal transduction and gene expression in keratinocytes and that low micromolar concentrations of arsenite stimulate key signaling pathways upon extended exposure. Stimulation of MAP kinase cascades by arsenic and subsequent regulation of genes including c-fos, c-jun, and the matrix degrading proteases may play an important

  1. Perm-seq: Mapping Protein-DNA Interactions in Segmental Duplication and Highly Repetitive Regions of Genomes with Prior-Enhanced Read Mapping

    PubMed Central

    Zeng, Xin; Li, Bo; Welch, Rene; Rojo, Constanza; Zheng, Ye; Dewey, Colin N.; Keleş, Sündüz

    2015-01-01

    Segmental duplications and other highly repetitive regions of genomes contribute significantly to cells’ regulatory programs. Advancements in next generation sequencing enabled genome-wide profiling of protein-DNA interactions by chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq). However, interactions in highly repetitive regions of genomes have proven difficult to map since short reads of 50–100 base pairs (bps) from these regions map to multiple locations in reference genomes. Standard analytical methods discard such multi-mapping reads and the few that can accommodate them are prone to large false positive and negative rates. We developed Perm-seq, a prior-enhanced read allocation method for ChIP-seq experiments, that can allocate multi-mapping reads in highly repetitive regions of the genomes with high accuracy. We comprehensively evaluated Perm-seq, and found that our prior-enhanced approach significantly improves multi-read allocation accuracy over approaches that do not utilize additional data types. The statistical formalism underlying our approach facilitates supervising of multi-read allocation with a variety of data sources including histone ChIP-seq. We applied Perm-seq to 64 ENCODE ChIP-seq datasets from GM12878 and K562 cells and identified many novel protein-DNA interactions in segmental duplication regions. Our analysis reveals that although the protein-DNA interactions sites are evolutionarily less conserved in repetitive regions, they share the overall sequence characteristics of the protein-DNA interactions in non-repetitive regions. PMID:26484757

  2. Perm-seq: Mapping Protein-DNA Interactions in Segmental Duplication and Highly Repetitive Regions of Genomes with Prior-Enhanced Read Mapping.

    PubMed

    Zeng, Xin; Li, Bo; Welch, Rene; Rojo, Constanza; Zheng, Ye; Dewey, Colin N; Keleş, Sündüz

    2015-10-01

    Segmental duplications and other highly repetitive regions of genomes contribute significantly to cells' regulatory programs. Advancements in next generation sequencing enabled genome-wide profiling of protein-DNA interactions by chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq). However, interactions in highly repetitive regions of genomes have proven difficult to map since short reads of 50-100 base pairs (bps) from these regions map to multiple locations in reference genomes. Standard analytical methods discard such multi-mapping reads and the few that can accommodate them are prone to large false positive and negative rates. We developed Perm-seq, a prior-enhanced read allocation method for ChIP-seq experiments, that can allocate multi-mapping reads in highly repetitive regions of the genomes with high accuracy. We comprehensively evaluated Perm-seq, and found that our prior-enhanced approach significantly improves multi-read allocation accuracy over approaches that do not utilize additional data types. The statistical formalism underlying our approach facilitates supervising of multi-read allocation with a variety of data sources including histone ChIP-seq. We applied Perm-seq to 64 ENCODE ChIP-seq datasets from GM12878 and K562 cells and identified many novel protein-DNA interactions in segmental duplication regions. Our analysis reveals that although the protein-DNA interactions sites are evolutionarily less conserved in repetitive regions, they share the overall sequence characteristics of the protein-DNA interactions in non-repetitive regions. PMID:26484757

  3. Space Weather Activities of IONOLAB Group: TEC Mapping

    NASA Astrophysics Data System (ADS)

    Arikan, F.; Yilmaz, A.; Arikan, O.; Sayin, I.; Gurun, M.; Akdogan, K. E.; Yildirim, S. A.

    2009-04-01

    Being a key player in Space Weather, ionospheric variability affects the performance of both communication and navigation systems. To improve the performance of these systems, ionosphere has to be monitored. Total Electron Content (TEC), line integral of the electron density along a ray path, is an important parameter to investigate the ionospheric variability. A cost-effective way of obtaining TEC is by using dual-frequency GPS receivers. Since these measurements are sparse in space, accurate and robust interpolation techniques are needed to interpolate (or map) the TEC distribution for a given region in space. However, the TEC data derived from GPS measurements contain measurement noise, model and computational errors. Thus, it is necessary to analyze the interpolation performance of the techniques on synthetic data sets that can represent various ionospheric states. By this way, interpolation performance of the techniques can be compared over many parameters that can be controlled to represent the desired ionospheric states. In this study, Multiquadrics, Inverse Distance Weighting (IDW), Cubic Splines, Ordinary and Universal Kriging, Random Field Priors (RFP), Multi-Layer Perceptron Neural Network (MLP-NN), and Radial Basis Function Neural Network (RBF-NN) are employed as the spatial interpolation algorithms. These mapping techniques are initially tried on synthetic TEC surfaces for parameter and coefficient optimization and determination of error bounds. Interpolation performance of these methods are compared on synthetic TEC surfaces over the parameters of sampling pattern, number of samples, the variability of the surface and the trend type in the TEC surfaces. By examining the performance of the interpolation methods, it is observed that both Kriging, RFP and NN have important advantages and possible disadvantages depending on the given constraints. It is also observed that the determining parameter in the error performance is the trend in the Ionosphere

  4. Performance Benchmarking Tsunami Models for NTHMP's Inundation Mapping Activities

    NASA Astrophysics Data System (ADS)

    Horrillo, Juan; Grilli, Stéphan T.; Nicolsky, Dmitry; Roeber, Volker; Zhang, Joseph

    2015-03-01

    The coastal states and territories of the United States (US) are vulnerable to devastating tsunamis from near-field or far-field coseismic and underwater/subaerial landslide sources. Following the catastrophic 2004 Indian Ocean tsunami, the National Tsunami Hazard Mitigation Program (NTHMP) accelerated the development of public safety products for the mitigation of these hazards. In response to this initiative, US coastal states and territories speeded up the process of developing/enhancing/adopting tsunami models that can be used for developing inundation maps and evacuation plans. One of NTHMP's requirements is that all operational and inundation-based numerical (O&I) models used for such purposes be properly validated against established standards to ensure the reliability of tsunami inundation maps as well as to achieve a basic level of consistency between parallel efforts. The validation of several O&I models was considered during a workshop held in 2011 at Texas A&M University (Galveston). This validation was performed based on the existing standard (OAR-PMEL-135), which provides a list of benchmark problems (BPs) covering various tsunami processes that models must meet to be deemed acceptable. Here, we summarize key approaches followed, results, and conclusions of the workshop. Eight distinct tsunami models were validated and cross-compared by using a subset of the BPs listed in the OAR-PMEL-135 standard. Of the several BPs available, only two based on laboratory experiments are detailed here for sake of brevity; since they are considered as sufficiently comprehensive. Average relative errors associated with expected parameters values such as maximum surface amplitude/runup are estimated. The level of agreement with the reference data, reasons for discrepancies between model results, and some of the limitations are discussed. In general, dispersive models were found to perform better than nondispersive models, but differences were relatively small, in part

  5. Full-sky Astrometric Mapping Explorer (FAME) Rescope Activities

    NASA Astrophysics Data System (ADS)

    Johnston, K.; Dorland, B.; Gaume, R.; Hajian, A.; Harris, F.; Harris, H.; Hennessy, G.; Kaplan, G.; Levine, S.; Monet, D.; Munn, J.; Murison, M.; Pier, J.; Urban, S.; Zacharias, N.; Seidelmann, P. K.; Lee, J.; Makarov, V.; Olling, R.; Codella, T.; Geary, J.; Latham, D.; Phillips, J.; Johnson, M.; Vassar, R.; Horner, S.

    2001-12-01

    The Full-sky Astrometric Mapping Explorer (FAME) will measure the positions, proper motions, parallaxes, and photometry for 40 million stars between 5 and 15 magnitude, with accuracies of 50 microarcseconds at 9th magnitude and degraded accuracy for fainter stars as faint as 15 magnitude. The mission is planned for an October 2004 launch and a five-year duration. The FAME mission has progressed in its development. Due to budget, weight, and power limitations, the FAME architecture has been rescoped so that it will satisfy the budget limitations and retain the basic science objectives of the mission. The new design and characteristics, the status of developments and tests, and the scientific baseline and minimum mission requirements are presented.

  6. Cloning of three novel neuronal Cdk5 activator binding proteins.

    PubMed

    Ching, Y P; Qi, Z; Wang, J H

    2000-01-25

    Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana. PMID:10721722

  7. A System and Method for Online High-Resolution Mapping of Gastric Slow-Wave Activity

    PubMed Central

    Bull, Simon H.; O’Grady, Gregory; Du, Peng

    2015-01-01

    High-resolution (HR) mapping employs multielectrode arrays to achieve spatially detailed analyses of propagating bioelectrical events. A major current limitation is that spatial analyses must currently be performed “off-line” (after experiments), compromising timely recording feedback and restricting experimental interventions. These problems motivated development of a system and method for “online” HR mapping. HR gastric recordings were acquired and streamed to a novel software client. Algorithms were devised to filter data, identify slow-wave events, eliminate corrupt channels, and cluster activation events. A graphical user interface animated data and plotted electrograms and maps. Results were compared against off-line methods. The online system analyzed 256-channel serosal recordings with no unexpected system terminations with a mean delay 18 s. Activation time marking sensitivity was 0.92; positive predictive value was 0.93. Abnormal slow-wave patterns including conduction blocks, ectopic pacemaking, and colliding wave fronts were reliably identified. Compared to traditional analysis methods, online mapping had comparable results with equivalent coverage of 90% of electrodes, average RMS errors of less than 1 s, and CC of activation maps of 0.99. Accurate slow-wave mapping was achieved in near real-time, enabling monitoring of recording quality and experimental interventions targeted to dysrhythmic onset. This work also advances the translation of HR mapping toward real-time clinical application. PMID:24860024

  8. Empirical Maps For The Calculation of Amide I Vibrational Spectra of Proteins From Classical Molecular Dynamics Simulations

    PubMed Central

    2015-01-01

    New sets of parameters (maps) for calculating amide I vibrational spectra for proteins through a vibrational exciton model are proposed. The maps are calculated as a function of electric field and van der Waals forces on the atoms of peptide bonds, taking into account the full interaction between peptide bonds and the surrounding environment. The maps are designed to be employed using data obtained from standard all-atom molecular simulations without any additional constraints on the system. Six proteins representing a wide range of sizes and secondary structure complexity were chosen as a test set. Spectra calculated for these proteins reproduce experimental data both qualitatively and quantitatively. The proposed maps lead to spectra that capture the weak second peak observed in proteins containing β-sheets, allowing for clear distinction between α-helical and β-sheet proteins. While the parametrization is specific to the CHARMM force field, the methodology presented can be readily applied to any empirical force field. PMID:24654732

  9. Empirical maps for the calculation of amide I vibrational spectra of proteins from classical molecular dynamics simulations.

    PubMed

    Małolepsza, Edyta; Straub, John E

    2014-07-17

    New sets of parameters (maps) for calculating amide I vibrational spectra for proteins through a vibrational exciton model are proposed. The maps are calculated as a function of electric field and van der Waals forces on the atoms of peptide bonds, taking into account the full interaction between peptide bonds and the surrounding environment. The maps are designed to be employed using data obtained from standard all-atom molecular simulations without any additional constraints on the system. Six proteins representing a wide range of sizes and secondary structure complexity were chosen as a test set. Spectra calculated for these proteins reproduce experimental data both qualitatively and quantitatively. The proposed maps lead to spectra that capture the weak second peak observed in proteins containing β-sheets, allowing for clear distinction between α-helical and β-sheet proteins. While the parametrization is specific to the CHARMM force field, the methodology presented can be readily applied to any empirical force field. PMID:24654732

  10. Pestivirus NS3 (p80) protein possesses RNA helicase activity.

    PubMed Central

    Warrener, P; Collett, M S

    1995-01-01

    The pestivirus bovine viral diarrhea virus (BVDV) p80 protein (referred to here as the NS3 protein) contains amino acid sequence motifs predictive of three enzymatic activities: serine proteinase, nucleoside triphosphatase, and RNA helicase. We have previously demonstrated that the former two enzymatic activities are associated with this protein. Here, we show that a purified recombinant BVDV NS3 protein derived from baculovirus-infected insect cells possesses RNA helicase activity. BVDV NS3 RNA helicase activity was specifically inhibited by monoclonal antibodies to the p80 protein. The activity was dependent on the presence of nucleoside triphosphate and divalent cation, with a preference for ATP and Mn2+. Hydrolysis of the nucleoside triphosphate was necessary for strand displacement. The helicase activity required substrates with an un-base-paired region on the template strand 3' of the duplex region. As few as three un-base-paired nucleotides were sufficient for efficient oligonucleotide displacement. However, the enzyme did not act on substrates having a single-stranded region only to the 5' end of the duplex or on substrates lacking single-stranded regions altogether (blunt-ended duplex substrates), suggesting that the directionality of the BVDV RNA helicase was 3' to 5' with respect to the template strand. The BVDV helicase activity was able to displace both RNA and DNA oligonucleotides from RNA template strands but was unable to release oligonucleotides from DNA templates. The possible role of this activity in pestivirus replication is discussed. PMID:7853509

  11. Activation of an Endoribonuclease by Non-intein Protein Splicing.

    PubMed

    Campbell, Stephen J; Stern, David B

    2016-07-29

    The Chlamydomonas reinhardtii chloroplast-localized poly(A)-binding protein RB47 is predicted to contain a non-conserved linker (NCL) sequence flanked by highly conserved N- and C-terminal sequences, based on the corresponding cDNA. RB47 was purified from chloroplasts in association with an endoribonuclease activity; however, protein sequencing failed to detect the NCL. Furthermore, while recombinant RB47 including the NCL did not display endoribonuclease activity in vitro, versions lacking the NCL displayed strong activity. Both full-length and shorter forms of RB47 could be detected in chloroplasts, with conversion to the shorter form occurring in chloroplasts isolated from cells grown in the light. This conversion could be replicated in vitro in chloroplast extracts in a light-dependent manner, where epitope tags and protein sequencing showed that the NCL was excised from a full-length recombinant substrate, together with splicing of the flanking sequences. The requirement for endogenous factors and light differentiates this protein splicing from autocatalytic inteins, and may allow the chloroplast to regulate the activation of RB47 endoribonuclease activity. We speculate that this protein splicing activity arose to post-translationally repair proteins that had been inactivated by deleterious insertions or extensions. PMID:27311716

  12. Iron-chelating activity of chickpea protein hydrolysate peptides.

    PubMed

    Torres-Fuentes, Cristina; Alaiz, Manuel; Vioque, Javier

    2012-10-01

    Chickpea-chelating peptides were purified and analysed for their iron-chelating activity. These peptides were purified after affinity and gel filtration chromatography from a chickpea protein hydrolysate produced with pepsin and pancreatin. Iron-chelating activity was higher in purified peptide fractions than in the original hydrolysate. Histidine contents were positively correlated with the iron-chelating activity. Hence fractions with histidine contents above 20% showed the highest chelating activity. These results show that iron-chelating peptides are generated after chickpea protein hydrolysis with pepsin plus pancreatin. These peptides, through metal chelation, may increase iron solubility and bioavailability and improve iron absorption. PMID:25005984

  13. Mapping of the Sequences Directing Localization of the Drosophila Germ Cell-Expressed Protein (GCE)

    PubMed Central

    Greb-Markiewicz, Beata; Sadowska, Daria; Surgut, Natalia; Godlewski, Jakub; Zarębski, Mirosław; Ożyhar, Andrzej

    2015-01-01

    Drosophila melanogaster germ cell-expressed protein (GCE) belongs to the family of bHLH-PAS transcription factors that are the regulators of gene expression networks that determine many physiological and developmental processes. GCE is a homolog of D. melanogaster methoprene tolerant protein (MET), a key mediator of anti-metamorphic signaling in insects and the putative juvenile hormone receptor. Recently, it has been shown that the functions of MET and GCE are only partially redundant and tissue specific. The ability of bHLH-PAS proteins to fulfill their function depends on proper intracellular trafficking, determined by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). Nevertheless, until now no data has been published on the GCE intracellular shuttling and localization signals. We performed confocal microscopy analysis of the subcellular distribution of GCE fused with yellow fluorescent protein (YFP) and YFP-GCE derivatives which allowed us to characterize the details of the subcellular traffic of this protein. We demonstrate that GCE possess specific pattern of localization signals, only partially consistent with presented previously for MET. The presence of a strong NLS in the C-terminal part of GCE, seems to be unique and important feature of this protein. The intracellular localization of GCE appears to be determined by the NLSs localized in PAS-B domain and C-terminal fragment of GCE, and NESs localized in PAS-A, PAS-B domains and C-terminal fragment of GCE. NLSs activity can be modified by juvenile hormone (JH) and other partners, likely 14-3-3 proteins. PMID:26186223

  14. Inclusion bodies and purification of proteins in biologically active forms.

    PubMed

    Mukhopadhyay, A

    1997-01-01

    Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved. PMID:8939059

  15. Activators of G Protein Signaling in the Kidney

    PubMed Central

    2015-01-01

    Heterotrimeric G proteins play a crucial role in regulating signal processing to maintain normal cellular homeostasis, and subtle perturbations in its activity can potentially lead to the pathogenesis of renal disorders or diseases. Cell-surface receptors and accessory proteins, which normally modify and organize the coupling of individual G protein subunits, contribute to the regulation of heterotrimeric G protein activity and their convergence and/or divergence of downstream signaling initiated by effector systems. Activators of G protein signaling (AGS) are a family of accessory proteins that intervene at multiple distinct points during the activation–inactivation cycle of G proteins, even in the absence of receptor stimulation. Perturbations in the expression of individual AGS proteins have been reported to modulate signal transduction pathways in a wide array of diseases and disorders within the brain, heart, immune system, and more recently, the kidney. This review will provide an overview of the expression profile, localization, and putative biologic role of the AGS family in the context of normal and diseased states of the kidney. PMID:25628392

  16. A novel approach for targeted elimination of CSPG4-positive triple-negative breast cancer cells using a MAP tau-based fusion protein.

    PubMed

    Amoury, Manal; Mladenov, Radoslav; Nachreiner, Thomas; Pham, Anh-Tuan; Hristodorov, Dmitrij; Di Fiore, Stefano; Helfrich, Wijnand; Pardo, Alessa; Fey, Georg; Schwenkert, Michael; Thepen, Theophilus; Kiessling, Fabian; Hussain, Ahmad F; Fischer, Rainer; Kolberg, Katharina; Barth, Stefan

    2016-08-15

    Chondroitin sulfate proteoglycan 4 (CSPG4) has been identified as a highly promising target antigen for immunotherapy of triple-negative breast cancer (TNBC). TNBC represents a highly aggressive heterogeneous group of tumors lacking expression of estrogen, progesterone and human epidermal growth factor receptor 2. TNBC is particularly prevalent among young premenopausal women. No suitable targeted therapies are currently available and therefore, novel agents for the targeted elimination of TNBC are urgently needed. Here, we present a novel cytolytic fusion protein (CFP), designated αCSPG4(scFv)-MAP, that consists of a high affinity CSPG4-specific single-chain antibody fragment (scFv) genetically fused to a functionally enhanced form of the human microtubule-associated protein (MAP) tau. Our data indicate that αCSPG4(scFv)-MAP efficiently targets CSPG4(+) TNBC-derived cell lines MDA-MB-231 and Hs 578T and potently inhibits their growth with IC50 values of ∼200 nM. Treatment with αCSPG(scFv)-MAP resulted in induction of the mitochondrial stress pathway by activation of caspase-9 as well as endonuclease G translocation to the nucleus, while induction of the caspase-3 apoptosis pathway was not detectable. Importantly, in vivo studies in mice bearing human breast cancer xenografts revealed efficient targeting to and accumulation of αCSPG4(scFv)-MAP at tumor sites resulting in prominent tumor regression. Taken together, this preclinical proof of concept study confirms the potential clinical value of αCSPG4(scFv)-MAP as a novel targeted approach for the elimination of CSPG4-positive TNBC. PMID:27037627

  17. Structural and kinetic analysis of protein-aggregate strains in vivo using binary epitope mapping

    PubMed Central

    Bergh, Johan; Zetterström, Per; Andersen, Peter M.; Brännström, Thomas; Graffmo, Karin S.; Jonsson, P. Andreas; Lang, Lisa; Danielsson, Jens; Oliveberg, Mikael; Marklund, Stefan L.

    2015-01-01

    Despite considerable progress in uncovering the molecular details of protein aggregation in vitro, the cause and mechanism of protein-aggregation disease remain poorly understood. One reason is that the amount of pathological aggregates in neural tissue is exceedingly low, precluding examination by conventional approaches. We present here a method for determination of the structure and quantity of aggregates in small tissue samples, circumventing the above problem. The method is based on binary epitope mapping using anti-peptide antibodies. We assessed the usefulness and versatility of the method in mice modeling the neurodegenerative disease amyotrophic lateral sclerosis, which accumulate intracellular aggregates of superoxide dismutase-1. Two strains of aggregates were identified with different structural architectures, molecular properties, and growth kinetics. Both were different from superoxide dismutase-1 aggregates generated in vitro under a variety of conditions. The strains, which seem kinetically under fragmentation control, are associated with different disease progressions, complying with and adding detail to the growing evidence that seeding, infectivity, and strain dependence are unifying principles of neurodegenerative disease. PMID:25802384

  18. Epitope mapping of monoclonal antibody 4C8 recognizing the protein huntingtin.

    PubMed

    Cong, Shu-Yan; Pepers, Barry A; Roos, Raymund A C; Van Ommen, Gert-Jan B; Dorsman, Josephine C

    2005-10-01

    Huntington's disease is a dominantly inherited, devastating neurodegenerative disorder, caused by a polyglutamine expansion (>37) in the N-terminal region of huntingtin, a protein of unknown function. In patients and normal individuals, N-terminal fragments of huntingtin are found, and the N-terminal fragments of mutant huntingtin are cytotoxic. The functions of wild-type huntingtin and the mechanisms underlying the toxic effects of mutant huntingtin are still ill defined. To get more insight into these topics, monoclonal antibodies (MAbs) are indispensable tools. Antibodies raised against the N-terminus are especially important. Among these, the 4C8 mouse MAb has been extensively used in various approaches. In this study, we have mapped the epitope of 4C8 to a 15-amino acid (aa) region spanning from aa 443 to 457 of the human protein, and found that mutation of three consecutive glutamic acids present in this region disrupts the recognition by 4C8. These results allow a more accurate interpretation of the results obtained by usage of the 4C8 antibody and broaden the utility of this antibody. PMID:16225422

  19. Structural and kinetic analysis of protein-aggregate strains in vivo using binary epitope mapping.

    PubMed

    Bergh, Johan; Zetterström, Per; Andersen, Peter M; Brännström, Thomas; Graffmo, Karin S; Jonsson, P Andreas; Lang, Lisa; Danielsson, Jens; Oliveberg, Mikael; Marklund, Stefan L

    2015-04-01

    Despite considerable progress in uncovering the molecular details of protein aggregation in vitro, the cause and mechanism of protein-aggregation disease remain poorly understood. One reason is that the amount of pathological aggregates in neural tissue is exceedingly low, precluding examination by conventional approaches. We present here a method for determination of the structure and quantity of aggregates in small tissue samples, circumventing the above problem. The method is based on binary epitope mapping using anti-peptide antibodies. We assessed the usefulness and versatility of the method in mice modeling the neurodegenerative disease amyotrophic lateral sclerosis, which accumulate intracellular aggregates of superoxide dismutase-1. Two strains of aggregates were identified with different structural architectures, molecular properties, and growth kinetics. Both were different from superoxide dismutase-1 aggregates generated in vitro under a variety of conditions. The strains, which seem kinetically under fragmentation control, are associated with different disease progressions, complying with and adding detail to the growing evidence that seeding, infectivity, and strain dependence are unifying principles of neurodegenerative disease. PMID:25802384

  20. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    SciTech Connect

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

  1. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    DOE PAGESBeta

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; et al

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function.more » We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.« less

  2. SARConnect: A Tool to Interrogate the Connectivity Between Proteins, Chemical Structures and Activity Data

    PubMed Central

    Eriksson, Mats; Nilsson, Ingemar; Kogej, Thierry; Southan, Christopher; Johansson, Martin; Tyrchan, Christian; Muresan, Sorel; Blomberg, Niklas; Bjäreland, Marcus

    2012-01-01

    Abstract The access and use of large-scale structure-activity relationships (SAR) is increasing as the range of targets and availability of bioactive compound-to-protein mappings expands. However, effective exploitation requires merging and normalisation of activity data, mappings to target classifications as well as visual display of chemical structure relationships. This work describes the development of the application “SARConnect” to address these issues. We discuss options for delivery and analysis of large-scale SAR data together with a set of use-cases to illustrate the design choices and utility. The main activity sources of ChEMBL,1 GOSTAR2 and AstraZeneca’s internal system IBIS, had already been integrated in Chemistry Connect.3 For target relationships we selected human UniProtKB/Swiss-Prot4 as our primary source of a heuristic target classification. Similarly, to explore chemical relationships we combined several methods for framework and scaffold analysis into a unified, hierarchical classification where ease of navigation was the primary goal. An application was built on TIBCO Spotfire to retrieve data for visual display. Consequently, users can explore relationships between target, activity and structure across internal, external and commercial sources that encompass approximately 3 million compounds, 2000 human proteins and 10 million activity values. Examples showing the utility of the application are given. PMID:23308082

  3. Fine-structure analyses of lipid-protein and protein-protein interactions of gag protein p19 of the avian sarcoma and leukemia viruses by cyanogen bromide mapping.

    PubMed Central

    Pepinsky, R B; Vogt, V M

    1984-01-01

    In avian sarcoma and leukemia viruses, the gag protein p19 functions structurally as a matrix protein, connecting internal components with the viral envelope. We have used a combination of in situ cross-linking and peptide mapping to localize within p19 the regions responsible for two major interactions in this complex, p19 with lipid and p19 with p19. Lipid-protein cross-links were localized near the amino terminus within the first 35 amino acids of the polypeptide. Homotypic protein-protein disulfide bridges were found to originate from near the carboxy terminus of p19, from cysteine residues at amino acids 111 and 153. These results suggest that p19 is divided into domains with distinct functions. The peptide maps constructed for p19, and for the related proteins p23 in avian sarcoma and leukemia viruses and p19 beta in recombinant avian sarcoma viruses, should serve as useful tools for other types of studies involving these proteins. Images PMID:6090691

  4. Notum deacylates Wnt proteins to suppress signalling activity.

    PubMed

    Kakugawa, Satoshi; Langton, Paul F; Zebisch, Matthias; Howell, Steven A; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O'Reilly, Nicola; Snijders, Ambrosius P; Jones, E Yvonne; Vincent, Jean-Paul

    2015-03-12

    Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase. PMID:25731175

  5. Identification of a common protein association region in the neuronal Cdk5 activator.

    PubMed

    Wang, X; Ching, Y P; Lam, W H; Qi, Z; Zhang, M; Wang, J H

    2000-10-13

    Cyclin-dependent protein kinase 5 (Cdk5) depends on the association with neuronal Cdk5 activator (Nck5a) for kinase activity. A variety of cellular proteins have been shown to undergo high affinity association with Nck5a, including three novel proteins, C42, C48, and C53 found by a yeast two-hybrid screen (Ching, Y. P., Qi, Z., and Wang, J. H. (2000) Gene 242, 285-294). The three proteins show competitive binding to Nck5a suggesting that they bind at a common site. The binding site has been mapped to a region of 26 amino acid residues (residues 145 to 170) at the N-terminal boundary of the kinase activation domain of Nck5a. This region of Nck5a contains an amphipathic alpha-helix whose hydrophobic face is involved in Cdk5 activation (Chin, K. T., Ohki, S, Tang, D., Cheng, H. C., Wang, J. H. , and Zhang, M. (1999) J. Biol. Chem. 274, 7120-7127). Several lines of evidence suggest that Nck5a interacts with the binding proteins at the hydrophilic face of the amphipathic alpha-helix. First, the Nck5a-(145-170) peptide can bind Cdk5 and Nck5a-binding proteins simultaneously. Second, the association of Nck5a-(145-170) to C48 can be markedly reduced by high ionic strength whereas the interaction between Nck5a and Cdk5 is not affected. Third, substitution of Glu(157) by glutamine in Nck5a-(145-170) abolishes the peptide's ability to bind to the three Nck5a-binding proteins without diminishing its Cdk5 binding activity. PMID:10915792

  6. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  7. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

  8. Activation of a human chromosomal replication origin by protein tethering

    PubMed Central

    Chen, Xiaomi; Liu, Guoqi; Leffak, Michael

    2013-01-01

    The specification of mammalian chromosomal replication origins is incompletely understood. To analyze the assembly and activation of prereplicative complexes (pre-RCs), we tested the effects of tethered binding of chromatin acetyltransferases and replication proteins on chromosomal c-myc origin deletion mutants containing a GAL4-binding cassette. GAL4DBD (DNA binding domain) fusions with Orc2, Cdt1, E2F1 or HBO1 coordinated the recruitment of the Mcm7 helicase subunit, the DNA unwinding element (DUE)-binding protein DUE-B and the minichromosome maintenance (MCM) helicase activator Cdc45 to the replicator, and restored origin activity. In contrast, replication protein binding and origin activity were not stimulated by fusion protein binding in the absence of flanking c-myc DNA. Substitution of the GAL4-binding site for the c-myc replicator DUE allowed Orc2 and Mcm7 binding, but eliminated origin activity, indicating that the DUE is essential for pre-RC activation. Additionally, tethering of DUE-B was not sufficient to recruit Cdc45 or activate pre-RCs formed in the absence of a DUE. These results show directly in a chromosomal background that chromatin acetylation, Orc2 or Cdt1 suffice to recruit all downstream replication initiation activities to a prospective origin, and that chromosomal origin activity requires singular DNA sequences. PMID:23658226

  9. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  10. The GAAIN Entity Mapper: An Active-Learning System for Medical Data Mapping

    PubMed Central

    Ashish, Naveen; Dewan, Peehoo; Toga, Arthur W.

    2016-01-01

    This work is focused on mapping biomedical datasets to a common representation, as an integral part of data harmonization for integrated biomedical data access and sharing. We present GEM, an intelligent software assistant for automated data mapping across different datasets or from a dataset to a common data model. The GEM system automates data mapping by providing precise suggestions for data element mappings. It leverages the detailed metadata about elements in associated dataset documentation such as data dictionaries that are typically available with biomedical datasets. It employs unsupervised text mining techniques to determine similarity between data elements and also employs machine-learning classifiers to identify element matches. It further provides an active-learning capability where the process of training the GEM system is optimized. Our experimental evaluations show that the GEM system provides highly accurate data mappings (over 90% accuracy) for real datasets of thousands of data elements each, in the Alzheimer's disease research domain. Further, the effort in training the system for new datasets is also optimized. We are currently employing the GEM system to map Alzheimer's disease datasets from around the globe into a common representation, as part of a global Alzheimer's disease integrated data sharing and analysis network called GAAIN1. GEM achieves significantly higher data mapping accuracy for biomedical datasets compared to other state-of-the-art tools for database schema matching that have similar functionality. With the use of active-learning capabilities, the user effort in training the system is minimal. PMID:26793094

  11. Map2k4 Functions as a Tumor Suppressor in Lung Adenocarcinoma and Inhibits Tumor Cell Invasion by Decreasing Peroxisome Proliferator-Activated Receptor γ2 Expression ▿

    PubMed Central

    Ahn, Young-Ho; Yang, Yanan; Gibbons, Don L.; Creighton, Chad J.; Yang, Fei; Wistuba, Ignacio I.; Lin, Wei; Thilaganathan, Nishan; Alvarez, Cristina A.; Roybal, Jonathon; Goldsmith, Elizabeth J.; Tournier, Cathy; Kurie, Jonathan M.

    2011-01-01

    MAP2K4 encodes a dual-specificity kinase (mitogen-activated protein kinase kinase 4, or MKK4) that is mutated in a variety of human malignancies, but the biochemical properties of the mutant kinases and their roles in tumorigenesis have not been fully elucidated. Here we showed that 8 out of 11 cancer-associated MAP2K4 mutations reduce MKK4 protein stability or impair its kinase activity. On the basis of findings from bioinformatic studies on human cancer cell lines with homozygous MAP2K4 loss, we posited that MKK4 functions as a tumor suppressor in lung adenocarcinomas that develop in mice owing to expression of mutant Kras and Tp53. Conditional Map2k4 inactivation in the bronchial epithelium of mice had no discernible effect alone but increased the multiplicity and accelerated the growth of incipient lung neoplasias induced by oncogenic Kras. MKK4 suppressed the invasion and metastasis of Kras-Tp53-mutant lung adenocarcinoma cells. MKK4 deficiency increased peroxisomal proliferator-activated receptor γ2 (PPARγ2) expression through noncanonical MKK4 substrates, and PPARγ2 enhanced tumor cell invasion. We conclude that Map2k4 functions as a tumor suppressor in lung adenocarcinoma and inhibits tumor cell invasion by decreasing PPARγ2 levels. PMID:21896780

  12. Rapamycin enhances eIF4E phosphorylation by activating MAP kinase-interacting kinase 2a (Mnk2a).

    PubMed

    Stead, Rebecca L; Proud, Christopher G

    2013-08-19

    Eukaryotic initiation factor eIF4E and its phosphorylation play key roles in cell transformation and tumorigenesis. eIF4E is phosphorylated by the Mnks (MAP (mitogen-activated protein) kinase-interacting kinases). Rapamycin increases eIF4E phosphorylation in cancer cells, potentially limiting their anti-cancer effects. Here we show that the rapamycin-induced increase in eIF4E phosphorylation reflects increased activity of Mnk2 but not Mnk1. This activation requires a novel phosphorylation site in Mnk2a, Ser437. Our findings have potentially important implications for the use of rapamycin and its analogues in cancer therapy, suggesting that inhibitors of mTOR and Mnk (or Mnk2) may be more efficacious than rapalogs alone. PMID:23831578

  13. Mapping membrane protein backbone dynamics: a comparison of site-directed spin labeling with NMR 15N-relaxation measurements.

    PubMed

    Lo, Ryan H; Kroncke, Brett M; Solomon, Tsega L; Columbus, Linda

    2014-10-01

    The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR (15)N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R1 relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins. PMID:25296323

  14. G Protein Activation without a GEF in the Plant Kingdom

    PubMed Central

    Wang, Hao; Matthews, Melissa; Bradford, William; Bennetzen, Jeffrey L.; Jones, Alan M.

    2012-01-01

    Animal heterotrimeric G proteins are activated by guanine nucleotide exchange factors (GEF), typically seven transmembrane receptors that trigger GDP release and subsequent GTP binding. In contrast, the Arabidopsis thaliana G protein (AtGPA1) rapidly activates itself without a GEF and is instead regulated by a seven transmembrane Regulator of G protein Signaling (7TM-RGS) protein that promotes GTP hydrolysis to reset the inactive (GDP-bound) state. It is not known if this unusual activation is a major and constraining part of the evolutionary history of G signaling in eukaryotes. In particular, it is not known if this is an ancestral form or if this mechanism is maintained, and therefore constrained, within the plant kingdom. To determine if this mode of signal regulation is conserved throughout the plant kingdom, we analyzed available plant genomes for G protein signaling components, and we purified individually the plant components encoded in an informative set of plant genomes in order to determine their activation properties in vitro. While the subunits of the heterotrimeric G protein complex are encoded in vascular plant genomes, the 7TM-RGS genes were lost in all investigated grasses. Despite the absence of a Gα-inactivating protein in grasses, all vascular plant Gα proteins examined rapidly released GDP without a receptor and slowly hydrolyzed GTP, indicating that these Gα are self-activating. We showed further that a single amino acid substitution found naturally in grass Gα proteins reduced the Gα-RGS interaction, and this amino acid substitution occurred before the loss of the RGS gene in the grass lineage. Like grasses, non-vascular plants also appear to lack RGS proteins. However, unlike grasses, one representative non-vascular plant Gα showed rapid GTP hydrolysis, likely compensating for the loss of the RGS gene. Our findings, the loss of a regulatory gene and the retention of the “self-activating” trait, indicate the existence of divergent

  15. Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity.

    PubMed

    Yamaguchi, Fuminori; Tsuchiya, Mitsumasa; Shimamoto, Seiko; Fujimoto, Tomohito; Tokumitsu, Hiroshi; Tokuda, Masaaki; Kobayashi, Ryoji

    2016-01-01

    Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium-binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis. PMID:27600583

  16. Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells.

    PubMed

    Graham, A; McLees, A; Kennedy, C; Gould, G W; Plevin, R

    1996-03-01

    1. We have investigated the characteristics of activation of the 42kDa isoform of mitogen-activated protein (MAP) kinase in response to various nucleotides in the endothelial cell line EAhy 926. 2. Adenosine 5'-triphosphate (ATP) in the concentration range 0.1-100 microM stimulated the rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peaked at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cells. 3. Uridine 5' triphosphate (UTP) also stimulated the 42 kDa isoform of MAP kinase with similar potency to ATP (EC50 values 5.1 +/- 0.2 microM for UTP; 2.9 +/- 0.8 microM for ATP), whilst the selective P2Y-purinoceptor agonist, 2-methylthioATP (2-meSATP) was without effect up to concentrations of 100 microM. In bovine aortic endothelial cells however, UTP and 2-meSATP both stimulated MAP kinase. 4. Pretreatment of cells for 24 h with 12-O tetradecanoyl phorbol 13-acetate resulted in the loss of the alpha and epsilon isoforms of protein kinase C (PKC) and virtual abolition of nucleotide-stimulated MAP kinase activity (> 90% inhibition). 5. Preincubation for 30 min with the PKC inhibitor, Ro-31 8220 (10 microM) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6. Removal of extracellular calcium in the presence of EGTA reduced the MAP kinase activation in response to UTP by approximately 30-50%. 7. Pretreatment with pertussis toxin (18 h, 50 ng ml-1) did not significantly affect the UTP-mediated activation of pp42 MAP kinase. 8. These results show that in the EAhy 926 endothelial cell line, nucleotides stimulate activation of MAP kinase in a protein kinase C-dependent manner through interaction with a P2U-purinoceptor. PMID:8882634

  17. Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells.

    PubMed Central

    Graham, A.; McLees, A.; Kennedy, C.; Gould, G. W.; Plevin, R.

    1996-01-01

    1. We have investigated the characteristics of activation of the 42kDa isoform of mitogen-activated protein (MAP) kinase in response to various nucleotides in the endothelial cell line EAhy 926. 2. Adenosine 5'-triphosphate (ATP) in the concentration range 0.1-100 microM stimulated the rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peaked at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cells. 3. Uridine 5' triphosphate (UTP) also stimulated the 42 kDa isoform of MAP kinase with similar potency to ATP (EC50 values 5.1 +/- 0.2 microM for UTP; 2.9 +/- 0.8 microM for ATP), whilst the selective P2Y-purinoceptor agonist, 2-methylthioATP (2-meSATP) was without effect up to concentrations of 100 microM. In bovine aortic endothelial cells however, UTP and 2-meSATP both stimulated MAP kinase. 4. Pretreatment of cells for 24 h with 12-O tetradecanoyl phorbol 13-acetate resulted in the loss of the alpha and epsilon isoforms of protein kinase C (PKC) and virtual abolition of nucleotide-stimulated MAP kinase activity (> 90% inhibition). 5. Preincubation for 30 min with the PKC inhibitor, Ro-31 8220 (10 microM) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6. Removal of extracellular calcium in the presence of EGTA reduced the MAP kinase activation in response to UTP by approximately 30-50%. 7. Pretreatment with pertussis toxin (18 h, 50 ng ml-1) did not significantly affect the UTP-mediated activation of pp42 MAP kinase. 8. These results show that in the EAhy 926 endothelial cell line, nucleotides stimulate activation of MAP kinase in a protein kinase C-dependent manner through interaction with a P2U-purinoceptor. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:8882634

  18. Mapping Active Faults and Tectonic Geomorphology offshore central California

    NASA Astrophysics Data System (ADS)

    Johnson, S. Y.; Watt, J. T.; Hart, P. E.; Sliter, R. W.; Wong, F. L.

    2009-12-01

    In June 2008, and July 2009, the USGS conducted two high-resolution, marine, seismic-reflection surveys across the continental shelf and upper slope between Piedras Blancas and Point Sal, central California, in order to better characterize regional earthquake sources. More than 1,300 km of single-channel seismic data were acquired aboard the USGS R/V Parke Snavely using a 500-joule mini-sparker source fired at a 0.5-second shot interval and recorded with a 15-meter streamer. Most tracklines were run perpendicular to the coast at 800-meter spacing, extending from the nearshore (~ 10-15 m water depth) to as far as 20 km offshore. Sub-bottom imaging varies with substrate, ranging from outstanding (100 to 150 m of penetration) in inferred Quaternary shallow marine, shelf and upper slope deposits to poor (0 to 10 m) in the Mesozoic basement rocks. Marine magnetic data were collected simultaneously on this survey, and both data sets are being integrated with new aeromagnetic data, publicly available industry seismic-reflection data, onshore geology, seismicity, and high-resolution bathymetry. Goals of the study are to map geology, structure, and sediment distribution; to document fault location, length, segmentation, shallow geometry and structure; and to identify possible sampling targets for constraining fault slip rates, earthquake recurrence, and tsunami hazard potential. The structure and tectonic geomorphology of the >100-km-long, right-lateral, Hosgri fault zone and its connections to the Los Osos, Pecho, Oceano and other northwest-trending inboard faults are the focus of this ongoing work. The Hosgri fault forms the eastern margin of the offshore Santa Maria basin and coincides in places with the outer edge of the narrow (5- to 15-km-wide), structurally complex continental shelf. The Hosgri is imaged as a relatively continuous, vertical fault zone that extends upward to the seafloor; varies significantly and rapidly along strike; and incorporates numerous

  19. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  20. Specific degradation of the mucus adhesion-promoting protein (MapA) of Lactobacillus reuteri to an antimicrobial peptide.

    PubMed

    Bøhle, Liv Anette; Brede, Dag Anders; Diep, Dzung B; Holo, Helge; Nes, Ingolf F

    2010-11-01

    The intestinal flora of mammals contains lactic acid bacteria (LAB) that may provide positive health effects for the host. Such bacteria are referred to as probiotic bacteria. From a pig, we have isolated a Lactobacillus reuteri strain that produces an antimicrobial peptide (AMP). The peptide was purified and characterized, and it was unequivocally shown that the AMP was a well-defined degradation product obtained from the mucus adhesion-promoting protein (MapA); it was therefore termed AP48-MapA. This finding demonstrates how large proteins might inherit unexpected pleiotropic functions by conferring antimicrobial capacities on the producer. The MapA/AP48-MapA system is the first example where a large protein of an intestinal LAB is shown to give rise to such an AMP. It is also of particular interest that the protein that provides this AMP is associated with the binding of the bacterium producing it to the surface/lining of the gut. This finding gives us new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota. PMID:20833791

  1. Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus.

    PubMed

    Soares, Alexandra Martins dos Santos; de Araújo, Sandra Alves; Lopes, Suzana Gomes; Costa Junior, Livio Martins

    2015-01-01

    The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts. PMID:26689178

  2. Heterozygous Mutations in MAP3K7, Encoding TGF-β-Activated Kinase 1, Cause Cardiospondylocarpofacial Syndrome.

    PubMed

    Le Goff, Carine; Rogers, Curtis; Le Goff, Wilfried; Pinto, Graziella; Bonnet, Damien; Chrabieh, Maya; Alibeu, Olivier; Nistchke, Patrick; Munnich, Arnold; Picard, Capucine; Cormier-Daire, Valérie

    2016-08-01

    Cardiospondylocarpofacial (CSCF) syndrome is characterized by growth retardation, dysmorphic facial features, brachydactyly with carpal-tarsal fusion and extensive posterior cervical vertebral synostosis, cardiac septal defects with valve dysplasia, and deafness with inner ear malformations. Whole-exome sequencing identified heterozygous MAP3K7 mutations in six distinct CSCF-affected individuals from four families and ranging in age from 5 to 37 years. MAP3K7 encodes transforming growth factor β (TGF-β)-activated kinase 1 (TAK1), which is involved in the mitogen-activated protein kinase (MAPK)-p38 signaling pathway. MAPK-p38 signaling was markedly altered when expression of non-canonical TGF-β-driven target genes was impaired. These findings support the loss of transcriptional control of the TGF-β-MAPK-p38 pathway in fibroblasts obtained from affected individuals. Surprisingly, although TAK1 is located at the crossroad of inflammation, immunity, and cancer, this study reports MAP3K7 mutations in a developmental disorder affecting mainly cartilage, bone, and heart. PMID:27426734

  3. Comparing Computer-Supported Dynamic Modeling and "Paper & Pencil" Concept Mapping Technique in Students' Collaborative Activity

    ERIC Educational Resources Information Center

    Komis, Vassilis; Ergazaki, Marida; Zogza, Vassiliki

    2007-01-01

    This study aims at highlighting the collaborative activity of two high school students (age 14) in the cases of modeling the complex biological process of plant growth with two different tools: the "paper & pencil" concept mapping technique and the computer-supported educational environment "ModelsCreator". Students' shared activity in both cases…

  4. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  5. Prototype active scanner for nighttime oil spill mapping and classification

    NASA Technical Reports Server (NTRS)

    Sandness, G. A.; Ailes, S. B.

    1977-01-01

    A prototype, active, aerial scanner system was constructed for nighttime water pollution detection and nighttime multispectral imaging of the ground. An arc lamp was used to produce the transmitted light and four detector channels provided a multispectral measurement capability. The feasibility of the design concept was demonstrated by laboratory and flight tests of the prototype system.

  6. Liposomal packaging generates Wnt protein with in vivo biological activity.

    PubMed

    Morrell, Nathan T; Leucht, Philipp; Zhao, Ludan; Kim, Jae-Beom; ten Berge, Derk; Ponnusamy, Karthik; Carre, A Lyonel; Dudek, Henryk; Zachlederova, Marie; McElhaney, Michael; Brunton, Shirley; Gunzner, Janet; Callow, Marinella; Polakis, Paul; Costa, Mike; Zhang, Xiaoyan M; Helms, Jill A; Nusse, Roel

    2008-01-01

    Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context. PMID:18698373

  7. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  8. The human renin-binding protein gene (RENBP) maps in Xq28

    SciTech Connect

    Ouweland, A.M.W. van der; Verdijk, M.; Oost, B.A. van ); Kiochis, P.; Poustka, A. )

    1994-05-01

    The authors report here the successful application of the method by which cDNA libraries are screened with positionally identified genomic clones. Human cosmid clones were selected from a cosmid library derived from the Q1Z cell line. This Q1Z cell line is a hamster-human somatic cell hybrid that contains the Xq28 region as its sole human component. To search for kidney-expressed genes, they screened a kidney cDNA library purchased from Clontech with cosmid-derived probes. Based on the physical mapping of the vasopressin V2 receptor gene close to the L1CAM gene, they analyzed cosmids derived from this region. One of the cosmids was 12B2, located 50 kb from the L1CAM gene. A 20-kb EcoRI subclone from the 12B2 cosmid was used as probe. This fragment did not hybridize to the probe 2-55 in contrast to the whole cosmid 12B2. Screening of 200,000 cDNA clones resulted in the identification of two positive clones. After sequence determination, it appeared that one of the positive cDNA clones contained Escherichia coli DNA as insert (data not shown). The other cDNA (pMV24) contained an open reading frame corresponding to the 243 amino-terminal amino acids of the human renin binding protein. The RENBP gene maps to interval 3 between the loci for DX52 and G-6-PD. This is the same interval as that for the color blindness gene, DXS707, and the AVPR2, L1CAM, and QM genes. This result confirms that the isolated RENBP cDNA originates from the same location as that from which the parental cosmid clone was derived. 28 refs., 1 fig.

  9. RNAi screens reveal novel metabolic regulators: RIP140, MAP4k4 and the lipid droplet associated fat specific protein (FSP) 27.

    PubMed

    Puri, V; Virbasius, J V; Guilherme, A; Czech, M P

    2008-01-01

    Adipose tissue modulates whole body metabolism and insulin sensitivity by controlling circulating lipid levels and producing molecules that can regulate fatty acid metabolism in such tissues as muscle and liver. We have developed RNA interference (RNAi) screens to identify genes in cultured adipocytes that regulate insulin signalling and key metabolic pathways. These short interfering RNA (siRNA)-based screens identified the transcriptional corepressor receptor interacting protein 140 (RIP140) (J Clin Invest 116: 125, 2006) and the mitogen-activated protein kinase (MAP4k4) (Proc Natl Acad Sci USA 103: 2087, 2006) as negative regulators of insulin-responsive hexose uptake and oxidative metabolism. Gene expression profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, tricarboxylic acid cycle, fatty acid oxidation, mitochondrial biogenesis and oxidative phosphorylation. RIP140-null mice resist weight gain on a high-fat diet and display enhanced glucose tolerance. MAP4k4 depletion in adipocytes increases many of the RIP140-sensitive genes, increases adipogenesis and mediates some actions of tumour necrosis factor-alpha (TNF-alpha). Remarkably, another hit in our RNAi screens was fat specific protein 27 (FSP27), a highly expressed isoform of Cidea. We discovered that FSP27 unexpectedly associates specifically with lipid droplets and regulates fat storage. We conclude that RIP140, MAP4k4 and the novel lipid droplet protein FSP27 are powerful regulators of adipose tissue metabolism and are potential therapeutic targets for controlling metabolic disease. The discovery of these novel proteins validates the power of RNAi screening for discovery of new therapeutic approaches to type 2 diabetes and obesity. PMID:18171433

  10. The role of photogeologic mapping in traverse planning: Lessons from DRATS 2010 activities

    NASA Astrophysics Data System (ADS)

    Skinner, , James A.; Fortezzo, Corey M.

    2013-10-01

    We produced a 1:24,000 scale photogeologic map of the Desert Research and Technology Studies (DRATS) 2010 simulated lunar mission traverse area and surrounding environments located within the northeastern part of the San Francisco Volcanic Field (SFVF), north-central Arizona. To mimic an exploratory mission, we approached the region "blindly" by rejecting prior knowledge or preconceived notions of the regional geologic setting and focused instead only on image and topographic base maps that were intended to be equivalent to pre-cursor mission "orbital returns". We used photogeologic mapping techniques equivalent to those employed during the construction of modern planetary geologic maps. Based on image and topographic base maps, we identified 4 surficial units (talus, channel, dissected, and plains units), 5 volcanic units (older cone, younger cone, older flow, younger flow, and block field units), and 5 basement units (grey-toned mottled, red-toned platy, red-toned layered, light-toned slabby, and light-toned layered units). Comparison of our remote-based map units with published field-based map units indicates that the two techniques yield pervasively similar results of contrasting detail, with higher accuracies linked to remote-based units that have high topographic relief and tonal contrast relative to adjacent units. We list key scientific questions that remained after photogeologic mapping and prior to DRATS activities and identify 13 specific observations that the crew and science team would need to make in order to address those questions and refine the interpreted geologic context. We translated potential observations into 62 recommended sites for visitation and observation during the mission traverse. The production and use of a mission-specific photogeologic map for DRATS 2010 activities resulted in strategic and tactical recommendations regarding observational context and hypothesis tracking over the course of an exploratory mission.

  11. The role of photogeologic mapping in traverse planning: lessons learned from DRATS 2010 activities

    USGS Publications Warehouse

    Skinner, James A.; Fortezzo, Corey M.

    2013-01-01

    We produced a 1:24,000 scale photogeologic map of the Desert Research and Technology Studies (DRATS) 2010 simulated lunar mission traverse area and surrounding environments located within the northeastern part of the San Francisco Volcanic Field (SFVF), north-central Arizona. To mimic an exploratory mission, we approached the region "blindly" by rejecting prior knowledge or preconceived notions of the regional geologic setting and focused instead only on image and topographic base maps that were intended to be equivalent to pre-cursor mission "orbital returns". We used photogeologic mapping techniques equivalent to those employed during the construction of modern planetary geologic maps. Based on image and topographic base maps, we identified 4 surficial units (talus, channel, dissected, and plains units), 5 volcanic units (older cone, younger cone, older flow, younger flow, and block field units), and 5 basement units (grey-toned mottled, red-toned platy, red-toned layered, light-toned slabby, and light-toned layered units). Comparison of our remote-based map units with published field-based map units indicates that the two techniques yield pervasively similar results of contrasting detail, with higher accuracies linked to remote-based units that have high topographic relief and tonal contrast relative to adjacent units. We list key scientific questions that remained after photogeologic mapping and prior to DRATS activities and identify 13 specific observations that the crew and science team would need to make in order to address those questions and refine the interpreted geologic context. We translated potential observations into 62 recommended sites for visitation and observation during the mission traverse. The production and use of a mission-specific photogeologic map for DRATS 2010 activities resulted in strategic and tactical recommendations regarding observational context and hypothesis tracking over the course of an exploratory mission.

  12. A 32-kDa protein associated with phospholipase A2-inhibitory activity from human placenta.

    PubMed

    Hayashi, H; Owada, M K; Sonobe, S; Kakunaga, T; Kawakatsu, H; Yano, J

    1987-11-01

    Two monomeric 32-kDa proteins, termed 32K-I (pI 5.8) and 32K-II (pI 5.1), were isolated from human placenta, which was solubilized by a Ca2+-chelator. Only 32K-I was associated with PLA2-inhibitory activity. CNBr peptide mapping indicated that 32K-I was distinct from 32K-II and two 36-kDa proteins, called calpactin I and II or lipocortin II and I, which have been shown to possess PLA2-inhibitory activity. 32K-I bound to PS in a Ca2+-dependent manner. 32K-I was detected in many tissues except brain, cardiac and skeletal muscle. PMID:3666152

  13. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    PubMed

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research. PMID:16302727

  14. Mapping of the local environmental changes in proteins by cysteine scanning

    PubMed Central

    Yamazaki, Yoichi; Nagata, Tomoko; Terakita, Akihisa; Kandori, Hideki; Shichida, Yoshinori; Imamoto, Yasushi

    2014-01-01

    Protein conformational changes, which regulate the activity of proteins, are induced by the alternation of intramolecular interactions. Therefore, the detection of the local environmental changes around the key amino acid residues is essential to understand the activation mechanisms of functional proteins. Here we developed the methods to scan the local environmental changes using the vibrational band of cysteine S-H group. We validated the sensitivity of this method using bathorhodopsin, a photoproduct of rhodopsin trapped at liquid nitrogen temperature, which undergoes little conformational changes from the dark state as shown by the X-ray crystallography. The cysteine residues were individually introduced into 15 positions of Helix III, which contains several key amino acid residues for the light-induced conformational changes of rhodopsin. The shifts of S-H stretching modes of these cysteine residues and native cysteine residues upon the formation of bathorhodopsin were measured by Fourier transform infrared spectroscopy. While most of cysteine residues demonstrated no shift of S-H stretching mode, cysteine residues introduced at positions 117, 118, and 122, which are in the vicinity of the chromophore, demonstrated the significant changes. The current results are consistent with the crystal structure of bathorhodopsin, implying that the cysteine scanning is sensitive enough to detect the tiny conformational changes.

  15. Mapping a Dynamic Innate Immunity Protein Interaction Network Regulating Type I Interferon Production

    PubMed Central

    Li, Shitao; Wang, Lingyan; Berman, Michael; Kong, Young-Yun; Dorf, Martin E.

    2011-01-01

    SUMMARY To systematically investigate innate immune signaling networks regulating production of type I interferon, we analyzed protein complexes formed after microbial recognition. Fifty-eight baits were associated with 260 interacting proteins forming a human innate immunity interactome for type I interferon (HI5) of 401 unique interactions; 21% of interactions were modulated by RNA, DNA, or LPS. Overexpression and depletion analyses identified 22 unique genes that regulated NF-κB and ISRE reporter activity, viral replication, or virus-induced interferon production. Detailed mechanistic analysis defined a role for mind bomb (MIB) E3 ligases in K63-linked ubiquitination of TBK1, a kinase that phosphorylates IRF transcription factors controlling interferon production. Mib genes selectively controlled responses to cytosolic RNA. MIB deficiency reduced antiviral activity, establishing the role of MIB proteins as positive regulators of antiviral responses. The HI5 provides a dynamic physical and regulatory network that serves as a resource for mechanistic analysis of innate immune signaling. PMID:21903422

  16. Mapping of the local environmental changes in proteins by cysteine scanning.

    PubMed

    Yamazaki, Yoichi; Nagata, Tomoko; Terakita, Akihisa; Kandori, Hideki; Shichida, Yoshinori; Imamoto, Yasushi

    2014-01-01

    Protein conformational changes, which regulate the activity of proteins, are induced by the alternation of intramolecular interactions. Therefore, the detection of the local environmental changes around the key amino acid residues is essential to understand the activation mechanisms of functional proteins. Here we developed the methods to scan the local environmental changes using the vibrational band of cysteine S-H group. We validated the sensitivity of this method using bathorhodopsin, a photoproduct of rhodopsin trapped at liquid nitrogen temperature, which undergoes little conformational changes from the dark state as shown by the X-ray crystallography. The cysteine residues were individually introduced into 15 positions of Helix III, which contains several key amino acid residues for the light-induced conformational changes of rhodopsin. The shifts of S-H stretching modes of these cysteine residues and native cysteine residues upon the formation of bathorhodopsin were measured by Fourier transform infrared spectroscopy. While most of cysteine residues demonstrated no shift of S-H stretching mode, cysteine residues introduced at positions 117, 118, and 122, which are in the vicinity of the chromophore, demonstrated the significant changes. The current results are consistent with the crystal structure of bathorhodopsin, implying that the cysteine scanning is sensitive enough to detect the tiny conformational changes. PMID:27493492

  17. Counteracting Protein Kinase Activity in the Heart: The Multiple Roles of Protein Phosphatases

    PubMed Central

    Weber, Silvio; Meyer-Roxlau, Stefanie; Wagner, Michael; Dobrev, Dobromir; El-Armouche, Ali

    2015-01-01

    Decades of cardiovascular research have shown that variable and flexible levels of protein phosphorylation are necessary to maintain cardiac function. A delicate balance between phosphorylated and dephosphorylated states of proteins is guaranteed by a complex interplay of protein kinases (PKs) and phosphatases. Serine/threonine phosphatases, in particular members of the protein phosphatase (PP) family govern dephosphorylation of the majority of these cardiac proteins. Recent findings have however shown that PPs do not only dephosphorylate previously phosphorylated proteins as a passive control mechanism but are capable to actively control PK activity via different direct and indirect signaling pathways. These control mechanisms can take place on (epi-)genetic, (post-)transcriptional, and (post-)translational levels. In addition PPs themselves are targets of a plethora of proteinaceous interaction partner regulating their endogenous activity, thus adding another level of complexity and feedback control toward this system. Finally, novel approaches are underway to achieve spatiotemporal pharmacologic control of PPs which in turn can be used to fine-tune misleaded PK activity in heart disease. Taken together, this review comprehensively summarizes the major aspects of PP-mediated PK regulation and discusses the subsequent consequences of deregulated PP activity for cardiovascular diseases in depth. PMID:26617522

  18. Fast clearance of lipid droplets through MAP1S-activated autophagy suppresses clear cell renal cell carcinomas and promotes patient survival

    PubMed Central

    Liu, Xian-De; Yue, Fei; Li, Wenjiao; Li, Xun; He, Yongzhong; Jiang, Xianhan; Huang, Hai; Chen, Qi; Jonasch, Eric; Liu, Leyuan

    2016-01-01

    Clear cell renal cell carcinoma (ccRCC) is composed of cells whose cytoplasm filled with lipid droplets, subcellular organelles coated with adipocyte differentiation-related protein (ADFP) for the storage of triacylglycerol converted from excess free fatty acids. Mammalian cells primarily use the autophagy-lysosome system to degrade misfolded/aggregated proteins and dysfunctional organelles such as lipid droplets. MAP1S (originally named C19ORF5) is an autophagy activator and promotes the biogenesis and degradation of autophagosomes. Previously, we reported that MAP1S suppresses hepatocellular carcinogenesis in a mouse model and promoted the survival of patients with prostate adenocarcinomas by increasing the degradation of aggregated proteins and dysfunctional mitochondria. Here we show that a suppression of MAP1S in renal cells causes an impairment of autophagic clearance of lipid droplets. In contrast, an overexpression of MAP1S causes an activation of autophagy flux and a reduction of lipid droplets so less DNA double strand breakage is induced. The levels of MAP1S in normal renal cells are dramatically higher than those in the ccRCC tissues and cell lines derived from renal cell carcinomas. High levels of MAP1S are associated with a reduced malignancy and metastasis of ccRCC and predict a better survival of ccRCC patients. Therefore, autophagy defects in the degradation of lipid droplets triggered by the MAP1S deficiency may enhance the initiation and development of ccRCC and reduce the survival of ccRCC patients. PMID:26701856

  19. Reverberation Mapping of the Dusty Tori in Active Galactic Nuclei

    NASA Astrophysics Data System (ADS)

    Richmond, Michael; Batcheldor, Daniel; Buchanan, Catherine; Capetti, Alessandro; Moshe, Elitzur; Gallimore, Jack; Horne, Keith; Kishimoto, Makoto; Marconi, Alessandro; Mason, Rachel; Maiolino, Robert; Netzer, Hagai; Packham, Christopher; Perez, Enrique; Peterson, Brad; Tadhunter, Clive; Robinson, Andrew; Stirpe, Giovanna; Storchi-Bergmann, Thaisa

    2012-12-01

    Our current understanding of the size and structure of AGN tori is weak, despite their central role in AGN unification models and their importance for studies of supermassive black hole demographics. We propose to use the warm phase of Spitzer to determine the sizes of circum-nuclear dust tori in AGN. To accomplish this we will extend an existing Spitzer monitoring campaign, coordinated with ground-based observations, to measure the 'light echo' as the dust emission responds to variations in the AGN optical/UV continuum. We have selected a sample of 12 bright type 1 nuclei in close proximity to the Spitzer Continuous Viewing Zone which can be observed for at least 70% of the 365 day cycle. We will observe each AGN every 30 days for the whole of Cycle 9, roughly doubling our existing baseline of one year, permitting us to identify optical-IR time lags of many months. We will continue our current ground based monitoring program using a variety of telescopes to determine the AGN light-curves in the optical. These observations will sample the torus more faithfully than previous measurements made in the K-band. Such high fidelity, continuously sampled IR light curves covering ~years cannot be obtained from the ground, and are needed because the expected reverberation timescales are hundreds of days. We will apply well developed techniques to determine the reverberation lag and therefore obtain the characteristic size of the torus in this sample which spans a range of black hole mass and Eddington ratio. Our team contains many leading experts in reverberation mapping of AGN and in the observational study and theoretical modeling of the physics of the dusty torus. We are requesting a total of 14 hours in the cycle to perform our observations. These observations will provide a stringent observational test of current models for the obscuring torus in AGN. The required measurements - long timescales, continuous monitoring in the near-infrared - are possible only with the

  20. The effects of cardamonin on lipopolysaccharide-induced inflammatory protein production and MAP kinase and NFκB signalling pathways in monocytes/macrophages

    PubMed Central

    Hatziieremia, S; Gray, A I; Ferro, V A; Paul, A; Plevin, R

    2006-01-01

    Background and purpose: In this study we examined the effect of the natural product cardamonin, upon lipopolysaccharide (LPS)-induced inflammatory gene expression in order to attempt to pinpoint the mechanism of action. Experimental approaches: Cardamonin was isolated from the Greek plant A. absinthium L. Its effects were assessed on LPS-induced nitrite release and iNOS and COX-2 protein expression in two macrophage cell lines. Western blotting was used to investigate its effects on phosphorylation of the mitogen activated protein (MAP) kinases, ERK, JNK and p38 MAP kinase, and activation of the NFκB pathway, at the level of IκBα degradation and phosphorylation of NFκB. Also its effects on NFκB and GAS/GAF-DNA binding were assessed by EMSA. Key results: Cardamonin concentration-dependently inhibited both NO release and iNOS expression but had no effect on COX-2 expression. It did not affect phosphorylation of the MAP kinases, degradation of IκBα or phosphorylation of NFκB. However, it inhibited NFκB DNA-binding in both LPS-stimulated cells and nuclear extracts of the cells (in vitro). It also inhibited IFNγ-stimulated iNOS induction and GAS/GAF-DNA binding. Conclusions and Implications: These results show that the inhibitory effect of cardamonin on LPS-induced iNOS induction is not mediated via effects on the initial activation of the NFκB or MAP kinase pathways but is due to a direct effect on transcription factor binding to DNA. However, although some selectivity in cardamonin's action is implicated by its inability to affect COX-2 expression, its exact mechanism(s) of action has yet to be identified. PMID:16894344

  1. Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

    PubMed Central

    Samuels, M; Deshpande, G; Schedl, P

    1998-01-01

    The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain. PMID:9592147

  2. Elicitor- and A23187-induced expression of WCK-1, a gene encoding mitogen-activated protein kinase in wheat.

    PubMed

    Takezawa, D

    1999-08-01

    Wheat cultured cells were used to study the role of Ca2+ in regulating protein kinases during the induction of defense-related genes by fungal elicitor treatments. Manipulation of intracellular Ca2+ concentrations by treatment with calcium ionophore A23187 in the presence of high extracellular Ca2+ resulted in the induction of mRNA expression of WCK-1, a gene encoding mitogen-activated protein (MAP) kinase. The induction of WCK-1 mRNA by A23187 did not occur when extracellular Ca2+ was chelated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The WCK-1 mRNA was also induced by Typhula ishikariensis-derived elicitors, suggesting a possible involvement of WCK-1 in the plant defense response against pathogens. BAPTA and a calcium channel blocker, La3+, inhibited the elicitor-induced expression of the WCK-1 mRNA. A recombinant fusion protein of WCK-1 (GST-WCK-1) autophosphorylated at the Tyr residue and exhibited an autophosphorylation-dependent protein kinase activity towards myelin basic protein. Alteration of Tyr-196 in the conserved 'TEY' motif in GST-WCK-1 to Phe by site-directed mutagenesis abolished the autophosphorylation. The GST-WCK-1 protein was activated by elicitor-treated wheat cell extracts but not by the control extract. These results suggest that fungal elicitors activate WCK-1, a specific MAP kinase in wheat. Furthermore, the results suggest a possible involvement of Ca2+ in enhancing the MAP kinase signaling cascade in plants by controlling the levels of the MAP kinase transcripts. PMID:10527417

  3. Evaluation of feasibility of mapping seismically active faults in Alaska

    NASA Technical Reports Server (NTRS)

    Gedney, L. D. (Principal Investigator); Vanwormer, J. D.

    1973-01-01

    The author has identified the following significant results. ERTS-1 imagery is proving to be exceptionally useful in delineating structural features in Alaska which have never been recognized on the ground. Previously unmapped features such as seismically active faults and major structural lineaments are especially evident. Among the more significant results of this investigation is the discovery of an active strand of the Denali fault. The new fault has a history of scattered activity and was the scene of a magnitude 4.8 earthquake on October 1, 1972. Of greater significance is the disclosure of a large scale conjugate fracture system north of the Alaska Range. This fracture system appears to result from compressive stress radiating outward from around Mt. McKinley. One member of the system was the scene of a magnitude 6.5 earthquake in 1968. The potential value of ERTS-1 imagery to land use planning is reflected in the fact that this earthquake occurred within 10 km of the site which was proposed for the Rampart Dam, and the fault on which it occurred passes very near the proposed site for the bridge and oil pipeline crossing of the Yukon River.

  4. Dependency of microtubule-associated proteins (MAPs) for tubulin stability and assembly; use of estramustine phosphate in the study of microtubules.

    PubMed

    Fridén, B; Wallin, M

    1991-07-10

    Microtubule-associated proteins (MAPs) were separated from tubulin with several different methods. The ability of the isolated MAPs to reinduce assembly of phosphocellulose purified tubulin differed markedly between the different methods. MAPs isolated by addition of 0.35 M NaCl to taxol-stabilized microtubules stimulated tubulin assembly most effectively, while addition of 0.6 M NaCl produced MAPs with a substantially lower ability to stimulate tubulin assembly. The second best preparation was achieved with phosphocellulose chromatographic separation of MAPs with 0.6 M NaCl elution. The addition of estramustine phosphate to microtubules reconstituted of MAPs prepared by 0.35 M NaCl or phosphocellulose chromatography, induced less disassembly than for microtubules assembled from unseparated proteins, and was almost without effect on microtubules reconstituted from MAPs prepared by taxol and 0.6 M NaCl. Estramustine phosphate binds to the tubulin binding part of the MAPs, and the results do therefore indicate that the MAPs are altered by the separation methods. Since the MAPs are regarded as highly stable molecules, one probable alteration could be aggregation of the MAPs, as also indicated by the results. The purified tubulin itself seemed not to be affected by the phosphocellulose purification, since the microtubule proteins were unchanged by the low buffer strenght used during the cromatography. However, the assembly competence after a prolonged incubation of the microtubule proteins at 4 degrees C was dependent on intact bindings between the tubulin and MAPs. PMID:1681420

  5. Heated Proteins are Still Active in a Functionalized Nanoporous Support

    SciTech Connect

    Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Lei, Chenghong; Liu, Jun

    2013-07-08

    We report that even under the heated condition, the conformation and activity of a protein can be hoarded in a functionalized nanoporous support via non-covalent interaction, although the hoarded protein was not exhibiting the full protein activity, the protein released subsequently still maintained its native conformation and activity. Glucose oxidase (GOX) was spontaneously and largely entrapped in aminopropyl-functionalized mesoporous silica (NH2-FMS) at 20 oC via a dominant electrostatic interaction. Although FMS-GOX displayed 45% activity of the free enzyme in solution, the GOX released from FMS exhibited its 100% activity prior to the entrapment. Surprisingly, the released GOX from FMS still maintained 89% of its initial activity prior to the entrapment after FMS-GOX was incubated at 60 oC for 1 h prior to release, while the free GOX in solution lost nearly all activity under the same incubation. Intrinsic fluorescence emission of GOX and native electrophoresis demonstrated that the heating resulted in significant conformational changes and oligomeric structures of the free GOX, but FMS efficiently maintained the thermal stability of GOX therein and resisted the thermal denaturation and oligomeric aggregation.

  6. Probing heterotrimeric G protein activation: applications to biased ligands

    PubMed Central

    Denis, Colette; Saulière, Aude; Galandrin, Ségolène; Sénard, Jean-Michel; Galés, Céline

    2012-01-01

    Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of “ligand-directed trafficking” has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting activation of specific signals and thus generating textured responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligand responses. However, considering that a single receptor has pleiotropic signalling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even if some G protein-independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discuss them in the context of G protein biased -ligands. PMID:22229559

  7. Bryostatins activate protein kinase C in intact human platelets

    SciTech Connect

    Smith, J.B.; Tallant, E.A.; Pettit, G.R.; Wallace, R.W.

    1986-05-01

    Bryostatins, macrocyclic lactones isolated from a marine bryozoan, have antineoplastic activity in the P388 lymphocytic leukemia system. These compounds also stimulate growth in Swiss 3T3 cells, induce secretion in leukocytes, inhibit phorbol dibutyrate binding to a high affinity receptor, and activate the C-kinase in vitro. In human platelets, phorbol esters induce aggregation and activate protein kinase C, resulting in phosphorylation of a 47K protein and the 20K myosin light chain. The authors now show that bryostatin 7 (B-7) triggers platelet aggregation to the same rate and extent as phorbol 12-myristate 13-acetate (PMA). B-7 also causes the in vivo activation of the C-kinase, resulting in phosphorylation of both the 47K and the 20K proteins; the time courses and dose-responses of these B-7-induced phosphorylations were similar to those found with PMA. In addition, B-7 increases the level of /sup 32/P-incorporation into the platelet polyphosphoinositides, which also occurs in response to PMA. Bryostatin 3 (B-3), which has been shown to be much less potent than B-7 in mimicking other PMA effects, was much less effective than PMA or B-7 in inducing platelet aggregation and in stimulating /sup 32/P-incorporation into both proteins and the phosphoinositides. These results demonstrate that, intact human platelets, bryostatins mimic the phorbol esters tumor promoters and directly activate protein kinase C.

  8. Probing heterotrimeric G protein activation: applications to biased ligands.

    PubMed

    Denis, Colette; Saulière, Aude; Galandrin, Segolene; Sénard, Jean-Michel; Galés, Céline

    2012-01-01

    Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of "ligand-directed trafficking" has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting the activation of specific signals and thus generating texture responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligands. However, considering that a single receptor has pleiotropic signaling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even, if some G protein independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discussed them in the context of G protein biased-ligands. PMID:22229559

  9. Brominated Flame Retardants, Tetrabromobisphenol A and Hexabromocyclododecane, Activate Mitogen-Activated Protein Kinases (MAPKs) in Human Natural Killer Cells

    PubMed Central

    Cato, Anita; Celada, Lindsay; Kibakaya, Esther Caroline; Simmons, Nadia; Whalen, Margaret M.

    2014-01-01

    NK cells provide a vital surveillance against virally infected cells, tumor cells, and antibody-coated cells through the release of cytolytic mediators and gamma interferon (IFN-γ). Hexabromocyclododecane (HBCD) is a brominated flame retardant used primarily in expanded (EPS) and extruded (XPS) polystyrene foams for thermal insulation in the building and construction industry. Tetrabromobisphenol A (TBBPA) is used both as a reactive and an additive flame retardant in a variety of materials. HBCD and TBBPA contaminate the environment and are found in human blood samples. In previous studies, we have shown that other environmental contaminants, such as the dibutyltin (DBT) and tributyltin (TBT), decrease NK lytic function by activating mitogen-activated protein kinases (MAPKs) in the NK cells. HBCD and TBBPA also interfere with NK cell(s) lytic function. The current study evaluates whether HBCD and/or TBBPA have the capacity to activate MAPKs and MAPK kinases (MAP2Ks). The effects of concentrations of HBCD and TBBPA that inhibited lytic function on the phosphorylation state and total levels of the MAPKs (p44/42, p38, and JNK) and the phosphorylation and total levels of the MAP2Ks (MEK1/2 and MKK3/6) were examined. Results indicate that exposure of human NK cells to 10-0.5 µM HBCD or TBBPA activate MAPKs and MAP2Ks. This HBCD and TBBPA-induced activation of MAPKs may leave them unavailable for activation by virally infected or tumor target cells and thus contributes to the observed decreases in lytic function seen in NK cells exposed to HBCD and TBBPA. PMID:25341744

  10. Are High-Coherent Concept Maps Better for Prior Knowledge Activation? Differential Effects of Concept Mapping Tasks on High School vs. University Students

    ERIC Educational Resources Information Center

    Gurlitt, J.; Renkl, A.

    2008-01-01

    We investigated whether and how prior knowledge activation improves learning outcomes for high school (less experienced learners) and university students (experienced learners) in a hypertext environment. Map coherence was defined as the extent to which relationships between the concepts in the map were made explicit. Therefore, we classified the…

  11. NALP3 inflammasome activation in protein misfolding diseases.

    PubMed

    Shi, Fushan; Kouadir, Mohammed; Yang, Yang

    2015-08-15

    Protein-misfolding diseases, such as Alzheimer's disease, type 2 diabetes, Prion diseases, and Parkinson's disease, are characterized by inflammatory reactions. In all these diseases, IL-1β (Interlukine-1β) has been shown to be an important regulator, and the misfolded proteins are proved to be triggers of the release of IL-1β. Recently, several reports demonstrated that the inflammasome activation is involved in the progress of the misfolded protein diseases, and that the inflammasome can recognize pathogenic proteins leading to the release of IL-1β. In this review, we discuss the role of inflammasome in the pathogenesis of misfolded protein diseases and the potential of inflammasome-targeting therapeutic interventions in the management of these diseases. PMID:26037399

  12. Peptides and proteins with antimicrobial activity

    PubMed Central

    Coutinho, Henrique Douglas Melo; Lôbo, Katiuscia Menezes; Bezerra, Denise Aline Casimiro; Lôbo, Inalzuir

    2008-01-01

    The increase of microbial resistance to antibiotics has led to a continuing search for newer and more effective drugs. Antimicrobial peptides are generally found in animals, plants, and microorganisms and are of great interest to medicine, pharmacology, and the food industry. These peptides are capable of inhibiting pathogenic microorganisms. They can attack parasites, while causing little or no harm to the host cells. The defensins are peptides found in granules in the polymorphonuclear neutrophils (PMNs) and are responsible for the defense of the organism. Several animal defensins, like dermaseptin, antileukoprotease, protegrin, and others, have had their activities and efficacy tested and been shown to be effective against bacteria, fungi, and protists; there are also specific defensins from invertebrates, e.g., drosomycin and heliomicin; from plants, e.g., the types A and B; and the bacteriocins, e.g., acrocin, marcescin, etc. The aim of the present work was to compile a comprehensive bibliographic review of the diverse potentially antimicrobial peptides in an effort to systematize the current knowledge on these substances as a contribution for further researches. The currently available bibliography does not give a holistic approach on this subject. The present work intends to show that the mechanism of defense represented by defensins is promising from the perspective of its application in the treatment of infectious diseases in human, animals and plants. PMID:21264153

  13. Chemical labelling of active serum thioester proteins for quantification.

    PubMed

    Holm, Lotta; Ackland, Gareth L; Edwards, Mark R; Breckenridge, Ross A; Sim, Robert B; Offer, John

    2012-02-01

    The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum. PMID:21852021

  14. Mapping Muscles Activation to Force Perception during Unloading

    PubMed Central

    Toma, Simone; Lacquaniti, Francesco

    2016-01-01

    It has been largely proved that while judging a force humans mainly rely on the motor commands produced to interact with that force (i.e., sense of effort). Despite of a large bulk of previous investigations interested in understanding the contributions of the descending and ascending signals in force perception, very few attempts have been made to link a measure of neural output (i.e., EMG) to the psychophysical performance. Indeed, the amount of correlation between EMG activity and perceptual decisions can be interpreted as an estimate of the contribution of central signals involved in the sensation of force. In this study we investigated this correlation by measuring the muscular activity of eight arm muscles while participants performed a quasi-isometric force detection task. Here we showed a method to quantitatively describe muscular activity (“muscle-metric function”) that was directly comparable to the description of the participants' psychophysical decisions about the stimulus force. We observed that under our experimental conditions, muscle-metric absolute thresholds and the shape of the muscle-metric curves were closely related to those provided by the psychophysics. In fact a global measure of the muscles considered was able to predict approximately 60% of the perceptual decisions total variance. Moreover the inter-subjects differences in psychophysical sensitivity showed high correlation with both participants' muscles sensitivity and participants' joint torques. Overall, our findings gave insights into both the role played by the corticospinal motor commands while performing a force detection task and the influence of the gravitational muscular torque on the estimation of vertical forces. PMID:27032087

  15. Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis

    PubMed Central

    Urano, Daisuke; Phan, Nguyen; Jones, Janice C.; Yang, Jing; Huang, Jirong; Grigston, Jeffrey; Taylor, J. Philip; Jones, Alan M.

    2012-01-01

    Signal transduction typically begins by ligand-dependent activation of a concomitant partner which is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (Regulator of G protein Signaling 1), which is the prototype of a seven transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required both for G protein-mediated sugar signaling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis. PMID:22940907

  16. Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

    PubMed Central

    Serrano, M; Gutiérrez, J; Prieto, I; Hermoso, J M; Salas, M

    1989-01-01

    Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication. Images PMID:2767056

  17. Automated Gastric Slow Wave Cycle Partitioning and Visualization for High-resolution Activation Time Maps

    PubMed Central

    Erickson, Jonathan C.; O’Grady, Greg; Du, Peng; Egbuji, John U.; Pullan, Andrew J.; Cheng, Leo K.

    2014-01-01

    High-resolution (HR) multi-electrode mapping has become an important technique for evaluating gastrointestinal (GI) slow wave (SW) behaviors. However, the application and uptake of HR mapping has been constrained by the complex and laborious task of analyzing the large volumes of retrieved data. Recently, a rapid and reliable method for automatically identifying activation times (ATs) of SWs was presented, offering substantial efficiency gains. To extend the automated data-processing pipeline, novel automated methods are needed for partitioning identified ATs into their propagation cycles, and for visualizing the HR spatiotemporal maps. A novel cycle partitioning algorithm (termed REGROUPS) is presented. REGROUPS employs an iterative REgion GROwing procedure and incorporates a Polynomial-surface-estimate Stabilization step, after initiation by an automated seed selection process. Automated activation map visualiza