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Sample records for single tyrosine residue

  1. The carboxyl terminal tyrosine 417 residue of NOK has an autoinhibitory effect on NOK-mediated signaling transductions

    International Nuclear Information System (INIS)

    Li Yinghua; Zhong Shan; Rong Zhili; Ren Yongming; Li Zhiyong; Zhang Shuping; Chang Zhijie; Liu Li

    2007-01-01

    Receptor protein tyrosine kinases (RPTKs) are essential mediators of cell growth, differentiation, migration, and metabolism. Recently, a novel RPTK named NOK has been cloned and characterized. In current study, we investigated the role of the carboxyl terminal tyrosine 417 residue of NOK in the activations of different signaling pathways. A single tyrosine to phenylalanine point mutation at Y417 site (Y417 F) not only dramatically enhanced the NOK-induced activation of extracellular signal-regulated kinase (ERK), but also markedly promoted the NOK-mediated activation of both signal transducer and activator of transcription 1 and 3 (STAT1 and 3). Moreover, the proliferation potential of NIH3T3-NOK (Y417F) stable cells were significantly elevated as compared with that of NIH3T3-NOK. Overall, our results demonstrate that the tyrosine Y417 residue at the carboxyl tail of NOK exhibits an autoinhibitory role in NOK-mediated signaling transductions

  2. Ionization of tyrosine residues in human serum albumin and in its complexes with bilirubin and laurate

    DEFF Research Database (Denmark)

    Honoré, B; Brodersen, R

    1992-01-01

    Spectrophotometric titration of human serum albumin indicates that ionization of the 18 tyrosine residues takes place between pH 9 and 12.7. A Hill plot indicates that protons dissociate co-operatively from tyrosine residues, in pure albumin between pH 11.0 and 11.4 with a Hill coefficient 1.7, a...

  3. A Multifeatures Fusion and Discrete Firefly Optimization Method for Prediction of Protein Tyrosine Sulfation Residues.

    Science.gov (United States)

    Guo, Song; Liu, Chunhua; Zhou, Peng; Li, Yanling

    2016-01-01

    Tyrosine sulfation is one of the ubiquitous protein posttranslational modifications, where some sulfate groups are added to the tyrosine residues. It plays significant roles in various physiological processes in eukaryotic cells. To explore the molecular mechanism of tyrosine sulfation, one of the prerequisites is to correctly identify possible protein tyrosine sulfation residues. In this paper, a novel method was presented to predict protein tyrosine sulfation residues from primary sequences. By means of informative feature construction and elaborate feature selection and parameter optimization scheme, the proposed predictor achieved promising results and outperformed many other state-of-the-art predictors. Using the optimal features subset, the proposed method achieved mean MCC of 94.41% on the benchmark dataset, and a MCC of 90.09% on the independent dataset. The experimental performance indicated that our new proposed method could be effective in identifying the important protein posttranslational modifications and the feature selection scheme would be powerful in protein functional residues prediction research fields.

  4. Roles of the C-terminal tyrosine residues of LAT in GPVI-induced platelet activation: insights into the mechanism of PLC gamma 2 activation.

    Science.gov (United States)

    Ragab, Ashraf; Séverin, Sonia; Gratacap, Marie-Pierre; Aguado, Enrique; Malissen, Marie; Jandrot-Perrus, Martine; Malissen, Bernard; Ragab-Thomas, Jeannie; Payrastre, Bernard

    2007-10-01

    Linker for activation of T cells (LAT) is an adaptor protein required for organization of the signaling machinery downstream of the platelet collagen receptor, the glycoprotein VI (GPVI). Here, we investigated the effect of LAT mutations on specific signaling pathways and on platelet functions in response to GPVI triggering by convulxin (Cvx). Using mice containing tyrosine to phenylalanine mutations of the adaptor, we show the crucial role played by the tyrosine residues at positions 175, 195, and 235 in the phosphorylation of LAT and in the whole pattern of protein tyrosine phosphorylation in response to Cvx. These 3 C-terminal tyrosine residues are important to recruit the tyrosine kinase Fyn, which may be involved in LAT phosphorylation. Efficient phosphoinositide 3-kinase (PI3K) activation requires the 3 C-terminal tyrosine residues of LAT but not its tyrosine 136. Interestingly, single mutation of the tyrosine 136 results in the loss of phospholipase C gamma2 (PLCgamma2) activation without affecting its PI3K-dependent membrane association, and is sufficient to impair platelet responses to Cvx. Thus, activation of PLCgamma2 via GPVI is dependent on 2 complementary events: its interaction with the tyrosine 136 of LAT and its membrane location, which itself requires events mediated by the 3 C-terminal tyrosines of LAT.

  5. Protein-bound glycogen is linked to tyrosine residues.

    OpenAIRE

    Aon, M A; Curtino, J A

    1985-01-01

    Tyrosine-glycogen obtained from retina proteoglycogen by exhaustive proteolytic digestion was radiolabelled with 125I. The 125I-labelled tyrosine-glycogen was degraded by amylolytic digestion to a very small radioactive product, which was identified as iodotyrosine by h.p.l.c. The amylolytic mixture used released glucose and maltose that were alpha-linked to the phenolic hydroxy group of p-nitrophenol. No free iodotyrosine was found before or after the intact [125I]iodotyrosine-glycogen was s...

  6. Direct observation of spin-injection in tyrosinate-functionalized single-wall carbon nanotubes

    NARCIS (Netherlands)

    Tsoufis, Theodoros; Ampoumogli, Asem; Gournis, Dimitrios; Georgakilas, Vasilios; Jankovic, Lubos; Christoforidis, Konstantinos C.; Deligiannakis, Yiannis; Mavrandonakis, Andreas; Froudakis, George E.; Maccallini, Enrico; Rudolf, Petra; Mateo-Alonso, Aurelio; Prato, Maurizio

    In this work, we report on the interaction of a tyrosinate radical with single wall carbon nanotubes (CNT). The tyrosinate radical was formed from tyrosine (ester) by Fenton's reagent and, reacted in situ with carbon nanotubes resulting in novel tyrosinated carbon nanotube derivatives. The covalent

  7. Requirement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor for selected GH-stimulated function

    DEFF Research Database (Denmark)

    Lobie, P E; Allevato, G; Norstedt, G

    1995-01-01

    We have examined the involvement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor in the cellular response to GH. Stable Chinese hamster ovary (CHO) cell clones expressing a receptor with tyrosine residues at position 333 and 338 of the receptor substituted for phenylalanine (...

  8. Importance of the content and localization of tyrosine residues for thyroxine formation within the N-terminal part of human thyroglobulin

    NARCIS (Netherlands)

    den Hartog, M. T.; Sijmons, C. C.; Bakker, O.; Ris-Stalpers, C.; de Vijlder, J. J.

    1995-01-01

    Thyroxine (T4) is formed by coupling of iodinated tyrosine residues within thyroglobulin (TG). In mature TG, some iodinated tyrosine residues are involved preferentially in T4 formation. In order to investigate the specific role of various tyrosine residues in T4 formation, N-terminal TG fragments

  9. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  10. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Macek, B

    2006-01-01

    by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

  11. Manganese superoxide dismutase (MnSOD catalyzes NO-dependent tyrosine residue nitration

    Directory of Open Access Journals (Sweden)

    SRDJAN STOJANOVIC

    2005-04-01

    Full Text Available The peroxynitrite-induced nitration of manganese superoxide dismutase (MnSOD tyrosine residue, which causes enzyme inactivation, is well established. This led to suggestions that MnSOD nitration and inactivation in vivo, detected in various diseases associated with oxidative stress and overproduction of nitric monoxide (NO, conditions which favor peroxynitrite formation, is also caused by peroxynitrite. However, our previous in vitro study demonstrated that exposure of MnSOD to NO led to NO conversion into nitrosonium (NO+ and nitroxyl (NO– species, which caused enzyme modifications and inactivation. Here it is reported that MnSOD is tyrosine nitrated upon exposure to NO, as well as that MnSOD nitration contributes to inactivation of the enzyme. Collectively, these observations provide a compelling argument supporting the generation of nitrating species in MnSOD exposed to NO and shed a new light on MnSOD tyrosine nitration and inactivation in vivo. This may represent a novel mechanism by which MnSOD protects cell from deleterious effects associated with overproduction of NO. However, extensive MnSOD modification and inactivation associated with prolonged exposure to NO will amplify the toxic effects caused by increased cell superoxide and NO levels.

  12. The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues

    DEFF Research Database (Denmark)

    Cook, Naomi L.; Moeke, Cassidy H.; Fantoni, Luca I.

    2016-01-01

    Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (S...

  13. C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein.

    Science.gov (United States)

    Popa, Andreea; Pager, Cara Teresia; Dutch, Rebecca Ellis

    2011-02-15

    The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus, and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the virus-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in the extent of cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F protein fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in the extent of fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of addition of the HA tag varied with other fusion proteins, as parainfluenza virus 5 F-HA showed a decreased level of surface expression and no stimulation of fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope-tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in the modulation of the membrane fusion reaction promoted by these viral glycoproteins.

  14. Requirement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor for selected GH-stimulated function

    DEFF Research Database (Denmark)

    Lobie, P E; Allevato, G; Norstedt, G

    1995-01-01

    We have examined the involvement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor in the cellular response to GH. Stable Chinese hamster ovary (CHO) cell clones expressing a receptor with tyrosine residues at position 333 and 338 of the receptor substituted for phenylalanine...... (CHO-GHR1-638 Y333F, Y338F) were generated by cDNA transfection. Compared with the wild type receptor the Y333F,Y338F mutant possessed normal high affinity ligand binding, hormone internalization, and ligand-induced receptor down-regulation. GH activation of mitogen-associated protein kinase was also...... similar in CHO clones expressing similar wild type and Y333F,Y338F receptor number. However, two GH-regulated cellular events (lipogenesis, and protein synthesis) were deficient in the tyrosine substituted receptor. In contrast, transcriptional regulation by GH (as evidenced by chloramphenicol...

  15. Replacement of insulin receptor tyrosine residues 1162 and 1163 does not alter the mitogenic effect of the hormone

    International Nuclear Information System (INIS)

    Debant, A.; Clauser, E.; Ponzio, G.; Filloux, C.; Auzan, C.; Contreres, J.O.; Rossi, B.

    1988-01-01

    Chinese hamster ovary transfectants that express insulin receptors in which tyrosine residues 1162 and 1163 were replaced by phenylalanine exhibit a total inhibition of the insulin-mediated tyrosine kinase activity toward exogenous substrates; this latter activity is associated with total inhibition of the hypersensitivity reported for insulin in promoting 2-deoxyglucose uptake. The authors now present evidence that the twin tyrosines also control the insulin-mediated stimulation of glycogen synthesis. Surprisingly, this type of Chinese hamster ovary transfectant is as hypersensitive to insulin for its mitogenic effect as are Chinese hamster ovary cells expressing many intact insulin receptors. Such data suggest that (i) the insulin mitogenic effect routes through a different pathway than insulin uses to activate the transport and metabolism of glucose and (ii) the mitogenic effect of insulin is not controlled by the twin tyrosines. At the molecular level, the solubilized mutated receptor has not insulin-dependent tyrosine kinase activity, whereas this receptor displays measurable insulin-stimulated phosphorylation of its β subunit in 32 P-labeled cells. The authors therefore propose that the autocatalytic phosphorylating activity of the receptor reports a cryptic tyrosine kinase activity that cannot be visualized by the use of classical exogenous substrates

  16. Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, L. H.; Wang, X.; Kopchick, J J

    1996-01-01

    phosphorylation in intracellular signaling, we constructed GH receptors in which combinations of tyrosines were mutated to phenylalanines. We identified three tyrosine residues at positions 534, 566, and 627 that were required for activation of GH-stimulated transcription of the serine protease inhibitor (Spi) 2...

  17. Oxidation of protein tyrosine or methionine residues: From the amino acid to the peptide

    Energy Technology Data Exchange (ETDEWEB)

    Berges, J [Universite Pierre et Marie Curie, UMR 7616, Laboratoire de Chimie Theorique, 75005 Paris (France); Trouillas, P [EA 4021 Faculte de Pharmacie, 2 Rue du Dr. Marcland, 87025 Limoges Cedex (France); Houee-Levin, C, E-mail: jb@lct.jussieu.fr, E-mail: patrick.trouillas@unilim.fr, E-mail: chantal.houee@u-psud.fr [Universite Paris Sud, UMR 8000, Laboratoire de Chimie Physique, 91405 Orsay (France) (France)

    2011-01-01

    Methionine and tyrosine are competing targets of oxidizing free radicals in peptides or proteins. The first step is the addition of OH radicals either on the sulphur atom of methionine, followed by OH{sup -} elimination, or on the aromatic cycle of tyrosine. The next step can be stabilization of methionine radical cation by a two centre-three electron bond, or intramolecular electron transfer from tyrosine to the methionine radical cation. In this latter case a tyrosine radical is formed, which appears deprotonated. In a first step we have compared the stability of the OH radical adducts on Methionine or on Tyrosine. In agreement with experimental results, the thermodynamical data indicate that the OH adduct on Tyrosine and the radical cation are more stable than those on methionine. In a second step we have investigated the stabilization of the radical cations of Methionine by formation of intramolecular S:X two-center three-electron bond (X=S, N, O). Finally we have compared the spin densities on separated amino acids to that in a radical pentapeptide, methionine enkephalin. One observes a delocalisation of the orbital of the odd electron on the sulfur atom of Met and on the cycle of Tyr. The peptidic chain is also concerned.

  18. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

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    Choi Yoo

    2012-10-01

    Full Text Available Abstract Background In nature, mussel adhesive proteins (MAPs show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate

  19. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

    Science.gov (United States)

    2012-01-01

    Background In nature, mussel adhesive proteins (MAPs) show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa) and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate the use of functional MAPs in

  20. Exposure of tropoelastin to peroxynitrous acid gives high yields of nitrated tyrosine residues, di-tyrosine cross-links and altered protein structure and function.

    Science.gov (United States)

    Degendorfer, Georg; Chuang, Christine Y; Mariotti, Michele; Hammer, Astrid; Hoefler, Gerald; Hägglund, Per; Malle, Ernst; Wise, Steven G; Davies, Michael J

    2018-02-01

    Elastin is an abundant extracellular matrix protein in elastic tissues, including the lungs, skin and arteries, and comprises 30-57% of the aorta by dry mass. The monomeric precursor, tropoelastin (TE), undergoes complex processing during elastogenesis to form mature elastic fibres. Peroxynitrous acid (ONOOH), a potent oxidising and nitrating agent, is formed in vivo from superoxide and nitric oxide radicals. Considerable evidence supports ONOOH formation in the inflamed artery wall, and a role for this species in the development of human atherosclerotic lesions, with ONOOH-damaged extracellular matrix implicated in lesion rupture. We demonstrate that TE is highly sensitive to ONOOH, with this resulting in extensive dimerization, fragmentation and nitration of Tyr residues to give 3-nitrotyrosine (3-nitroTyr). This occurs with equimolar or greater levels of oxidant and increases in a dose-dependent manner. Quantification of Tyr loss and 3-nitroTyr formation indicates extensive Tyr modification with up to two modified Tyr per protein molecule, and up to 8% conversion of initial ONOOH to 3-nitroTyr. These effects were modulated by bicarbonate, an alternative target for ONOOH. Inter- and intra-protein di-tyrosine cross-links have been characterized by mass spectrometry. Examination of human atherosclerotic lesions shows colocalization of 3-nitroTyr with elastin epitopes, consistent with TE or elastin modification in vivo, and also an association of 3-nitroTyr containing proteins and elastin with lipid deposits. These data suggest that exposure of TE to ONOOH gives marked chemical and structural changes to TE and altered matrix assembly, and that such damage accumulates in human arterial tissue during the development of atherosclerosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Single Tyrosine Mutation in AAV8 Vector Capsid Enhances Gene Lung Delivery and Does Not Alter Lung Morphofunction in Mice

    Directory of Open Access Journals (Sweden)

    Sabrina V. Martini

    2014-08-01

    Full Text Available Background/Aims: Vectors derived from adeno-associated viruses (AAVs are important gene delivery tools for treating pulmonary diseases. Phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. We evaluated the pulmonary transduction efficiency of AAV8 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Male C57BL/6 mice (20-25 g, n=24 were randomly assigned into three groups: control group animals received intratracheal (i.t. instillation of saline (50 μl, wild-type AAV8 group, and capsid mutant Y733F AAV8 group, which received (i.t. AAV8 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP. Four weeks after instillation, lung mechanics and morphometry, vector transduction (immunohistochemistry and mRNA expression of eGFP, and inflammatory cytokines and growth factor expression were analyzed. Results: Tyrosine-mutant AAV8 vectors displayed significantly increased transduction efficiency in the lung compared with their wild-type counterparts. No significant differences were observed in lung mechanics and morphometry between experimental groups. There was no evidence of inflammatory response in any group. Conclusion: AAV8 vectors may be useful for new therapeutic strategies for the treatment of pulmonary diseases.

  2. Redox-active tyrosine residue in the microcin J25 molecule

    Energy Technology Data Exchange (ETDEWEB)

    Chalon, Miriam C. [Departamento de Bioquimica de la Nutricion, Instituto Superior de Investigaciones Biologicas (Consejo Nacional de Investigaciones Cientificas y Tecnicas-Universidad Nacional de Tucuman) and Instituto de Quimica Biologica ' Dr Bernabe Bloj' , Chacabuco 461, 4000 San Miguel de Tucuman, Tucuman (Argentina); Wilke, Natalia [CIQUIBIC, Dpto. de Quimica Biologica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Pabellon Argentina, Ciudad Universitaria, X5000HUA, Cordoba (Argentina); Pedersen, Jens; Rufini, Stefano [Department of Biology, University of Rome Tor Vergata 00133, Rome (Italy); Morero, Roberto D.; Cortez, Leonardo; Chehin, Rosana N.; Farias, Ricardo N. [Departamento de Bioquimica de la Nutricion, Instituto Superior de Investigaciones Biologicas (Consejo Nacional de Investigaciones Cientificas y Tecnicas-Universidad Nacional de Tucuman) and Instituto de Quimica Biologica ' Dr Bernabe Bloj' , Chacabuco 461, 4000 San Miguel de Tucuman, Tucuman (Argentina); Vincent, Paula A., E-mail: pvincent@fbqf.unt.edu.ar [Departamento de Bioquimica de la Nutricion, Instituto Superior de Investigaciones Biologicas (Consejo Nacional de Investigaciones Cientificas y Tecnicas-Universidad Nacional de Tucuman) and Instituto de Quimica Biologica ' Dr Bernabe Bloj' , Chacabuco 461, 4000 San Miguel de Tucuman, Tucuman (Argentina)

    2011-03-18

    Research highlights: {yields} Cyclic voltammetry measurements showed irreversible oxidation of MccJ25 and MccJ25 (Y9F). {yields} Infrared spectroscopy studies showed that only Tyr9 could be deprotonated upon chemical oxidation. {yields} Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H{sub 2}O{sub 2} was demonstrated. {yields} Tyr9 but not Tyr20 can be easily oxidized and form a tyrosyl radical. -- Abstract: Microcin J25 (MccJ25) is a 21 amino acid lasso-peptide antibiotic produced by Escherichia coli and composed of an 8-residues ring and a terminal 'tail' passing through the ring. We have previously reported two cellular targets for this antibiotic, bacterial RNA polymerase and the membrane respiratory chain, and shown that Tyr9 is essential for the effect on the membrane respiratory chain which leads to superoxide overproduction. In the present paper we investigated the redox behavior of MccJ25 and the mutant MccJ25 (Y9F). Cyclic voltammetry measurements showed irreversible oxidation of both Tyr9 and Tyr20 in MccJ25, but infrared spectroscopy studies demonstrated that only Tyr9 could be deprotonated upon chemical oxidation in solution. Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H{sub 2}O{sub 2} was demonstrated by Electron Paramagnetic Resonance Spectroscopy; this radical was not detected when the reaction was carried out with the MccJ25 (Y9F) mutant. These results show that the essential Tyr9, but not Tyr20, can be easily oxidized and form a tyrosyl radical.

  3. Redox-active tyrosine residue in the microcin J25 molecule

    International Nuclear Information System (INIS)

    Chalon, Miriam C.; Wilke, Natalia; Pedersen, Jens; Rufini, Stefano; Morero, Roberto D.; Cortez, Leonardo; Chehin, Rosana N.; Farias, Ricardo N.; Vincent, Paula A.

    2011-01-01

    Research highlights: → Cyclic voltammetry measurements showed irreversible oxidation of MccJ25 and MccJ25 (Y9F). → Infrared spectroscopy studies showed that only Tyr9 could be deprotonated upon chemical oxidation. → Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H 2 O 2 was demonstrated. → Tyr9 but not Tyr20 can be easily oxidized and form a tyrosyl radical. -- Abstract: Microcin J25 (MccJ25) is a 21 amino acid lasso-peptide antibiotic produced by Escherichia coli and composed of an 8-residues ring and a terminal 'tail' passing through the ring. We have previously reported two cellular targets for this antibiotic, bacterial RNA polymerase and the membrane respiratory chain, and shown that Tyr9 is essential for the effect on the membrane respiratory chain which leads to superoxide overproduction. In the present paper we investigated the redox behavior of MccJ25 and the mutant MccJ25 (Y9F). Cyclic voltammetry measurements showed irreversible oxidation of both Tyr9 and Tyr20 in MccJ25, but infrared spectroscopy studies demonstrated that only Tyr9 could be deprotonated upon chemical oxidation in solution. Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H 2 O 2 was demonstrated by Electron Paramagnetic Resonance Spectroscopy; this radical was not detected when the reaction was carried out with the MccJ25 (Y9F) mutant. These results show that the essential Tyr9, but not Tyr20, can be easily oxidized and form a tyrosyl radical.

  4. Single low doses of MPTP decrease tyrosine hydroxylase expression in the absence of overt neuron loss.

    Science.gov (United States)

    Alam, Gelareh; Edler, Melissa; Burchfield, Shelbie; Richardson, Jason R

    2017-05-01

    Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a prototypical neurotoxicant used in mice to mimic primary features of PD pathology including striatal dopamine depletion and dopamine neuron loss in the substantia nigra pars compacta (SNc). In the literature, there are several experimental paradigms involving multiple doses of MPTP that are used to elicit dopamine neuron loss. However, a recent study reported that a single low dose caused significant loss of dopamine neurons. Here, we determined the effect of a single intraperitoneal injection of one of three doses of MPTP (0.1, 2 and 20mg/kg) on dopamine neurons, labeled by tyrosine hydroxylase (TH + ), and total neuron number (Nissl + ) in the SNc using unbiased stereological counting. Data reveal a significant loss of neurons in the SNc (TH + and Nissl + ) only in the group treated with 20mg/kg MPTP. Groups treated with lower dose of MPTP (0.1 and 2mg/kg) only showed significant loss of TH + neurons rather than TH + and Nissl + neurons. Striatal dopamine levels were decreased in the groups treated with 2 and 20mg/kg MPTP and striatal terminal markers including, TH and the dopamine transporter (DAT), were only decreased in the groups treated with 20mg/kg MPTP. These data demonstrate that lower doses of MPTP likely result in loss of TH expression rather than actual dopamine neuron loss in the SN. This finding reinforces the need to measure both total neuron number along with TH + cells in determining dopamine neuron loss. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. A Conserved Active Site Tyrosine Residue of Proline Dehydrogenase Helps Enforce the Preference for Proline over Hydroxyproline as the Substrate

    Energy Technology Data Exchange (ETDEWEB)

    Ostrander, E.L.; Larson, J.D.; Schuermann, J.P.; Tanner, J.J.; (Cornell); (Missouri)

    2009-03-02

    Proline dehydrogenase (PRODH) catalyzes the oxidation of L-proline to {Delta}-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-L-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analogue L-tetrahydro-2-furoic acid were determined at resolutions of 1.75, 1.90, and 1.85 {angstrom}, respectively. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline 3-fold and decreases the specificity for proline by factors of 20 (Y540S) and 50 (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding the substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate.

  6. Plastic roles of phenylalanine and tyrosine residues of TLS/FUS in complex formation with the G-quadruplexes of telomeric DNA and TERRA.

    Science.gov (United States)

    Kondo, Keiko; Mashima, Tsukasa; Oyoshi, Takanori; Yagi, Ryota; Kurokawa, Riki; Kobayashi, Naohiro; Nagata, Takashi; Katahira, Masato

    2018-02-12

    The length of a telomere is regulated via elongation and shortening processes. Telomeric DNA and telomeric repeat-containing RNA (TERRA), which both contain G-rich repeated sequences, form G-quadruplex structures. Previously, translocated in liposarcoma (TLS) protein, also known as fused in sarcoma (FUS) protein, was found to form a ternary complex with the G-quadruplex structures of telomeric DNA and TERRA. We then showed that the third RGG motif of TLS, the RGG3 domain, is responsible for the complex formation. However, the structural basis for their binding remains obscure. Here, NMR-based binding assaying revealed the interactions in the binary and ternary complexes of RGG3 with telomeric DNA or/and TERRA. In the ternary complex, tyrosine bound exclusively to TERRA, while phenylalanine bound exclusively to telomeric DNA. Thus, tyrosine and phenylalanine each play a central role in the recognition of TERRA and telomeric DNA, respectively. Surprisingly in the binary complexes, RGG3 used both tyrosine and phenylalanine residues to bind to either TERRA or telomeric DNA. We propose that the plastic roles of tyrosine and phenylalanine are important for RGG3 to efficiently form the ternary complex, and thereby regulate the telomere shortening.

  7. Structural perfection and residual electric resistance of tungsten single crystals

    International Nuclear Information System (INIS)

    Tagirova, D.M.; Dyakina, V.P.; Startsev, V.E.; Esin, V.O.

    1997-01-01

    A study was made into residual relative resistance (RRR) and structural perfection (SP) of tungsten single crystals, grown by electron beam zone melting using seeding crystals of several orientations, namely, , , , . The single crystals were of 99.98 and 99.9995 wt.% purity. The RRR value is found to depend on crystallographic orientation of an axis of crystal growth and to correlate with SP. Single crystals of different purity are differ in the nature of orientational dependences. It is shown that the correlation between RRR and SP of crystals is mainly due to conduction electron scattering by subgrain boundaries (internal size effect)

  8. Exploring oxidative modifications of tyrosine

    DEFF Research Database (Denmark)

    Houée-Lévin, C; Bobrowski, K; Horakova, L

    2015-01-01

    residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different...... effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor...... processes are important in vivo and can contribute to cellular pathology....

  9. Exposure of tropoelastin to peroxynitrous acid gives high yields of nitrated tyrosine residues, di-tyrosine cross-links and altered protein structure and function

    DEFF Research Database (Denmark)

    Degendorfer, Georg; Chuang, Christine Yu-Nung; Mariotti, Michele

    2018-01-01

    Elastin is an abundant extracellular matrix protein in elastic tissues, including the lungs, skin and arteries, and comprises 30–57% of the aorta by dry mass. The monomeric precursor, tropoelastin (TE), undergoes complex processing during elastogenesis to form mature elastic fibres. Peroxynitrous......-damaged extracellular matrix implicated in lesion rupture. We demonstrate that TE is highly sensitive to ONOOH, with this resulting in extensive dimerization, fragmentation and nitration of Tyr residues to give 3-nitrotyrosine (3-nitroTyr). This occurs with equimolar or greater levels of oxidant and increases in a dose...... with lipid deposits. These data suggest that exposure of TE to ONOOH gives marked chemical and structural changes to TE and altered matrix assembly, and that such damage accumulates in human arterial tissue during the development of atherosclerosis....

  10. Protein-tyrosine phosphatases in zebrafish gastrulation

    NARCIS (Netherlands)

    van Eekelen, M.J.L.

    2011-01-01

    Protein tyrosine phosphorylation plays a key role in relaying external stimuli and signals into the cell towards the appropriate responses. This process is mediated by protein-tyrosine kinases adding a phosphor group to a tyrosine residue and protein-tyrosine phosphatases removing a phosphor group

  11. A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9.

    Science.gov (United States)

    Demoulin, J B; Uyttenhove, C; Van Roost, E; DeLestré, B; Donckers, D; Van Snick, J; Renauld, J C

    1996-09-01

    Interleukin-9 (IL-9), a T-cell-derived cytokine, interacts with a specific receptor associated with the IL-2 receptor gamma chain. In this report, we analyze the functional domains of the human IL-9 receptor transfected into mouse lymphoid cell lines. Three different functions were examined: growth stimulation in factor-dependent pro-B Ba/F3 cells, protection against dexamethasone-induced apoptosis, and Ly-6A2 induction in BW5147 lymphoma cells. The results indicated that a single tyrosine, at position 116 in the cytoplasmic domain, was required for all three activities. In addition, we observed that human IL-9 reduced the proliferation rate of transfected BW5147 cells, an effect also dependent on the same tyrosine. This amino acid was necessary for IL-9-mediated tyrosine phosphorylation of the receptor and for STAT activation but not for IRS-2/4PS activation or for JAK1 phosphorylation, which depended on a domain closer to the plasma membrane. We also showed that JAK1 was constitutively associated with the IL-9 receptor. Activated STAT complexes induced by IL-9 were found to contain STAT1, STAT3, and STAT5 transcription factors. Moreover, sequence homologies between human IL-9 receptor tyrosine 116 and tyrosines (of other receptors activating STAT3 and STAT5 were observed. Taken together, these data indicate that a single tyrosine of the IL-9 receptor, required for activation of three different STAT proteins, is necessary for distinct activities of this cytokine, including proliferative responses.

  12. A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in Mycobacterium smegmatis.

    Science.gov (United States)

    Bansal, Ankita; Kar, Debasish; Pandey, Satya Deo; Matcha, Ashok; Kumar, N Ganesh; Nathan, Soshina; Ghosh, Anindya S

    2017-06-01

    Mycobacterial beta-lactamases are involved in exerting beta-lactam resistance, though many of these proteins remain uncharacterized. Here, we have characterized MSMEG_4455 of Mycobacterium smegmatis as a beta-lactamase using molecular, biochemical and mutational techniques. To elucidate its nature in vivo and in vitro, and to predict its structure-function relationship in silico analysis is done. The MSMEG_4455 is cloned and expressed ectopically in a beta-lactamase deficient Escherichia coli mutant to establish the in vivo beta-lactamase like nature via minimum inhibitory concentration (MIC) determination. Likewise the in vivo results, purified soluble form of MSMEG_4455 showed beta-lactam hydrolysis pattern similar to group 2a penicillinase. In silico analyses of MSMEG_4455 reveal glutamic acid (E)193 and tyrosine (Y)194 of omega-like loop might have importance in strengthening hydrogen bond network around the active-site, though involvement of tyrosine is rare for beta-lactamase activity. Accordingly, these residues are mutated to alanine (A) and phenylalanine (F), respectively. The mutated proteins have partially lost their ability to exert beta-lactamase activity both in vivo and in vitro. The Y194F mutation had more prominent effect on the enzymatic activity. Therefore, we infer that Y194 is the key for beta-lactamase activity of MSMEG_4455.

  13. Residue 259 in protein-tyrosine phosphatase PTP1B and PTPα determines the flexibility of glutamine 262

    DEFF Research Database (Denmark)

    Peters, Günther H.j.; Iversen, L.F.; Andersen, H.S.

    2004-01-01

    To study the flexibility of the substrate-binding site and in particular of Gln262, we have performed adiabatic conformational search and molecular dynamics simulations on the crystal structure of the catalytic domain of wild-type protein-tyrosine phosphatase (PTP) 1B, a mutant PTP1B(R47V),(D48N...... around Gln262 and the active site Cys215 reveals that the probability of finding a water molecule correctly positioned for catalysis is much larger in PTP1B than in PTP1B(R47V),(D48N),(M258C),(G259Q) and PTPalpha, in accordance with experiments....

  14. Activation of Bacillus subtilis Ugd by the BY-Kinase PtkA Proceeds via Phosphorylation of Its Residue Tyrosine 70

    DEFF Research Database (Denmark)

    Petranovic, Dina; Grangeasse, C.; Macek, B.

    2009-01-01

    The phosphorylation-dependent activation of bacterial UDP-glucose dehydrogenases by BY-kinases has been previously described in several bacterial model organisms, but the identity of phosphorylated tyrosine(s) and the exact activation mechanism remained unknown. A recent site-specific phosphoprot......The phosphorylation-dependent activation of bacterial UDP-glucose dehydrogenases by BY-kinases has been previously described in several bacterial model organisms, but the identity of phosphorylated tyrosine(s) and the exact activation mechanism remained unknown. A recent site...

  15. The Effect of Neighboring Methionine Residue on Tyrosine Nitration & Oxidation in Peptides Treated with MPO, H2O2, & NO2- or Peroxynitrite and Bicarbonate: Role of Intramolecular Electron-Transfer Mechanism?

    Science.gov (United States)

    Zhang, Hao; Zielonka, Jacek; Sikora, Adam; Joseph, Joy; Xu, Yingkai; Kalyanaraman, B.

    2009-01-01

    Recent reports suggest that intramolecular electron-transfer reactions can profoundly affect the site and specificity of tyrosyl nitration and oxidation in peptides and proteins. Here we investigated the effects of methionine on tyrosyl nitration and oxidation induced by myeloperoxidase (MPO), H2O2 and NO2- and peroxynitrite (ONOO-) or ONOO- and bicarbonate (HCO3-) in model peptides, tyrosylmethionine (YM), tyrosylphenylalanine (YF) and tyrosine. Nitration and oxidation products of these peptides were analysed by HPLC with UV/Vis and fluorescence detection, and mass spectrometry; radical intermediates were identified by electron paramagnetic resonance (EPR)-spin-trapping. We have previously shown (Zhang et al., J. Biol. Chem. (2005) 280, 40684-40698) that oxidation and nitration of tyrosyl residue was inhibited in tyrosylcysteine(YC)-type peptides as compared to free tyrosine. Here we show that methionine, another sulfur-containing amino acid, does not inhibit nitration and oxidation of a neighboring tyrosine residue in the presence of ONOO- (or ONOOCO2-) or MPO/H2O2/NO2- system. Nitration of tyrosyl residue in YM was actually stimulated under the conditions of in situ generation of ONOO- (formed by reaction of superoxide with nitric oxide during SIN-1 decomposition), as compared to YF, YC and tyrosine. The dramatic variations in tyrosyl nitration profiles caused by methionine and cysteine residues have been attributed to differences in the direction of intramolecular electron transfer mechanism in these peptides. Further confirmation of HPLC data analysis was obtained by steady-state radiolysis and photolysis experiments. Potential implications of the intramolecular electron-transfer mechanism in mediating selective nitration of protein tyrosyl groups are discussed. PMID:19056332

  16. Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, L. H.; Wang, X.; Kopchick, J J

    1996-01-01

    The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, -3, and -5. In order to study the role of GH receptor tyrosine phosphor...

  17. Tyrosine Modifications in Aging

    OpenAIRE

    Feeney, Maria B.; Schöneich, Christian

    2012-01-01

    Significance: The understanding of physiological and pathological processes involving protein oxidation, particularly under conditions of aging and oxidative stress, can be aided by proteomic identification of proteins that accumulate oxidative post-translational modifications only if these detected modifications are connected to functional consequences. The modification of tyrosine (Tyr) residues can elicit significant changes in protein structure and function, which, in some cases, may cont...

  18. Effect of residual stress on the nanoindentation response of (100) copper single crystal

    International Nuclear Information System (INIS)

    Zhu, Li-na; Xu, Bin-shi; Wang, Hai-dou; Wang, Cheng-biao

    2012-01-01

    Experimental measurements were used to investigate the effect of residual stress on the nanoindentation of (100) copper single crystal. Equi-biaxial tensile and compressive stresses were applied to the copper single crystal using a special designed apparatus. It was found that residual stresses greatly affected peak load, curvature of the loading curve, elastically recovered depth, residual depth, indentation work, pile-up amount and contact area. The Suresh and Giannakopoulos and Lee and Kwon methods were used to calculate the residual stresses from load-depth data and morphology observation of nanoindents using atomic force microscopy. Comparison of the obtained results with stress values from strain gage showed that the residual stresses analyzed from the Suresh and Giannakopoulos model agreed well with the applied stresses. -- Highlights: ► Residual stresses greatly affected various nanoindentation parameters. ► The contact area can be accurately measured from AFM observation. ► The residual stresses analyzed from the S and G model agreed well with applied stresses.

  19. Dityrosine formation is impaired by tyrosine phosphorylation.

    Science.gov (United States)

    Christian, S; Bernhard, G; Patrizia, R; Brigitte, M

    1992-10-15

    Using pure tyrosine and phosphotyrosine we have recently shown that phosphotyrosine is unable to form peroxidase catalyzed dimers (1989, FEBS Lett. 255, 395-397). In the present report, the effect of phosphotyrosine residues within a protein structure on dityrosine formation was studied using casein as a model protein. Dephosphorylation of casein resulted in a dose and time dependent increased synthesis of dityrosines following treatment with peroxidase/H2O2. The extent of crosslink formation was inversely related to the amount of phosphorylated tyrosine residues as quantitated by immunoblotting. Thus, phosphorylation of tyrosine residues could play a regulatory role in protein-crosslinking where dityrosine bonds are involved.

  20. Multiple tyrosine residues in the intracellular domain of the common ß subunit of the interleukin 5 receptor are involved in activation of STAT5

    NARCIS (Netherlands)

    Dijk, Thamar B. van; Caldenhoven, Eric; Raaijmakers, J.A.M.; Lammers, J.W.J.; Koenderman, L.; Groot, Rolf P. de

    1997-01-01

    Abstract In contrast to the general model of cytokine-induced JAK/STAT signaling, tyrosine phosphorylation of the IL-5R ß chain seems to be dispensable for STAT activation in cells overexpressing exogenous STAT proteins. In this study we expressed IL-5 receptor mutants in 293 cells and studied

  1. Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells.

    Science.gov (United States)

    Petit, V; Boyer, B; Lentz, D; Turner, C E; Thiery, J P; Vallés, A M

    2000-03-06

    Identification of signaling molecules that regulate cell migration is important for understanding fundamental processes in development and the origin of various pathological conditions. The migration of Nara Bladder Tumor II (NBT-II) cells was used to determine which signaling molecules are specifically involved in the collagen-mediated locomotion. We show here that paxillin is tyrosine phosphorylated after induction of motility on collagen. Overexpression of paxillin mutants in which tyrosine 31 and/or tyrosine 118 were replaced by phenylalanine effectively impaired cell motility. Moreover, stimulation of motility by collagen preferentially enhanced the association of paxillin with the SH2 domain of the adaptor protein CrkII. Mutations in both tyrosine 31 and 118 diminished the phosphotyrosine content of paxillin and prevented the formation of the paxillin-Crk complex, suggesting that this association is necessary for collagen-mediated NBT-II cell migration. Other responses to collagen, such as cell adhesion and spreading, were not affected by these mutations. Overexpression of wild-type paxillin or Crk could bypass the migration-deficient phenotype. Both the SH2 and the SH3 domains of CrkII are shown to play a critical role in this collagen-mediated migration. These results demonstrate the important role of the paxillin-Crk complex in the collagen-induced cell motility.

  2. Residual stresses measurement by neutron diffraction and theoretical estimation in a single weld bead

    International Nuclear Information System (INIS)

    Price, John W.H.; Paradowska, Anna; Joshi, Suraj; Finlayson, Trevor

    2006-01-01

    Welding residual stresses are important in pressure vessel and structural applications. However, residual stress remains the single largest unknown in industrial damage situations. They are difficult to measure or theoretically estimate and are often significant when compared with the in-service stresses on which they superimpose. High residual stresses lead to loss of performance in corrosion, fatigue and fracture. In this research, a measurement of residual stress by the neutron diffraction technique is compared to an analysis of the same geometry by theoretical finite element procedures. The results indicate good agreement but scope for further understanding of the details of modelling the welding heat source, heat transfer and variation of material properties with temperature

  3. Single-step immobilization of cell adhesive peptides on a variety of biomaterial substrates via tyrosine oxidation with copper catalyst and hydrogen peroxide.

    Science.gov (United States)

    Kakinoki, Sachiro; Yamaoka, Tetsuji

    2015-04-15

    Immobilization of biologically active peptides which were isolated from extracellular matrix proteins is a powerful strategy for the design and functionalization of biomaterial substrates. However, the method of peptide immobilization was restricted, that is, peptide is often immobilized through the reactive groups inherent in substrates with multistep reactions. Here, we report a single-step immobilization of fibronectin-derived cell adhesive peptide (Arg-Glu-Asp-Val; REDV) onto polymer materials by use of tyrosine oxidation with copper catalyst and hydrogen peroxide. REDV peptide was successfully immobilized on tissue culture polystyrene, poly(ethylene terephthalate), poly(vinyl chloride), expanded-poly(tetrafluoroethylene), and poly(l-lactic acid), resulting in enhanced adhesion of human umbilical vein endothelial cells. This method is a single-step reaction under very mild conditions and is available for the biological functionalization of various medical devices.

  4. Residual Stress State in Single-Edge Notched Tension Specimen Caused by the Local Compression Technique

    Directory of Open Access Journals (Sweden)

    Huang Yifan

    2016-12-01

    Full Text Available Three-dimensional (3D finite element analyses (FEA are performed to simulate the local compression (LC technique on the clamped single-edge notched tension (SE(T specimens. The analysis includes three types of indenters, which are single pair of cylinder indenters (SPCI, double pairs of cylinder indenters (DPCI and single pair of ring indenters (SPRI. The distribution of the residual stress in the crack opening direction in the uncracked ligament of the specimen is evaluated. The outcome of this study can facilitate the use of LC technique on SE(T specimens.

  5. Structure-based network analysis of activation mechanisms in the ErbB family of receptor tyrosine kinases: the regulatory spine residues are global mediators of structural stability and allosteric interactions.

    Directory of Open Access Journals (Sweden)

    Kevin A James

    Full Text Available The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced "superacceptor" activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD motif in the catalytic loop and the Asp-Phe-Gly (DFG motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not

  6. Contaminant Release from Residual Waste in Single Shell Tanks at the Hanford Site, Washington, USA - 9276

    International Nuclear Information System (INIS)

    Cantrell, Kirk J.; Krupka, Kenneth M.; Deutsch, William J.; Lindberg, Michael J.

    2009-01-01

    Determinations of elemental and solid-phase compositions, and contaminant release studies have been applied in an ongoing study of residual tank wastes (i.e., waste remaining after final retrieval operations) from five of 149 underground single-shell storage tanks (241-C-103, 241-C-106, 241-C-202, 241-C-203, and 241-S-112) at the U.S. Department of Energy's Hanford Site in Washington State. This work is being conducted to support performance assessments that will be required to evaluate long-term health and safety risks associated with tank site closure. The results of studies completed to date show significant variability in the compositions, solid phase properties, and contaminant release characteristics from these residual tank wastes. This variability is the result of differences in waste chemistry/composition of wastes produced from several different spent fuel reprocessing schemes, subsequent waste reprocessing to remove certain target constituents, tank farm operations that concentrated wastes and mixed wastes between tanks, and differences in retrieval processes used to remove the wastes from the tanks. Release models were developed based upon results of chemical characterization of the bulk residual waste, solid-phase characterization (see companion paper 9277 by Krupka et al.), leaching and extraction experiments, and geochemical modeling. In most cases empirical release models were required to describe contaminant release from these wastes. Release of contaminants from residual waste was frequently found to be controlled by the solubility of phases that could not be identified and/or for which thermodynamic data and/or dissolution rates have not been measured. For example, significant fractions of Tc-99, I-129, and Cr appear to be coprecipitated at trace concentrations in metal oxide phases that could not be identified unambiguously. In the case of U release from tank 241-C-103 residual waste, geochemical calculations indicated that leachate

  7. Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

    Science.gov (United States)

    Wu, Hao; Wang, Wei; Naiyer, Nada; Fichtenbaum, Eric; Qualley, Dominic F.; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic

  8. A Single Residue in Ebola Virus Receptor NPC1 Influences Cellular Host Range in Reptiles.

    Science.gov (United States)

    Ndungo, Esther; Herbert, Andrew S; Raaben, Matthijs; Obernosterer, Gregor; Biswas, Rohan; Miller, Emily Happy; Wirchnianski, Ariel S; Carette, Jan E; Brummelkamp, Thijn R; Whelan, Sean P; Dye, John M; Chandran, Kartik

    2016-01-01

    Filoviruses are the causative agents of an increasing number of disease outbreaks in human populations, including the current unprecedented Ebola virus disease (EVD) outbreak in western Africa. One obstacle to controlling these epidemics is our poor understanding of the host range of filoviruses and their natural reservoirs. Here, we investigated the role of the intracellular filovirus receptor, Niemann-Pick C1 (NPC1) as a molecular determinant of Ebola virus (EBOV) host range at the cellular level. Whereas human cells can be infected by EBOV, a cell line derived from a Russell's viper (Daboia russellii) (VH-2) is resistant to infection in an NPC1-dependent manner. We found that VH-2 cells are resistant to EBOV infection because the Russell's viper NPC1 ortholog bound poorly to the EBOV spike glycoprotein (GP). Analysis of panels of viper-human NPC1 chimeras and point mutants allowed us to identify a single amino acid residue in NPC1, at position 503, that bidirectionally influenced both its binding to EBOV GP and its viral receptor activity in cells. Significantly, this single residue change perturbed neither NPC1's endosomal localization nor its housekeeping role in cellular cholesterol trafficking. Together with other recent work, these findings identify sequences in NPC1 that are important for viral receptor activity by virtue of their direct interaction with EBOV GP and suggest that they may influence filovirus host range in nature. Broader surveys of NPC1 orthologs from vertebrates may delineate additional sequence polymorphisms in this gene that control susceptibility to filovirus infection. IMPORTANCE Identifying cellular factors that determine susceptibility to infection can help us understand how Ebola virus is transmitted. We asked if the EBOV receptor Niemann-Pick C1 (NPC1) could explain why reptiles are resistant to EBOV infection. We demonstrate that cells derived from the Russell's viper are not susceptible to infection because EBOV cannot bind to

  9. Effect of enzymatic desialylation of human serum amyloid P component on surface exposure of laser photo CIDNP (chemically induced dynamic nuclear polarization) - reactive histidine, tryptophan and tyrosine residues

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Siebert, H.-C; André, S.; Reuter, G.; Gabius, H.-J.; Kaptein, R.

    1995-01-01

    The human pentraxin serum amyloid P component (SAP) exhibits no microheterogeneity in its complex di-antennary glycan. To elucidate whether the removal of sialic acids from this glycoprotein might affect the accessibility of certain amino acid residues of the protein we employed the laser photo

  10. Dityrosine, 3,4-Dihydroxyphenylalanine (DOPA), and radical formation from tyrosine residues on milk proteins with globular and flexible structures as a result of riboflavin-mediated photo-oxidation

    DEFF Research Database (Denmark)

    Dalsgaard, Trine Kastrup; Nielsen, Jacob Holm; Brown, Bronwyn

    2011-01-01

    Riboflavin-mediated photo-oxidative damage to protein Tyr residues has been examined to determine whether protein structure influences competing protein oxidation pathways in single proteins and protein mixtures. EPR studies resulted in the detection of Tyr-derived o-semiquione radicals, with thi......Riboflavin-mediated photo-oxidative damage to protein Tyr residues has been examined to determine whether protein structure influences competing protein oxidation pathways in single proteins and protein mixtures. EPR studies resulted in the detection of Tyr-derived o-semiquione radicals...

  11. Comparison of mineral dust and droplet residuals measured with two single particle aerosol mass spectrometers

    Science.gov (United States)

    Wonaschütz, Anna; Ludwig, Wolfgang; Zawadowicz, Maria; Hiranuma, Naruki; Hitzenberger, Regina; Cziczo, Daniel; DeMott, Paul; Möhler, Ottmar

    2017-04-01

    Single Particle mass spectrometers are used to gain information on the chemical composition of individual aerosol particles, aerosol mixing state, and other valuable aerosol characteristics. During the Mass Spectrometry Intercomparison at the Fifth Ice Nucleation (FIN-01) Workshop, the new LAAPTOF single particle aerosol mass spectrometer (AeroMegt GmbH) was conducting simultaneous measurements together with the PALMS (Particle Analysis by Laser Mass Spectrometry) instrument. The aerosol particles were sampled from the AIDA chamber during ice cloud expansion experiments. Samples of mineral dust and ice droplet residuals were measured simultaneously. In this work, three expansion experiments are chosen for a comparison between the two mass spectrometers. A fuzzy clustering routine is used to group the spectra. Cluster centers describing the ensemble of particles are compared. First results show that while differences in the peak heights are likely due to the use of an amplifier in PALMS, cluster centers are comparable.

  12. Recent advances in understanding the role of protein-tyrosine phosphatases in development and disease

    NARCIS (Netherlands)

    Hale, Alexander James; Ter Steege, Eline; den Hertog, Jeroen

    2017-01-01

    Protein-tyrosine phosphatases (PTPs) remove phosphate groups from tyrosine residues, and thereby propagate or inhibit signal transduction, and hence influence cellular processes such as cell proliferation and differentiation. The importance of tightly controlled PTP activity is reflected by the

  13. Chemical composition of ambient aerosol, ice residues and cloud droplet residues in mixed-phase clouds: single particle analysis during the Cloud and Aerosol Characterization Experiment (CLACE 6

    Directory of Open Access Journals (Sweden)

    M. Kamphus

    2010-08-01

    Full Text Available Two different single particle mass spectrometers were operated in parallel at the Swiss High Alpine Research Station Jungfraujoch (JFJ, 3580 m a.s.l. during the Cloud and Aerosol Characterization Experiment (CLACE 6 in February and March 2007. During mixed phase cloud events ice crystals from 5–20 μm were separated from larger ice aggregates, non-activated, interstitial aerosol particles and supercooled droplets using an Ice-Counterflow Virtual Impactor (Ice-CVI. During one cloud period supercooled droplets were additionally sampled and analyzed by changing the Ice-CVI setup. The small ice particles and droplets were evaporated by injection into dry air inside the Ice-CVI. The resulting ice and droplet residues (IR and DR were analyzed for size and composition by the two single particle mass spectrometers: a custom-built Single Particle Laser-Ablation Time-of-Flight Mass Spectrometer (SPLAT and a commercial Aerosol Time-of-Flight Mass Spectrometer (ATOFMS, TSI Model 3800. During CLACE 6 the SPLAT instrument characterized 355 individual IR that produced a mass spectrum for at least one polarity and the ATOFMS measured 152 IR. The mass spectra were binned in classes, based on the combination of dominating substances, such as mineral dust, sulfate, potassium and elemental carbon or organic material. The derived chemical information from the ice residues is compared to the JFJ ambient aerosol that was sampled while the measurement station was out of clouds (several thousand particles analyzed by SPLAT and ATOFMS and to the composition of the residues of supercooled cloud droplets (SPLAT: 162 cloud droplet residues analyzed, ATOFMS: 1094. The measurements showed that mineral dust was strongly enhanced in the ice particle residues. Close to all of the SPLAT spectra from ice residues did contain signatures from mineral compounds, albeit connected with varying amounts of soluble compounds. Similarly, close to all of the ATOFMS IR spectra show a

  14. Tyrosine phosphorylation in signal transduction

    International Nuclear Information System (INIS)

    Roberts, T.M.; Kaplan, D.; Morgan, W.; Keller, T.; Mamon, H.; Piwnica-Worms, H.; Druker, B.; Whitman, M.; Morrison, D.; Cohen, B.; Schaffhausen, B.; Cantley, L.; Rapp, U.

    1988-01-01

    Recent work has focused on the elucidation of the mechanisms by which membrane-bound tyrosine kinases transmit signals within the cell. To examine the role of tyrosine phosphorylation the authors have employed the following strategy. First, they have utilized antibodies to phosphotyrosine (anti-P.Tyr) to identify candidate substrates of various tyrosine kinases, such as pp60 c-src , the CSF- receptor, or the platelet-derived growth factor (PDGF) receptor. Second, they have attempted to characterize the biochemical properties of the putative substrates and to determine in what manner these properties are modified by phosphorylation on tyrosine residues. In this endeavor, they are recapitulating the classic biochemical analysis used to study the effect of kinases on metabolism. The final portion of our work consists of using modern molecular biological strategies to clone the genes or cDNAs for the substrates and overproduce the relevant proteins for studies in vitro in defined systems. This paper describes the first and second aspects of this strategy, the identification and characterization of novel substrate molecules

  15. Analysis of morphology and residual porosity in selective laser melting of Fe powders using single track experiments

    Science.gov (United States)

    Shutov, I. V.; Gordeev, G. A.; Kharanzhevskiy, E. V.; Krivilyov, M. D.

    2017-04-01

    Morphology and residual porosity of single tracks obtained by pulse selective laser melting (SLM) of Fe powder have been studied by metallography. Multiple cross sections of the stainless substrate with the single tracks deposited by SLM are examined and classified depending on processing parameters. A sustainable scanning strategy to reduce residual porosity is suggested for pulse laser annealing. The developed method is suitable both for improvement of processing regimes in commercial SLM machines and validation of numerical models in additive manufacturing of metal parts. The effect of the beam radius, pulse energy, its frequency and duration on a shape of the single track and its adhesion to the substrate is revealed.

  16. Autophosphorylation of Specific Threonine and Tyrosine Residues in Arabidopsis CERK1 is Essential for the Activation of Chitin-Induced Immune Signaling.

    Science.gov (United States)

    Suzuki, Maruya; Shibuya, Masatoshi; Shimada, Hikaru; Motoyama, Noriko; Nakashima, Masato; Takahashi, Shohei; Suto, Kenkichi; Yoshida, Issei; Matsui, Saki; Tsujimoto, Natsumi; Ohnishi, Mihoko; Ishibashi, Yuko; Fujimoto, Zui; Desaki, Yoshitake; Kaku, Hanae; Kito, Keiji; Shibuya, Naoto

    2016-11-01

    Pattern recognition receptors on the plant cell surface mediate the recognition of microbe/damage-associated molecular patterns (MAMPs/DAMPs) and activate downstream immune signaling. Autophosphorylation of signaling receptor-like kinases is a critical event for the activation of downstream responses but the function of each phosphorylation site in the regulation of immune signaling is not well understood. In this study, 41 Ser/Thr/Tyr and 15 Ser/Thr residues were identified as in vitro and in vivo autophosphorylation sites of Arabidopsis CERK1, which is essential for chitin signaling. Comprehensive analysis of transgenic plants expressing mutated CERK1 genes for each phosphorylation site in the cerk1-2 background indicated that the phosphorylation of T479 in the activation segment and Y428 located upstream of the catalytic loop is important for the activation of chitin-triggered defense responses. Contribution of the phosphorylation of T573 to the chitin responses was also suggested. In vitro evaluation of kinase activities of mutated kinase domains indicated that the phosphorylation of T479 and T573 is directly involved in the regulation of kinase activity of CERK1 but the phosphorylation of Y428 regulates chitin signaling independently of the regulation of kinase activity. These results indicated that the phosphorylation of specific residues in the kinase domain contributes to the regulation of downstream signaling either through the regulation of kinase activity or the different mechanisms, e.g. regulation of protein-protein interactions. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  17. Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination.

    Science.gov (United States)

    Fan, Hsiu-Fang; Cheng, Yong-Song; Ma, Chien-Hui; Jayaram, Makkuni

    2015-03-31

    Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of 'non-productive', 'pre-synaptic' and 'synaptic' complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination

    Science.gov (United States)

    Fan, Hsiu-Fang; Cheng, Yong-Song; Ma, Chien-Hui; Jayaram, Makkuni

    2015-01-01

    Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of ‘non-productive’, ‘pre-synaptic’ and ‘synaptic’ complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations. PMID:25765648

  19. Analysis of cosmetic residues on a single human hair by ATR FT-IR microspectroscopy.

    Science.gov (United States)

    Pienpinijtham, Prompong; Thammacharoen, Chuchaat; Naranitad, Suwimol; Ekgasit, Sanong

    2018-05-15

    In this work, ATR FT-IR spectra of single human hair and cosmetic residues on hair surface are successfully collected using a homemade dome-shaped Ge μIRE accessary installed on an infrared microscope. By collecting ATR spectra of hairs from the same person, the spectral patterns are identical and superimposed while different spectral features are observed from ATR spectra of hairs collected from different persons. The spectral differences depend on individual hair characteristics, chemical treatments, and cosmetics on hair surface. The "Contact-and-Collect" technique that transfers remarkable materials on the hair surface to the tip of the Ge μIRE enables an identification of cosmetics on a single hair. Moreover, the differences between un-split and split hairs are also studied in this report. These highly specific spectral features can be employed for unique identification or for differentiation of hairs based on the molecular structures of hairs and cosmetics on hairs. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Identification and analysis of a novel protein-tyrosine kinase from bovine thymus

    International Nuclear Information System (INIS)

    Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

    1986-01-01

    A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn 2+ and [γ- 32 P]ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes

  1. Bruton's tyrosine kinase gene mutations in Turkish patients with X-linked agammaglobulinemia from a single center: Novel mutations in βTK gene

    NARCIS (Netherlands)

    Ç. Aydoǧmuş (Çiǧdem); Y. Camcioǧlu (Yildiz); M. van der Burg (Mirjam); H. Çokuǧraş (H.); N. Akçakaya (Necla); J.J.M. van Dongen (Jacques)

    2013-01-01

    textabstractObjective: X-linked agammaglobulinemia (XLA) is caused by a mutation in the Bruton's tyrosine kinase gene and is characterized by a delay in the maturation and differentiation of B lymphocytes. Patients with XLA have either absent or very low levels of circulating mature B cells (<1%),

  2. Regulatory Closure Options for the Residue in the Hanford Site Single-Shell Tanks

    International Nuclear Information System (INIS)

    Cochran, J.R.; Shyr, L.J.

    1998-01-01

    Liquid, mixed, high-level radioactive waste (HLW) has been stored in 149 single-shell tanks (SSTS) located in tank farms on the U.S. Department of Energy's (DOE's) Hanford Site. The DOE is developing technologies to retrieve as much remaining HLW as technically possible prior to physically closing the tank farms. In support of the Hanford Tanks Initiative, Sandia National Laboratories has addressed the requirements for the regulatory closure of the radioactive component of any SST residue that may remain after physical closure. There is significant uncertainty about the end state of each of the 149 SSTS; that is, the nature and amount of wastes remaining in the SSTS after retrieval is uncertain. As a means of proceeding in the face of these uncertainties, this report links possible end-states with associated closure options. Requirements for disposal of HLW and low-level radioactive waste (LLW) are reviewed in detail. Incidental waste, which is radioactive waste produced incidental to the further processing of HLW, is then discussed. If the low activity waste (LAW) fraction from the further processing of HLW is determined to be incidental waste, then DOE can dispose of that incidental waste onsite without a license from the U.S. Nuclear Regulatory Commissions (NRC). The NRC has proposed three Incidental Waste Criteria for determining if a LAW fraction is incidental waste. One of the three Criteria is that the LAW fraction should not exceed the NRC's Class C limits

  3. Computing H/D-Exchange rates of single residues from data of proteolytic fragments

    Directory of Open Access Journals (Sweden)

    Althaus Ernst

    2010-08-01

    Full Text Available Abstract Background Protein conformation and protein/protein interaction can be elucidated by solution-phase Hydrogen/Deuterium exchange (sHDX coupled to high-resolution mass analysis of the digested protein or protein complex. In sHDX experiments mutant proteins are compared to wild-type proteins or a ligand is added to the protein and compared to the wild-type protein (or mutant. The number of deuteriums incorporated into the polypeptides generated from the protease digest of the protein is related to the solvent accessibility of amide protons within the original protein construct. Results In this work, sHDX data was collected on a 14.5 T FT-ICR MS. An algorithm was developed based on combinatorial optimization that predicts deuterium exchange with high spatial resolution based on the sHDX data of overlapping proteolytic fragments. Often the algorithm assigns deuterium exchange with single residue resolution. Conclusions With our new method it is possible to automatically determine deuterium exchange with higher spatial resolution than the level of digested fragments.

  4. Regulatory Closure Options for the Residue in the Hanford Site Single-Shell Tanks

    Energy Technology Data Exchange (ETDEWEB)

    Cochran, J.R. Shyr, L.J.

    1998-10-05

    Liquid, mixed, high-level radioactive waste (HLW) has been stored in 149 single-shell tanks (SSTS) located in tank farms on the U.S. Department of Energy's (DOE's) Hanford Site. The DOE is developing technologies to retrieve as much remaining HLW as technically possible prior to physically closing the tank farms. In support of the Hanford Tanks Initiative, Sandia National Laboratories has addressed the requirements for the regulatory closure of the radioactive component of any SST residue that may remain after physical closure. There is significant uncertainty about the end state of each of the 149 SSTS; that is, the nature and amount of wastes remaining in the SSTS after retrieval is uncertain. As a means of proceeding in the face of these uncertainties, this report links possible end-states with associated closure options. Requirements for disposal of HLW and low-level radioactive waste (LLW) are reviewed in detail. Incidental waste, which is radioactive waste produced incidental to the further processing of HLW, is then discussed. If the low activity waste (LAW) fraction from the further processing of HLW is determined to be incidental waste, then DOE can dispose of that incidental waste onsite without a license from the U.S. Nuclear Regulatory Commissions (NRC). The NRC has proposed three Incidental Waste Criteria for determining if a LAW fraction is incidental waste. One of the three Criteria is that the LAW fraction should not exceed the NRC's Class C limits.

  5. Tyrosine modifications in aging.

    Science.gov (United States)

    Feeney, Maria B; Schöneich, Christian

    2012-12-01

    The understanding of physiological and pathological processes involving protein oxidation, particularly under conditions of aging and oxidative stress, can be aided by proteomic identification of proteins that accumulate oxidative post-translational modifications only if these detected modifications are connected to functional consequences. The modification of tyrosine (Tyr) residues can elicit significant changes in protein structure and function, which, in some cases, may contribute to biological aging and age-related pathologies, such as atherosclerosis, neurodegeneration, and cataracts. Studies characterizing proteins in which Tyr has been modified to 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, 3,3'-dityrosine and other cross-links, or 3-chlorotyrosine are reviewed, with an emphasis on structural and functional consequences. Distinguishing between inconsequential modifications and functionally significant ones requires careful biochemical and biophysical analysis of target proteins, as well as innovative methods for isolating the effects of the multiple modifications that often occur under oxidizing conditions. The labor-intensive task of isolating and characterizing individual modified proteins must continue, especially given the expanding list of known modifications. Emerging approaches, such as genetic and metabolic incorporation of unnatural amino acids, hold promise for additional focused studies of this kind.

  6. Protein tyrosine nitration in the cell cycle

    International Nuclear Information System (INIS)

    Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

    2011-01-01

    Highlights: → Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. → Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. → Nineteen identified proteins were previously described as regulators of cell proliferation. -- Abstract: Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.

  7. The Sulfinator: predicting tyrosine sulfation sites in protein sequences.

    Science.gov (United States)

    Monigatti, Flavio; Gasteiger, Elisabeth; Bairoch, Amos; Jung, Eva

    2002-05-01

    Protein tyrosine sulfation is an important post-translational modification of proteins that go through the secretory pathway. No clear-cut acceptor motif can be defined that allows the prediction of tyrosine sulfation sites in polypeptide chains. The Sulfinator is a software tool that can be used to predict tyrosine sulfation sites in protein sequences with an overall accuracy of 98%. Four different Hidden Markov Models were constructed, each of them specialized to recognize sulfated tyrosine residues depending on their location within the sequence: near the N-terminus, near the C-terminus, in the center of a window with a size of at least 25 amino acids, as well as in windows containing several tyrosine residues. The Sulfinator is accessible at (http://www.expasy.org/tools/sulfinator/). Sulfinator documentation is accessible at (http://www.expasy.org/tools/sulfinator/sulfinator-doc.html).

  8. Effect of increased exposure times on amount of residual monomer released from single-step self-etch adhesives.

    Science.gov (United States)

    Altunsoy, Mustafa; Botsali, Murat Selim; Tosun, Gonca; Yasar, Ahmet

    2015-10-16

    The aim of this study was to evaluate the effect of increased exposure times on the amount of residual Bis-GMA, TEGDMA, HEMA and UDMA released from single-step self-etch adhesive systems. Two adhesive systems were used. The adhesives were applied to bovine dentin surface according to the manufacturer's instructions and were polymerized using an LED curing unit for 10, 20 and 40 seconds (n = 5). After polymerization, the specimens were stored in 75% ethanol-water solution (6 mL). Residual monomers (Bis-GMA, TEGDMA, UDMA and HEMA) that were eluted from the adhesives (after 10 minutes, 1 hour, 1 day, 7 days and 30 days) were analyzed by high-performance liquid chromatography (HPLC). The data were analyzed using 1-way analysis of variance and Tukey HSD tests. Among the time periods, the highest amount of released residual monomers from adhesives was observed in the 10th minute. There were statistically significant differences regarding released Bis-GMA, UDMA, HEMA and TEGDMA between the adhesive systems (p<0.05). There were no significant differences among the 10, 20 and 40 second polymerization times according to their effect on residual monomer release from adhesives (p>0.05). Increasing the polymerization time did not have an effect on residual monomer release from single-step self-etch adhesives.

  9. The mining of toxin-like polypeptides from EST database by single residue distribution analysis

    Science.gov (United States)

    2011-01-01

    Background Novel high throughput sequencing technologies require permanent development of bioinformatics data processing methods. Among them, rapid and reliable identification of encoded proteins plays a pivotal role. To search for particular protein families, the amino acid sequence motifs suitable for selective screening of nucleotide sequence databases may be used. In this work, we suggest a novel method for simplified representation of protein amino acid sequences named Single Residue Distribution Analysis, which is applicable both for homology search and database screening. Results Using the procedure developed, a search for amino acid sequence motifs in sea anemone polypeptides was performed, and 14 different motifs with broad and low specificity were discriminated. The adequacy of motifs for mining toxin-like sequences was confirmed by their ability to identify 100% toxin-like anemone polypeptides in the reference polypeptide database. The employment of novel motifs for the search of polypeptide toxins in Anemonia viridis EST dataset allowed us to identify 89 putative toxin precursors. The translated and modified ESTs were scanned using a special algorithm. In addition to direct comparison with the motifs developed, the putative signal peptides were predicted and homology with known structures was examined. Conclusions The suggested method may be used to retrieve structures of interest from the EST databases using simple amino acid sequence motifs as templates. The efficiency of the procedure for directed search of polypeptides is higher than that of most currently used methods. Analysis of 39939 ESTs of sea anemone Anemonia viridis resulted in identification of five protein precursors of earlier described toxins, discovery of 43 novel polypeptide toxins, and prediction of 39 putative polypeptide toxin sequences. In addition, two precursors of novel peptides presumably displaying neuronal function were disclosed. PMID:21281459

  10. Correlation Between the Microstructural Defects and Residual Stress in a Single Crystal Nickel-Based Superalloy During Different Creep Stages

    Science.gov (United States)

    Mo, Fangjie; Wu, Erdong; Zhang, Changsheng; Wang, Hong; Zhong, Zhengye; Zhang, Jian; Chen, Bo; Hofmann, Michael; Gan, Weimin; Sun, Guangai

    2018-03-01

    The present work attempts to reveal the correlation between the microstructural defects and residual stress in the single crystal nickel-based superalloy, both of which play the significant role on properties and performance. Neutron diffraction was employed to investigate the microstructural defects and residual stresses in a single crystal (SC) nickel-based superalloy, which was subjected to creeping under 220 MPa and 1000 °C for different times. The measured superlattice and fundamental lattice reflections confirm that the mismatch and tetragonal distortions with c/a > 1 exist in the SC superalloy. At the initially unstrained state, there exists the angular distortion between γ and γ' phases with small triaxial compressive stresses, ensuring the structural stability of the superalloy. After creeping, the tetragonal distortion for the γ phase is larger than that for the γ' phase. With increasing the creeping time, the mismatch between γ and γ' phases increases to the maximum, then decreases gradually and finally remains unchanged. The macroscopic residual stress shows a similar behavior with the mismatch, indicating the correlation between them. Based on the model of shear and dislocations, the evolution of microstructural defects and residual stress are reasonably explained. The effect of shear is dominant at the primary creep stage, which greatly enlarges the mismatch and the residual stress. The dislocations weaken the effect of shear for the further creep stage, resulting in the decrease of the mismatch and relaxation of the residual stress. Those findings add some helpful understanding into the microstructure-performance relationship in the SC nickel-based superalloy, which might provide the insight to materials design and applications.

  11. Residual strains and microstructure development in single and sequential double sided friction stir welds in RQT-701 steel

    International Nuclear Information System (INIS)

    Barnes, S.J.; Steuwer, A.; Mahawish, S.; Johnson, R.; Withers, P.J.

    2008-01-01

    Single and double sided partial penetration friction stir butt welds, in a rolled, quenched and tempered steel (RQT-701), were produced at The Welding Institute (TWI) under controlled process conditions. The residual strain distributions in the longitudinal and transverse directions have been measured using energy dispersive synchrotron X-ray diffraction. The measured strains were indicative of longitudinal tensile residual stresses at levels greater than the 0.2% yield stress of the parent metal in both the single and double pass welds. In both cases, the maximum tensile strain was found in the parent metal at the boundary of the heat affected zone (HAZ). Microstructural analysis of the welds was carried out using optical microscopy and hardness variations were also mapped across the weld-plate cross-section. The maximum hardness was observed in the mixed bainite/martensite structure of the weld nugget on the advancing side of the stir zone. The minimum hardness was observed in the HAZ

  12. Candidate genes and single nucleotide polymorphisms associated with variation in residual feed intake in beef cattle.

    Science.gov (United States)

    Karisa, B K; Thomson, J; Wang, Z; Stothard, P; Moore, S S; Plastow, G S

    2013-08-01

    The candidate gene approach was used to identify genes associated with residual feed intake (RFI) in beef steers. The approach uses prior knowledge of gene functions to predict their biological role in the variation observed in a trait. It is suited to identify genes associated with complex traits where each gene has a relatively small effect. First, positional candidate genes were identified within the genomic positions of previously reported QTL associated with component traits related to RFI such as dry matter intake (DMI), growth, feed conversion ratio (FCR), average daily gain (ADG), and energy balance. Secondly, the positional candidate genes were prioritized into functional candidate genes according to their biological functions and their relationship with the biological processes associated with RFI including carbohydrate, fat and protein metabolism, thermoregulation, immunity and muscle activity. Single nucleotide polymorphisms (SNPs) located within the functional candidate genes were identified using mRNA sequences and prioritized into functional classes such as non-synonymous (nsSNP), synonymous (sSNP) or intronic SNP. A total of 117 nsSNP were considered as functional SNP and genotyped in steers at the University of Alberta ranch in Kinsella. Multiple marker association analysis in ASReml was performed using RFI data obtained from 531 beef steers. Twenty-five SNP were significantly associated with RFI (P < 0.05) accounting for 19.7% of the phenotypic variation. Using SIFT program to predict the effect of the SNP on the function of the corresponding protein, 3 of the 25 SNP were predicted to cause a significant effect on protein function (P < 0.05). One of the 3 SNP was located in the GHR gene and was also associated with a significant effect on the tertiary structure of the GHR protein (P < 0.05) as modeled using SWISSModel software. Least square means for each genotype were estimated and an over-dominance effect was observed for the SNP located in the

  13. A single residue mutation of 5-enoylpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate.

    Science.gov (United States)

    Liang, Aimin; Sha, Jiying; Lu, Wei; Chen, Ming; Li, Liang; Jin, Dan; Yan, Yongliang; Wang, Jin; Ping, Shuzhen; Zhang, Wei; Wang, Yiding; Lin, Min

    2008-08-01

    A novel class II 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) was identified from Pseudomonas stutzeri A1501 by complementation of an Escherichia coli auxotrophic aroA mutant. The single amino acid substitution of serine (Ser) for asparagine (Asn)-130 of the A1501 EPSPS enhanced resistance to 200 mM glyphosate. The mutated EPSPS had a 2.5-fold increase for IC(50) [glyphosate] value, a 2-fold increase for K (i) [glyphosate] value, but a K (m) [PEP] value similar to that of wild type. The effect of the single residue mutation on glyphosate resistance was also analyzed using a computer-based three-dimensional model.

  14. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    Energy Technology Data Exchange (ETDEWEB)

    Li, Bin, E-mail: binli@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Eyer, Peter, E-mail: peter.eyer@lrz.uni-muenchen.de [Walther-Straub-Institut Für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, 80336 München (Germany); Eddleston, Michael, E-mail: M.Eddleston@ed.ac.uk [Clinical Pharmacology Unit, University of Edinburgh, Edinburgh (United Kingdom); Jiang, Wei, E-mail: wjiang@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Schopfer, Lawrence M., E-mail: lmschopf@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Lockridge, Oksana, E-mail: olockrid@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States)

    2013-06-15

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

  15. Membranes fabricated with a deep single corrugation for package stress reduction and residual stress relief

    NARCIS (Netherlands)

    Spiering, V.L.; Spiering, V.L.; Bouwstra, S.; Bouwstra, S.; Burger, Johannes Faas; Elwenspoek, Michael Curt

    1993-01-01

    Thin square membranes including a deep circular corrugation are realized and tested for application in a strain-based pressure sensor. Package-induced stresses are reduced and relief of the residual stress is obtained, resulting in a larger pressure sensitivity and a reduced temperature sensitivity.

  16. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    International Nuclear Information System (INIS)

    Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.

    2008-01-01

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ∼ 68% and ∼ 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy

  17. Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain

    Science.gov (United States)

    Sheffler-Collins, Sean I.; Xia, Nan L.; Henderson, Nathan; Tillu, Dipti V.; Hassler, Shayne; Spellman, Daniel S.; Zhang, Guoan; Neubert, Thomas A.; Price, Theodore J.

    2017-01-01

    Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function. PMID:28719605

  18. Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.

    Science.gov (United States)

    Fang, Gang; Munera, Diana; Friedman, David I; Mandlik, Anjali; Chao, Michael C; Banerjee, Onureena; Feng, Zhixing; Losic, Bojan; Mahajan, Milind C; Jabado, Omar J; Deikus, Gintaras; Clark, Tyson A; Luong, Khai; Murray, Iain A; Davis, Brigid M; Keren-Paz, Alona; Chess, Andrew; Roberts, Richard J; Korlach, Jonas; Turner, Steve W; Kumar, Vipin; Waldor, Matthew K; Schadt, Eric E

    2012-12-01

    Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.

  19. Construction of a recombinant single chain antibody recognizing nonreducing terminal mannose residues applicable to immunohistochemistry.

    Science.gov (United States)

    Yuasa, Noriyuki; Iida, Noriko; Sakaue, Hiroyuki; Zhang, Wei; Wilczynski, Sharon; Fujita-Yamaguchi, Yoko

    2007-10-01

    We recently reported characterization of 25 clones isolated from a phage library displaying human scFvs using a neoglycolipid Man3-DPPE, which was synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE). Of those, 5A3 scFv was successfully expressed and purified as a humanized scFv-Fc form (Sakai et al., Biochemistry 46:253, 2007, Zhang et al. ibid 263). To carry out immunohistochemistry (IHC) in human tissues, a HA tag sequence was introduced to the 5A3 scFv-Fc gene and the resulting construct was transfected to murine myeloma NS0 cells. The 5A3 scFv-Fc protein expressed was affinity-purified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing and reducing conditions and enzyme-linked immunosorbent assay confirmed that 5A3 scFv-Fc protein is dimeric and retained the ability to recognize nonreducing terminal mannose residues. IHC staining of non-neoplastic tissues by this recombinant antibody revealed that no immunoreactivity was detectable in most of 16 tissues examined. Exceptions were found in IHC staining of kidney and pancreas, which demonstrated clear staining of proximal tubules and islet of Langerhans, respectively. These results demonstrated that nonreducing terminal mannose residues are not usually present under normal physiological conditions. This study thus provided a potentially useful tool for examination of the nonreducing terminal mannose residues, which may become exposed under certain pathophysiologycal conditions.

  20. Single particle measurements of the chemical composition of cirrus ice residue during CRYSTAL-FACE

    Science.gov (United States)

    Cziczo, D. J.; Murphy, D. M.; Hudson, P. K.; Thomson, D. S.

    2004-02-01

    The first real-time, in situ, investigation of the chemical composition of the residue of cirrus ice crystals was performed during July 2002. This study was undertaken on a NASA WB-57F high-altitude research aircraft as part of CRYSTAL-FACE, a field campaign which sought to further our understanding of the relation of clouds, water vapor, and climate by characterizing, among other parameters, anvil cirrus formed about the Florida peninsula. A counter flow virtual impactor (CVI) was used to separate cirrus ice from the unactivated interstitial aerosol particles and evaporate condensed-phase water. Residual material, on a crystal-by-crystal basis, was subsequently analyzed using the NOAA Aeronomy Laboratory's Particle Analysis by Laser Mass Spectrometry (PALMS) instrument. Sampling was performed from 5 to 15 km altitude and from 12° to 28° north latitude within cirrus originating over land and ocean. Chemical composition measurements provided several important results. Sea salt was often incorporated into cirrus, consistent with homogeneous ice formation by aerosol particles from the marine boundary layer. Size measurements showed that large particles preferentially froze over smaller ones. Meteoritic material was found within ice crystals, indicative of a relation between stratospheric aerosol particles and tropospheric clouds. Mineral dust was the dominant residue observed in clouds formed during a dust transport event from the Sahara, consistent with a heterogeneous freezing mechanism. These results show that chemical composition and size are important determinants of which aerosol particles form cirrus ice crystals.

  1. Single well push-pull CO2 injection experiment for evaluating in-situ residual trapping at Heletz, Israel

    Science.gov (United States)

    Niemi, Auli; Bensabat, Jacob; Fagerlund, Fritjof; Ronen, Rona; Goren, Yoni; Perez, Lily; Tsarfis, Igal; Joodaki, Saba; Yang, Zhibing; Liang, Tian; Sauter, Martin; Hassan, Jawad; Gouze, Philippe; Rasmusson, Kristina

    2017-04-01

    The Heletz sands is a depleted oil reservoir at 1.6 km depth with saline water at its edges. In the saline part of the reservoir a CO2 injection experiment site has been developed for the purpose of scientifically motivated injection experiments, especially in the context of EU FP7 projects MUSTANG and TRUST. This presentation describes the single-well CO2 injection experiment carried out in September 2016, with the objective of determining field scale values of key CO2 trapping mechanisms, the residual and dissolution trapping. The sequence consisted in creating a residually trapped CO2 zone as well as reference hydraulic and heater tests prior and after the establishment of the zone, in order to determine the in-situ residual trapping. Monitoring included down-hole pressure and temperature measurement, distributed temperature sensing along the well via an optical fiber (DTS), U-tube sampling and tracers. We here present the experimental sequence, the monitoring and sampling system, the key results as well as the first interpretations.

  2. A single conserved basic residue in the potassium channel filter region controls KCNQ1 insensitivity toward scorpion toxins.

    Science.gov (United States)

    Chen, Zongyun; Hu, Youtian; Wang, Bin; Cao, Zhijian; Li, Wenxin; Wu, Yingliang

    2015-09-01

    Although many studies concerning the sensitivity mechanism of scorpion toxin-potassium channel interactions have been reported, few have explored the biochemical insensitivity mechanisms of potassium channel receptors toward natural scorpion toxin peptides, such as the KCNQ1 channel. Here, by sequence alignment analyses of the human KCNQ1 channel and scorpion potassium channel MmKv2, which is completely insensitive to scorpion toxins, we proposed that the insensitivity mechanism of KCNQ1 toward natural scorpion toxins might involve two functional regions, the turret and filter regions. Based on this observation, a series of KCNQ1 mutants were constructed to study molecular mechanisms of the KCNQ1 channel insensitivity toward natural scorpion toxins. Electrophysiological studies of chimera channels showed that the channel filter region controls KCNQ1 insensitivity toward the classical scorpion toxin ChTX. Interestingly, further residue mutant experiments showed that a single basic residue in the filter region determined the insensitivity of KCNQ1 channels toward scorpion toxins. Our present work showed that amino acid residue diversification at common sites controls the sensitivity and insensitivity of potassium channels toward scorpion toxins. The unique insensitivity mechanism of KCNQ1 toward natural scorpion toxins will accelerate the rational design of potent peptide inhibitors toward this channel.

  3. UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 1: basic principles and properties of tyrosine chromophore.

    Science.gov (United States)

    Antosiewicz, Jan M; Shugar, David

    2016-06-01

    Spectroscopic properties of tyrosine residues may be employed in structural studies of proteins. Here we discuss several different types of UV-Vis spectroscopy, like normal, difference and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, and corresponding optical properties of the tyrosine chromophore, phenol, which are used to study protein structure.

  4. UV?Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 1: basic principles and properties of tyrosine chromophore

    OpenAIRE

    Antosiewicz, Jan M.; Shugar, David

    2016-01-01

    Spectroscopic properties of tyrosine residues may be employed in structural studies of proteins. Here we discuss several different types of UV?Vis spectroscopy, like normal, difference and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, and corresponding optical properties of the tyrosine chromophore, phenol, which are used to study protein structure.

  5. Tyrosine supplementation for phenylketonuria.

    Science.gov (United States)

    Webster, Diana; Wildgoose, Joanne

    2013-06-05

    Phenylketonuria is an inherited disease for which the main treatment is the dietary restriction of the amino acid phenylalanine. The diet has to be initiated in the neonatal period to prevent or reduce mental handicap. However, the diet is very restrictive and unpalatable and can be difficult to follow. A deficiency of the amino acid tyrosine has been suggested as a cause of some of the neuropsychological problems exhibited in phenylketonuria. Therefore, this review aims to assess the efficacy of tyrosine supplementation for phenylketonuria. To assess the effects of tyrosine supplementation alongside or instead of a phenylalanine-restricted diet for people with phenylketonuria, who commenced on diet at diagnosis and either continued on the diet or relaxed the diet later in life. To assess the evidence that tyrosine supplementation alongside, or instead of a phenylalanine-restricted diet improves intelligence, neuropsychological performance, growth and nutritional status, mortality rate and quality of life. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Trials Register which is comprised of references identified from comprehensive electronic database searches, handsearches of relevant journals and abstract books of conference proceedings. Additional studies were identified from handsearches of the Journal of Inherited Metabolic Disease (from inception in 1978 to 1998). The manufacturers of prescribable dietary products used in the treatment of phenylketonuria were also contacted for further references.Date of the most recent search of the Group's Inborn Errors of Metabolism Trials Register: 28 June 2012. All randomised or quasi-randomised trials investigating the use of tyrosine supplementation versus placebo in people with phenylketonuria in addition to, or instead of, a phenylalanine-restricted diet. People treated for maternal phenylketonuria were excluded. Two authors independently assessed the trial eligibility, methodological quality

  6. Single-Residue Sensitivity in Neutron Reflectivity and Resonant X-ray Reflectivity from Langmuir Monolayers of Synthetic Peptides

    Science.gov (United States)

    Strzalka, Joseph; Satija, Sushil; Dimasi, Elaine; Kuzmenko, Ivan; Gog, Thomas; Blasie, J. Kent

    2004-03-01

    Labeling groups with ^2H to distinguish them in the scattering length density (SLD) profile constitutes the chief advantage of neutron reflectivity (NR) in studying Langmuir monolayers (LM) of lipids and proteins. Solid phase synthesis (SPPS) permits the labeling of a single residue in a peptide. Recent work demonstrates the sensitivity of NR to single ^2H-labeled residues in LM of vectorially oriented α -helical bundle peptides. NR requires comparison of isomorphic samples of all-^1H and ^2H-labeled peptides. Alternately, resonant x-ray reflectivity (RXR) uses only one sample. RXR exploits energy-dependent changes in the scattering factor from heavy atoms to distinguish them within the SLD profile. Peptides may be labeled by SPPS (e.g. Br-Phe), or may have inherent labels (e.g. Fe in heme proteins). As test cases, we studied LM of Br-labeled lipids and peptides with RXR. Both approaches require a model-independent means of obtaining SLD profiles from the reflectivity data. We have applied box-refinement to obtain the gradient SLD profile. This is fit uniquely with a sum of Gaussians and integrated analytically [Blasie et al., PRB 67 224201 (2003)] to provide the SLD profile. Label positions can then be determined to sub-Ångstrom accuracy. This work supported by the NIH (GM55876).

  7. Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry

    DEFF Research Database (Denmark)

    Rand, Kasper D; Zehl, Martin; Jensen, Ole Nørregaard

    2009-01-01

    Because of unparalleled sensitivity and tolerance to protein size, mass spectrometry (MS) has become a popular method for measuring the solution hydrogen (1H/2H) exchange (HX) of biologically relevant protein states. While incorporated deuterium can be localized to different regions by pepsin...... proteolysis of the labeled protein, the assignment of deuteriums to individual residues is typically not obtained, thereby limiting a detailed understanding of HX and the dynamics of protein structure. Here we use gas-phase fragmentation of peptic peptides by electron transfer dissociation (ETD) to measure...... the HX of individual amide linkages in the amyloidogenic protein beta2-microglobulin. A comparison of the deuterium levels of 60 individual backbone amides of beta2-microglobulin measured by HX-ETD-MS analysis to the corresponding values measured by NMR spectroscopy shows an excellent correlation...

  8. The dopaminergic system in patients with functional dyspepsia analysed by single photon emission computed tomography (SPECT) and an alpha-methyl-para-tyrosine (AMPT) challenge test

    Energy Technology Data Exchange (ETDEWEB)

    Braak, Breg; Klooker, Tamira K. [Academic Medical Center, Department of Gastroenterology and Hepatology, Amsterdam (Netherlands); Booij, Jan [Academic Medical Center, Department of Nuclear Medicine, Amsterdam (Netherlands); Wijngaard, Rene M.J. van den [Academic Medical Center, Tytgat Institute of Liver and Intestinal Research, Amsterdam (Netherlands); Boeckxstaens, Guy E.E. [Academic Medical Center, Department of Gastroenterology and Hepatology, Amsterdam (Netherlands); University Hospital Leuven, Catholic University Leuven, Department of Gastroenterology, Leuven (Belgium)

    2012-04-15

    Functional dyspepsia (FD) is a chronic condition characterized by upper abdominal symptoms without an identifiable cause. While the serotonergic system is thought to play a key role in the regulation of gut physiology, the role of the dopaminergic system, which is important in the regulation of visceral pain and stress, is under-studied. Therefore, this study investigated the dopaminergic system and its relationship with drinking capacity and symptoms in FD patients. In FD patients and healthy volunteers (HV) the dopaminergic system was investigated by in-vivo assessment of central dopamine D2 receptors (D2Rs) with [{sup 123}I]IBZM SPECT and by an acute, but reversible, dopamine depletion alpha-methyl-para-tyrosine (AMPT) challenge test. A nutrient drink test was performed to investigate the association between maximal ingested volume, evoked symptoms, and D2Rs. The HV subjects comprised 12 women and 8 men (mean age 31 {+-} 3 years), and the FD patients comprised 5 women and 3 men (mean age 39 {+-} 5 years). The FD patients had a lower left plus right average striatal binding potential (BP{sub NP}) for the caudate nucleus (p = 0.02), but not for putamen (p = 0.15), which in the FD patients was correlated with maximal ingested volume (r = 0.756, p = 0.03). The D2R BP{sub NP} in the putamen was correlated with nausea (r = 0.857, p = 0.01). The acute dopamine depletion test, however, failed to reveal differences in prolactin release between the FD patients and the HV subjects. These preliminary data suggest that chronic rather than acute alterations in the dopaminergic system may be involved in the pathogenesis of FD. Further studies are required to reproduce our novel findings and to evaluate to what extent the dopaminergic changes may be secondary to abnormalities in serotonergic pathways. (orig.)

  9. Cessation of tyrosine kinase inhibitors in patients with chronic-phase chronic myelogenous leukemia following durable complete molecular response: a single center facing the dilemma.

    Science.gov (United States)

    Iliakis, Theodoros; Papadopoulou, Vasiliki; Diamantopoulos, Panagiotis T; Panayiotidis, Panayiotis; Zervakis, Konstantinos; Giannakopoulou, Nefeli; Tilimidos, Gerassimos; Angelopoulou, Maria; Siakantaris, Marina P; Pangalis, Gerassimos; Mantzourani, Marina; Variami, Eleni; Viniou, Nora Athina

    2013-08-01

    Tyrosine kinase inhibitors (TKIs), namely imatinib mesylate (IM) and recently approved second-generation TKIs dasatinib and nilotinib, are currently considered the treatment of choice for newly-diagnosed chronic phase chronic myelogenous leukemia (CP-CML). Although treatment with TKIs has not yet been proven curative, it certainly accomplishes a sustained control of the disease in the vast majority of patients. More than a decade after the successful launching of IM in first-line treatment of CP-CML and the subsequent introduction of second-generation TKIs in this setting, the question of the possibility of TKI cessation in a specific subset of patients has emerged. Side-effects of TKIs, along with some patients' wish to abandon the drugs and the rising financial burden upon healthcare systems, have led to the dilemma whether IM can be safely withdrawn after achieving deep molecular remissions and which patients are suitable for this discontinuation. We examined the data of our patients with CML in search of potential canditates for cessation of TKI therapy and identified their characteristics. We also performed a thorough review of the relevant literature. Eight out of fifty patients were discriminated on grounds of sustained complete molecular response (CMR) exceeding 12 months, most of them with a low or intermediate Sokal score at diagnosis. The median interval from IM initiation to CMR was almost 2 years and the median duration of detected CMR reached 6.5 years. Based on the promising results of prospective clinical trials reporting successful cessation of treatment with TKIs on selected subgroups of patients, we decided to proceed to interruption of therapy in the specific subset of our patients and closely monitor their response.

  10. Single-Use Sensor Strips for Reliable Field Analysis of Gunshot Residue

    Science.gov (United States)

    2013-10-13

    determined in a single GSR assay. Wearable textile -based printed electrodes were also examined towards a ’Lab-on-Sleeve’ forensic field analysis. New...Example of the different cyclic square-wave stripping voltammetric signals obtained with “swiping” samples at a bare SPCE electrode . Score plot of the...A) The Forensic Finger exhibiting the three electrode surface screen-printed onto a flexible nitrile finger cot (bottom left inset), as well as a

  11. Simulation investigation of thermal phase transformation and residual stress in single pulse EDM of Ti-6Al-4V

    Science.gov (United States)

    Tang, Jiajing; Yang, Xiaodong

    2018-04-01

    The thermal phase transformation and residual stress are ineluctable in the electrical discharge machining (EDM) process, and they will greatly affect the working performances of the machined surface. This paper presents a simulation study on the thermal phase transformation and residual stress in single-pulse EDM of Ti-6Al-4V, which is the most popular titanium alloy in fields such as aircraft engine and some other leading industries. A multi-physics model including thermal, hydraulic, metallography and structural mechanics was developed. Based on the proposed model, the thickness and metallographic structure of the recast layer and heat affected layer (HAZ) were investigated. The distribution and characteristics of residual stress around the discharge crater were obtained. The recast layer and HAZ at the center of crater are found to be the thinnest, and their thicknesses gradually increase approaching the periphery of the crater. The recast layer undergoes a complete α‧ (martensitic) transformation, while the HAZ is mainly composed by the α  +  β  +  α‧ three-phase microstructure. Along the depth direction of crater, the Von Mises stress increases first and then decreases, reaching its maximal value near the interface of recast layer and HAZ. In the recast layer, both compressive stress component and tensile stress component are observed. ANOVA results showed that the influence of discharge current on maximal tensile stress is more significant than that of pulse duration, while the pulse duration has more significant influence on average thickness of the recast layer and the depth location of the maximal tensile stress. The works conducted in this study will help to evaluate the quality and integrity of EDMed surface, especially when the non-destructive testing is difficult to achieve.

  12. Pharmacokinetics and egg residues after oral administration of a single dose of meloxicam in domestic chickens (Gallus domesticus).

    Science.gov (United States)

    Souza, Marcy J; Bergman, Joan B; White, Molly S; Gordon, Kristen I; Gerhardt, Lillian E; Cox, Sherry K

    2017-08-01

    OBJECTIVE To determine the pharmacokinetics of meloxicam in domestic hens and duration and quantity of drug residues in their eggs following PO administration of a single dose (1 mg of meloxicam/kg). ANIMALS 8 healthy adult White Leghorn hens. PROCEDURES Hens were administered 1 mg of meloxicam/kg PO once. A blood sample was collected immediately before and at intervals up to 48 hours after drug administration. The hens' eggs were collected for 3 weeks after drug administration. Samples of the hens' plasma, egg whites (albumen), and egg yolks were analyzed by high-performance liquid chromatography. RESULTS The half-life, maximum concentration, and time to maximum concentration of meloxicam in plasma samples were 2.8 hours, 7.21 μg/mL, and 2 hours, respectively. Following meloxicam administration, the drug was not detected after 4 days in egg whites and after 8 days in egg yolks. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that meloxicam administered at a dose of 1 mg/kg PO in chickens appears to maintain plasma concentrations equivalent to those reported to be therapeutic for humans for 12 hours. The egg residue data may be used to aid establishment of appropriate drug withdrawal time recommendations.

  13. Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis

    International Nuclear Information System (INIS)

    Ceridono, Mara; Belleudi, Francesca; Ceccarelli, Simona; Torrisi, Maria Rosaria

    2005-01-01

    Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCγ binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCγ as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR

  14. Tyrosine phosphorylation of Rab7 by Src kinase.

    Science.gov (United States)

    Lin, Xiaosi; Zhang, Jiaming; Chen, Lingqiu; Chen, Yongjun; Xu, Xiaohui; Hong, Wanjin; Wang, Tuanlao

    2017-07-01

    The small molecular weight GTPase Rab7 is a key regulator for late endosomal/lysosomal membrane trafficking, it was known that Rab7 is phosphorylated, but the corresponding kinase and the functional regulation of Rab7 phosphorylation remain unclear. We provide evidence here that Rab7 is a substrate of Src kinase, and is tyrosine-phosphorylated by Src, withY183 residue of Rab7 being the optimal phosphorylation site for Src. Further investigations demonstrated that the tyrosine phosphorylation of Rab7 depends on the guanine nucleotide binding activity of Rab7 and the activity of Src kinase. The tyrosine phosphorylation of Rab7 is physiologically induced by EGF, and impairs the interaction of Rab7 with RILP, consequently inhibiting EGFR degradation and sustaining Akt signaling. These results suggest that the tyrosine phosphorylation of Rab7 may be involved in coordinating membrane trafficking and cell signaling. Copyright © 2017. Published by Elsevier Inc.

  15. Study of radiolytic effect on residual ethylene oxide in perfusion devices for single use by using gas chromatography

    International Nuclear Information System (INIS)

    Wang Mingsuo; Teng Weifang; Liu Keliang; Feng Jixin; Su Liaoyuan

    1993-01-01

    The efficiency of the 60 Co γ-rays irradiation induced elimination reaction of residual ethylene oxide (RETO) in the perfusion devices for single use conducted with RETO gas chromatography has been studied. The observations show: 1. the splinting reaction of RETO absorbed by the infusion sets after ethylene oxide (ETO) sterilization occurred accompanied with 4 kGy irradiation, therefore the relative elimination percentage (RELP) calculated from RETO is over 90%. 2. the optimum radiation conditions are as following irradiation dose 4 kGy, dose rate 8 Gy/min, irradiation temperature 20 degree C. 3. the RETO content within medical infusion sets at 2 weeks storing time after radiation was probably controlled under 2.0 ppm. 4. the radiation chemistry mechanism of the RETO elimination reaction is discussed in relation to the primary and secondary actions of elimination reaction

  16. Contaminant Release from Residual Waste in Closed Single-Shell Tanks and Other Waste Forms Associated with the Tanks

    International Nuclear Information System (INIS)

    Deutsch, William J.

    2008-01-01

    This chapter describes the release of contaminants from the various waste forms that are anticipated to be associated with closure of the single-shell tanks. These waste forms include residual sludge or saltcake that will remain in the tanks after waste retrieval. Other waste forms include engineered glass and cementitious materials as well as contaminated soil impacted by previous tank leaks. This chapter also describes laboratory testing to quantify contaminant release and how the release data are used in performance/risk assessments for the tank waste management units and the onsite waste disposal facilities. The chapter ends with a discussion of the surprises and lessons learned to date from the testing of waste materials and the development of contaminant release models

  17. A residual-based a posteriori error estimator for single-phase Darcy flow in fractured porous media

    KAUST Repository

    Chen, Huangxin

    2016-12-09

    In this paper we develop an a posteriori error estimator for a mixed finite element method for single-phase Darcy flow in a two-dimensional fractured porous media. The discrete fracture model is applied to model the fractures by one-dimensional fractures in a two-dimensional domain. We consider Raviart–Thomas mixed finite element method for the approximation of the coupled Darcy flows in the fractures and the surrounding porous media. We derive a robust residual-based a posteriori error estimator for the problem with non-intersecting fractures. The reliability and efficiency of the a posteriori error estimator are established for the error measured in an energy norm. Numerical results verifying the robustness of the proposed a posteriori error estimator are given. Moreover, our numerical results indicate that the a posteriori error estimator also works well for the problem with intersecting fractures.

  18. Electron transfer dissociation facilitates the measurement of deuterium incorporation into selectively labeled peptides with single residue resolution

    DEFF Research Database (Denmark)

    Zehl, Martin; Rand, Kasper D; Jensen, Ole N

    2008-01-01

    Mass spectrometry is routinely applied to measure the incorporation of deuterium into proteins and peptides. The exchange of labile, heteroatom-bound hydrogens is mainly used to probe the structural dynamics of proteins in solution, e.g., by hydrogen-exchange mass spectrometry, but also to study...... the gas-phase structure and fragmentation mechanisms of polypeptide ions. Despite considerable effort in recent years, there is no widely established mass spectrometric method to localize the incorporated deuterium to single amino acid residues, and typically, only the overall deuterium content...... of peptides or proteins is obtained. The main reason for this is that CID and related techniques induce intramolecular migration of hydrogens ("hydrogen scrambling") upon vibrational excitation of the even-electron precursor ion, thus randomizing the positional distribution of the incorporated deuterium atoms...

  19. Protein residue linking in a single spectrum for magic-angle spinning NMR assignment

    Energy Technology Data Exchange (ETDEWEB)

    Andreas, Loren B.; Stanek, Jan; Marchand, Tanguy Le; Bertarello, Andrea; Paepe, Diane Cala-De; Lalli, Daniela; Krejčíková, Magdaléna; Doyen, Camille; Öster, Carl [Université de Lyon, Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques (CNRS, ENS Lyon, UCB Lyon 1) (France); Knott, Benno; Wegner, Sebastian; Engelke, Frank [Bruker Biospin (Germany); Felli, Isabella C.; Pierattelli, Roberta [University of Florence, Department of Chemistry “Ugo Schiff“and Magnetic Resonance Center (CERM) (Italy); Dixon, Nicholas E. [University of Wollongong, School of Chemistry (Australia); Emsley, Lyndon; Herrmann, Torsten; Pintacuda, Guido, E-mail: guido.pintacuda@ens-lyon.fr [Université de Lyon, Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques (CNRS, ENS Lyon, UCB Lyon 1) (France)

    2015-07-15

    Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample. We find that amide signals have a low chance of ambiguous linkage, which is further improved by linking in both forward and backward directions. The spectra obtained are amenable to automated resonance assignment using general-purpose software such as UNIO-MATCH.

  20. A Single Coxsackievirus B2 Capsid Residue Controls Cytolysis and Apoptosis in Rhabdomyosarcoma Cells

    DEFF Research Database (Denmark)

    Gullberg, M.; Tolf, C.; Jonsson, N.

    2010-01-01

    genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells......) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse....... These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions....

  1. Tyrosine nitration affects thymidylate synthase properties.

    Science.gov (United States)

    Dąbrowska-Maś, Elżbieta; Frączyk, Tomasz; Ruman, Tomasz; Radziszewska, Karolina; Wilk, Piotr; Cieśla, Joanna; Zieliński, Zbigniew; Jurkiewicz, Agata; Gołos, Barbara; Wińska, Patrycja; Wałajtys-Rode, Elżbieta; Leś, Andrzej; Nizioł, Joanna; Jarmuła, Adam; Stefanowicz, Piotr; Szewczuk, Zbigniew; Rode, Wojciech

    2012-01-14

    Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.

  2. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

    DEFF Research Database (Denmark)

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei

    2015-01-01

    of residue, i.e. serine, threonine, tyrosine and cysteine, is also quite common. The phosphorylation of the ester type (phospho-serine/threonine/tyrosine) is more stable than the aspartate phosphorylation of TCSs. The kinases which catalyse these phosphorylation events (Hanks-type serine/threonine protein...

  3. A single gp120 residue can affect HIV-1 tropism in macaques.

    Directory of Open Access Journals (Sweden)

    Gregory Q Del Prete

    2017-09-01

    Full Text Available Species-dependent variation in proteins that aid or limit virus replication determines the ability of lentiviruses to jump between host species. Identifying and overcoming these differences facilitates the development of animal models for HIV-1, including models based on chimeric SIVs that express HIV-1 envelope (Env glycoproteins, (SHIVs and simian-tropic HIV-1 (stHIV strains. Here, we demonstrate that the inherently poor ability of most HIV-1 Env proteins to use macaque CD4 as a receptor is improved during adaptation by virus passage in macaques. We identify a single amino acid, A281, in HIV-1 Env that consistently changes during adaptation in macaques and affects the ability of HIV-1 Env to use macaque CD4. Importantly, mutations at A281 do not markedly affect HIV-1 Env neutralization properties. Our findings should facilitate the design of HIV-1 Env proteins for use in non-human primate models and thus expedite the development of clinically relevant reagents for testing interventions against HIV-1.

  4. 2D IR cross peaks reveal hydrogen-deuterium exchange with single residue specificity.

    Science.gov (United States)

    Dunkelberger, Emily B; Woys, Ann Marie; Zanni, Martin T

    2013-12-12

    A form of chemical exchange, hydrogen-deuterium exchange (HDX), has long been used as a method for studying the secondary and tertiary structure of peptides and proteins using mass spectrometry and NMR spectroscopy. Using two-dimensional infrared (2D IR) spectroscopy, we resolve cross peaks between the amide II band and a (13)C(18)O isotope-labeled amide I band, which we show measures HDX with site-specific resolution. By rapidly scanning 2D IR spectra using mid-IR pulse shaping, we monitor the kinetics of HDX exchange on-the-fly. For the antimicrobial peptide ovispirin bound to membrane bilayers, we find that the amide II peak decays with a biexponential with rate constants of 0.54 ± 0.02 and 0.12 ± 0.01 min(-1), which is a measure of the overall HDX in the peptide. The cross peaks between Ile-10-labeled ovispirin and the amide II mode, which specifically monitor HDX kinetics at Ile-10, decay with a single rate constant of 0.36 ± 0.1 min(-1). Comparing this exchange rate to theoretically determined exchange rates of Ile-10 for ovispirin in a solution random coil configuration, the exchange rate at Ile-10 is at least 100 times slower, consistent with the known α-helix structure of ovispirin in bilayers. Because backbone isotope labels produce only a very small shift of the amide II band, site-specific HDX cannot be measured with FTIR spectroscopy, which is why 2D IR spectroscopy is needed for these measurements.

  5. Crucial Roles of Single Residues in Binding Affinity, Specificity, and Promiscuity in the Cellulosomal Cohesin-Dockerin Interface*

    Science.gov (United States)

    Slutzki, Michal; Reshef, Dan; Barak, Yoav; Haimovitz, Rachel; Rotem-Bamberger, Shahar; Lamed, Raphael; Bayer, Edward A.; Schueler-Furman, Ora

    2015-01-01

    Interactions between cohesin and dockerin modules play a crucial role in the assembly of multienzyme cellulosome complexes. Although intraspecies cohesin and dockerin modules bind in general with high affinity but indiscriminately, cross-species binding is rare. Here, we combined ELISA-based experiments with Rosetta-based computational design to evaluate the contribution of distinct residues at the Clostridium thermocellum cohesin-dockerin interface to binding affinity, specificity, and promiscuity. We found that single mutations can show distinct and significant effects on binding affinity and specificity. In particular, mutations at cohesin position Asn37 show dramatic variability in their effect on dockerin binding affinity and specificity: the N37A mutant binds promiscuously both to cognate (C. thermocellum) as well as to non-cognate Clostridium cellulolyticum dockerin. N37L in turn switches binding specificity: compared with the wild-type C. thermocellum cohesin, this mutant shows significantly increased preference for C. cellulolyticum dockerin combined with strongly reduced binding to its cognate C. thermocellum dockerin. The observation that a single mutation can overcome the naturally observed specificity barrier provides insights into the evolutionary dynamics of this system that allows rapid modulation of binding specificity within a high affinity background. PMID:25833947

  6. [Tyrosine hydroxylase of the blood leukocytes].

    Science.gov (United States)

    Mineeva, M F

    1987-07-01

    Tyrosine hydroxylase activity has been established in blood plasma leucocytes of rat, cat and man. Tyrosine precursors and some nuclear erythroid cells. GFU-GM did hydroxylase activity in leucocytes shows the Km for tyrosine inhibited by high concentrations of L6 tyrosine (substrate inhibition), alpha-methyl-para-tyrosine dopamine. The kinetic properties of leucocyte tyrosine hydroxylase are qualitatively similar to the properties of brain tyrosine hydroxylase.

  7. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    Unknown

    5, in Luria Bertani broth ..... bacteria has been used by Larose et al (1993) to identify residues in the platelet derived growth factor ... GCC by EphB1 within bacterial cells suggests that GCC may be a substrate for the Eph family of tyrosine ...

  8. Risk factors for poor outcome of a single Epley maneuver and residual positional vertigo in patients with benign paroxysmal positional vertigo.

    Science.gov (United States)

    Sato, Go; Sekine, Kazunori; Matsuda, Kazunori; Takeda, Noriaki

    2013-11-01

    The findings suggest that in patients with posterior semicircular canal (PSCC) benign paroxysmal positional vertigo (P-BPPV), head trauma and prolonged bedrest, but not inner ear disease, are risk factors for poor outcome of a single Epley maneuver and persistent residual positional vertigo. We first examined the efficacy of a single Epley maneuver and then assessed the time course in remission of residual positional vertigo in patients with idiopathic P-BPPV and secondary P-BPPV. A total of 157 patients with idiopathic P-BPPV and 40 patients with secondary P-BPPV (secondary to head trauma in 8 patients, to prolonged bedrest in 14 patients, and to inner ear disease in 18 patients) were treated with a single Epley maneuver. The negative rates of the Dix-Hallpike test on day 7 after a single Epley maneuver in both patients with P-BPPV secondary to head trauma (25%) and those with prolonged bedrest (36%) were significantly lower than that (73%) in patients with idiopathic P-BPPV. Additionally, the remission of residual positional vertigo in the former groups of patients was significantly delayed in comparison with that of the latter group. However, there were no significant differences in the efficacy of a single Epley maneuver and persistent residual positional vertigo between idiopathic P-BPPV and P-BPPV secondary to inner ear disease.

  9. Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors

    Science.gov (United States)

    Thompson, Robert E.; Liu, Xuyu; Ripoll-Rozada, Jorge; Alonso-García, Noelia; Parker, Benjamin L.; Pereira, Pedro José Barbosa; Payne, Richard J.

    2017-09-01

    Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification—tyrosine sulfation—of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

  10. Evolution and function of the Mycoplasma hyopneumoniae peroxiredoxin, a 2-Cys-like enzyme with a single Cys residue.

    Science.gov (United States)

    Gonchoroski, Taylor; Virginio, Veridiana G; Thompson, Claudia E; Paes, Jéssica A; Machado, Cláudio X; Ferreira, Henrique B

    2017-04-01

    The minimal genome of the mollicute Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia, encodes a limited repertoire of antioxidant enzymes that include a single and atypical peroxiredoxin (MhPrx), whose evolution and function were studied here. MhPrx has only one catalytic cysteine, in contrast with some of its possible ancestors (2-Cys peroxiredoxins), which have two. Although it is more similar to 2-Cys orthologs, MhPrx can still function with a single peroxidatic cysteine (Cys P ), using non-thiolic electron donors to reduce it. Therefore, MhPrx could be a representative of a possible group of 2-Cys peroxiredoxins, which have lost the resolving cysteine (Cys R ) residue without losing their catalytic properties. To further investigate MhPrx evolution, we performed a comprehensive phylogenetic analysis in the context of several bacterial families, including Prxs belonging to Tpx and AhpE families, shedding light on the evolutionary history of Mycoplasmataceae Prxs and giving support to the hypothesis of a relatively recent loss of the Cys R within this family. Moreover, mutational analyses provided insights into MhPrx function with one, two, or without catalytic cysteines. While removal of the MhPrx putative Cys P caused complete activity loss, confirming its catalytic role, the introduction of a second cysteine in a site correspondent to that of the Cys R of a 2-Cys orthologue, as in the MhPrx supposed ancestral form, was compatible with enzyme activity. Overall, our phylogenetic and mutational studies support that MhPrx recently diverged from a 2-Cys Prx ancestor and pave the way for future studies addressing structural, functional, and evolutive aspects of peroxiredoxin subfamilies in Mollicutes and other bacteria.

  11. A Single Residue Mutation in the Gαq Subunit of the G Protein Complex Causes Blindness in Drosophila

    Directory of Open Access Journals (Sweden)

    Jinguo Cao

    2018-01-01

    Full Text Available Heterotrimeric G proteins play central roles in many signaling pathways, including the phototransduction cascade in animals. However, the degree of involvement of the G protein subunit Gαq is not clear since animals with previously reported strong loss-of-function mutations remain responsive to light stimuli. We recovered a new allele of Gαq in Drosophila that abolishes light response in a conventional electroretinogram assay, and reduces sensitivity in whole-cell recordings of dissociated cells by at least five orders of magnitude. In addition, mutant eyes demonstrate a rapid rate of degeneration in the presence of light. Our new allele is likely the strongest hypomorph described to date. Interestingly, the mutant protein is produced in the eyes but carries a single amino acid change of a conserved hydrophobic residue that has been assigned to the interface of interaction between Gαq and its downstream effector, PLC. Our study has thus uncovered possibly the first point mutation that specifically affects this interaction in vivo.

  12. Critical role for a single leucine residue in leukemia induction by E2A-PBX1.

    Science.gov (United States)

    Bayly, Richard; Murase, Takayuki; Hyndman, Brandy D; Savage, Rachel; Nurmohamed, Salima; Munro, Kim; Casselman, Richard; Smith, Steven P; LeBrun, David P

    2006-09-01

    In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.

  13. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  14. Cloning and Characterization of Secretory Tyrosine Phosphatases of Mycobacterium tuberculosis

    Science.gov (United States)

    Koul, Anil; Choidas, Axel; Treder, Martin; Tyagi, Anil K.; Drlica, Karl; Singh, Yogendra; Ullrich, Axel

    2000-01-01

    Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of Mycobacterium tuberculosis H37Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17.5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while mptpA is present in slow-growing mycobacterial species as well as fast-growing saprophytes, mptpB was restricted to members of the M. tuberculosis complex. These phosphatases were present in both whole-cell lysates and culture filtrates of M. tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis. PMID:10986245

  15. Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis.

    Science.gov (United States)

    Koul, A; Choidas, A; Treder, M; Tyagi, A K; Drlica, K; Singh, Y; Ullrich, A

    2000-10-01

    Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of Mycobacterium tuberculosis H(37)Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17. 5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while mptpA is present in slow-growing mycobacterial species as well as fast-growing saprophytes, mptpB was restricted to members of the M. tuberculosis complex. These phosphatases were present in both whole-cell lysates and culture filtrates of M. tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis.

  16. Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Shuhei Noda

    Full Text Available To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC, which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

  17. Undetected residual cement on standard or individualized all-ceramic abutments with cemented zirconia single crowns - a prospective randomized pilot trial.

    Science.gov (United States)

    Kappel, Stefanie; Eiffler, Constantin; Lorenzo-Bermejo, Justo; Stober, Thomas; Rammelsberg, Peter

    2016-09-01

    To assess the frequency and amount of residual cement after attachment of monolithic zirconia crowns to standard and individualized ceramic abutments. Twenty patients (mean age 58.9 years at inclusion in the study; 30% male) were randomized to receive either a standard or an individualized abutment on a bone-level implant. Monolithic zirconia single crowns were attached to abutments by use of permanent glass-ionomer cement. Crowns were fabricated with an occlusal hole to enable unscrewing of the abutment-crown complex. Immediately after cementation, superstructures were removed and both the peri-implant soft tissue and the abutment-crown complex were photographed in a standardized manner, to detect residual cement. Photographs were analyzed using Corel Photo Paint X7, and residual cement-to-total abutment and residual cement-to-peri-implant soft tissue area ratios were calculated. Residual cement was observed for 9 of 10 (90%) individualized abutments, compared with 4 of 10 (40%) standard abutments (OR = 13.5, P = 0.049). Twenty-seven of 40 (68%) individualized abutment surfaces were affected, compared with 12 of 40 (30%) standard abutment surfaces. The probability of observing residual cement was approximately five times higher for the surfaces of individualized abutments than for those of standard abutments (P = 0.005). The mean amount of sulcus surface covered by cement was 1.17% (SD 2.85) for the individualized abutments and 3.78% (SD 7.40) for the standard abutments. The position of the margin significantly affected the amount of residual cement. Both individualized and standard all-ceramic abutments result in small amounts of subgingival residual cement on abutment and sulcus surfaces. However, use of individualized abutments does not guarantee complete avoidance of undetected cement rests. Undetected residual cement might be avoided by use of all-ceramic abutments with visible abutment shoulders. © 2015 John Wiley & Sons A/S. Published by John Wiley

  18. Single particle analysis of ice crystal residuals observed in orographic wave clouds over Scandinavia during INTACC experiment

    Directory of Open Access Journals (Sweden)

    A. C. Targino

    2006-01-01

    Full Text Available Individual ice crystal residual particles collected over Scandinavia during the INTACC (INTeraction of Aerosol and Cold Clouds experiment in October 1999 were analyzed by Scanning Electron Microscopy (SEM equipped with Energy-Dispersive X-ray Analysis (EDX. Samples were collected onboard the British Met Office Hercules C-130 aircraft using a Counterflow Virtual Impactor (CVI. This study is based on six samples collected in orographic clouds. The main aim of this study is to characterize cloud residual elemental composition in conditions affected by different airmasses. In total 609 particles larger than 0.1 μm diameter were analyzed and their elemental composition and morphology were determined. Thereafter a hierarchical cluster analysis was performed on the signal detected with SEM-EDX in order to identify the major particle classes and their abundance. A cluster containing mineral dust, represented by aluminosilicates, Fe-rich and Si-rich particles, was the dominating class of particles, accounting for about 57.5% of the particles analyzed, followed by low-Z particles, 23.3% (presumably organic material and sea salt (6.7%. Sulfur was detected often across all groups, indicating ageing and in-cloud processing of particles. A detailed inspection of samples individually unveiled a relationship between ice crystal residual composition and airmass origin. Cloud residual samples from clean airmasses (that is, trajectories confined to the Atlantic and Arctic Oceans and/or with source altitude in the free troposphere were dominated primarily by low-Z and sea salt particles, while continentally-influenced airmasses (with trajectories that originated or traveled over continental areas and with source altitude in the continental boundary layer contained mainly mineral dust residuals. Comparison of residual composition for similar cloud ambient temperatures around –27°C revealed that supercooled clouds are more likely to persist in conditions where

  19. NET TG1: Residual stress assessment by neutron diffraction and finite element modeling on a single bead weld on a steel plate

    International Nuclear Information System (INIS)

    Ohms, C.; Wimpory, R.C.; Katsareas, D.E.; Youtsos, A.G.

    2009-01-01

    In the context of the efforts of Task Group 1 (TG1) of the European Network on Neutron Techniques Standardization for Structural Integrity (NET), the Joint Research Centre (JRC) participated in the experimental round robin campaign for residual stress analysis on a single weld bead on a steel plate. In parallel, the University of Patras (UP), in collaboration with the JRC, contributed to the corresponding numerical analysis round robin exercise. Neutron diffraction measurements were performed on a specimen, designated as A12, using the residual stress diffractometer at beam tube HB5 at the High Flux Reactor (HFR) in Petten, The Netherlands. Several line scans of strains and stresses were performed in accordance with an experimental protocol devised for this exercise and their results are presented in this paper. Two scans were made along the weld longitudinal direction beneath the upper surface of the plate, three were made in the weld transverse direction, and three through the thickness of the plate. The measured residual stresses are presented in detail. The measurements confirm that the stress distribution around this single weld bead on a plate is intrinsically 3-dimensional. The procedure followed by UP in the numerical assessment of the problem is presented in detail. The numerical results are presented in direct comparison to the JRC measurement data

  20. Impact of single annual treatment and four-monthly treatment for hookworm and Ascaris lumbricoides, and factors associated with residual infection among Kenyan school children.

    Science.gov (United States)

    Kepha, Stella; Mwandawiro, Charles S; Anderson, Roy M; Pullan, Rachel L; Nuwaha, Fred; Cano, Jorge; Njenga, Sammy M; Odiere, Maurice R; Allen, Elizabeth; Brooker, Simon J; Nikolay, Birgit

    2017-02-09

    School-based deworming is widely implemented in various countries to reduce the burden of soil-transmitted helminths (STHs), however, the frequency of drug administration varies in different settings. In this study, we compared the impact of a single annual treatment and 4-monthly treatment over a follow-up among Kenyan school children, and investigated the factors associated with residual infection. We performed a secondary analysis of data from a randomized trial investigating whether deworming for STHs alters risk of acquiring malaria. Children received either a single treatment or 4-monthly albendazole treatments were followed longitudinally from February 2014 to October 2014. The relative impact of treatment and factors associated with residual infections were investigated using mixed-effects regression models. Predisposition to infection was assessed based on Spearman's rank and Kendall's Tau correlation coefficients. In the 4-monthly treatment group, the proportion of children infected with hookworm decreased from 59.9 to 5.7%, while Ascaris lumbricoides infections dropped from 55.7 to 6.2%. In the single treatment group, hookworm infections decreased over the same time period from 58.7 to 18.3% (12.6% absolute difference in reduction, 95% CI: 8.9-16.3%), and A. lumbricoides from 56.7 to 23.3% (17.1% absolute difference in reduction, 95% CI: 13.1-21.1%). There was strong evidence for predisposition to both STH types. Residual hookworm infection among children on 4-monthly treatment were associated with male sex and baseline nutritional status, whereas A. lumbricoides infection was associated with individual and school-level infection at baseline, latrine cleanliness at schools. This study found that 4-monthly treatment w more effective than single annual treatment. Repeated treatments led to dramatic reductions in the intensities of STHs, but did not completely clear infections among school children in Kenya, a presumed reflection of reinfection in a setting

  1. Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding

    DEFF Research Database (Denmark)

    Sap, J; Jiang, Y P; Friedlander, D

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y...

  2. Insight into the interactions, residue snorkeling, and membrane disordering potency of a single antimicrobial peptide into different lipid bilayers.

    Directory of Open Access Journals (Sweden)

    Majid Jafari

    Full Text Available Pardaxin, with a bend-helix-bend-helix structure, is a membrane-active antimicrobial peptide that its membrane activity depends on the lipid bilayer composition. Herein, all-atom molecular dynamics (MD simulations were performed to provide further molecular insight into the interactions, structural dynamics, orientation behavior, and cationic residues snorkeling of pardaxin in the DMPC, DPPC, POPC, POPG, POPG/POPE (3:1, and POPG/POPE (1:3 lipid bilayers. The results showed that the C-terminal helix of the peptide was maintained in all six types of the model-bilayers and pardaxin was tilted into the DMPC, DPPC, and POPG/POPE mixed bilayers more than the POPC and POPG bilayers. As well as, the structure of zwitterionic membranes was more affected by the peptide than the anionic bilayers. Taken together, the study demonstrated that the cationic residues of pardaxin snorkeled toward the interface of lipid bilayers and all phenylalanine residues of the peptide played important roles in the peptide-membrane interactions. We hope that this work will provide a better understanding of the interactions of antimicrobial peptides with the membranes.

  3. Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants

    Energy Technology Data Exchange (ETDEWEB)

    Schenck, Craig A.; Holland, Cynthia K.; Schneider, Matthew R.; Men, Yusen; Lee, Soon Goo; Jez, Joseph M.; Maeda , Hiroshi A. (UW); (WU)

    2017-06-26

    L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrAp) or arogenate dehydrogenase (ADH, also known as TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that defines TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.

  4. Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants.

    Science.gov (United States)

    Schenck, Craig A; Holland, Cynthia K; Schneider, Matthew R; Men, Yusen; Lee, Soon Goo; Jez, Joseph M; Maeda, Hiroshi A

    2017-09-01

    L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrA p ) or arogenate dehydrogenase (ADH, also known as TyrA a ), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that defines TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.

  5. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    phosphorylation. Protein-tyrosine phosphorylation in bacteria is particular with respect to very low occupancy of phosphorylation sites in vivo; this has represented a major challenge for detection techniques. Only the recent breakthroughs in gel-free high resolution mass spectrometry allowed the systematic...... detection of phosphorylated tyrosines by phosphoprotomics studies in bacteria. Other pioneering studies conducted in recent years, such as the first structures of BY-kinases and biochemical and phyiological studies of new BY-kinase substrates significantly furthered our understanding of these enzymes...

  6. Food for thought: association between dietary tyrosine and cognitive performance in younger and older adults.

    Science.gov (United States)

    Kühn, Simone; Düzel, Sandra; Colzato, Lorenza; Norman, Kristina; Gallinat, Jürgen; Brandmaier, Andreas M; Lindenberger, Ulman; Widaman, Keith F

    2017-12-18

    The fact that tyrosine increases dopamine availability that, in turn, may enhance cognitive performance has led to numerous studies on healthy young participants taking tyrosine as a food supplement. As a result of this dietary intervention, participants show performance increases in working memory and executive functions. However, the potential association between habitual dietary tyrosine intake and cognitive performance has not been investigated to date. The present study aims at clarifying the association of episodic memory (EM), working memory (WM) and fluid intelligence (Gf), and tyrosine intake in younger and older adults. To this end, we acquired habitual tyrosine intake (food frequency questionnaire) from 1724 participants of the Berlin Aging Study II (1383 older adults, 341 younger adults) and modelled its relations to cognitive performance assessed in a broad battery of cognitive tasks using structural equation modeling. We observed a significant association between tyrosine intake and the latent factor capturing WM, Gf, and EM in the younger and the older sample. Due to partial strong factorial invariance between age groups for a confirmatory factor analysis on cognitive performance, we were able to compare the relationship between tyrosine and cognition between age groups and found no difference. Above and beyond previous studies on tyrosine food supplementation the present result extend this to a cross-sectional association between habitual tyrosine intake levels in daily nutrition and cognitive performance (WM, Gf, and EM). This corroborates nutritional recommendations that are thus far derived from single-dose administration studies.

  7. Tyrosine phosphorylation in human lymphomas

    NARCIS (Netherlands)

    Haralambieva, E; Jones, M.; Roncador, GM; Cerroni, L; Lamant, L; Ott, G; Rosenwald, A; Sherman, C; Thorner, P; Kusec, R; Wood, KM; Campo, E; Falini, B; Ramsay, A; Marafioti, T; Stein, H; Kluin, PM; Pulford, K; Mason, DY

    2002-01-01

    In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated

  8. 3D-TROSY-based backbone and ILV-methyl resonance assignments of a 319-residue homodimer from a single protein sample

    Energy Technology Data Exchange (ETDEWEB)

    Krejcirikova, Anna; Tugarinov, Vitali, E-mail: vitali@umd.edu [University of Maryland, Department of Chemistry and Biochemistry (United States)

    2012-10-15

    The feasibility of practically complete backbone and ILV methyl chemical shift assignments from a single [U-{sup 2}H,{sup 15}N,{sup 13}C; Ile{delta}1-{l_brace}{sup 13}CH{sub 3}{r_brace}; Leu,Val-{l_brace}{sup 13}CH{sub 3}/{sup 12}CD{sub 3}{r_brace}]-labeled protein sample of the truncated form of ligand-free Bst-Tyrosyl tRNA Synthetase (Bst-{Delta}YRS), a 319-residue predominantly helical homodimer, is established. Protonation of ILV residues at methyl positions does not appreciably detract from the quality of TROSY triple resonance data. The assignments are performed at 40 Degree-Sign C to improve the sensitivity of the measurements and alleviate the overlap of {sup 1}H-{sup 15}N correlations in the abundant {alpha}-helical segments of the protein. A number of auxiliary approaches are used to assist in the assignment process: (1) selection of {sup 1}H-{sup 15}N amide correlations of certain residue types (Ala, Thr/Ser) that simplifies 2D {sup 1}H-{sup 15}N TROSY spectra, (2) straightforward identification of ILV residue types from the methyl-detected 'out-and-back' HMCM(CG)CBCA experiment, and (3) strong sequential HN-HN NOE connectivities in the helical regions. The two subunits of Bst-YRS were predicted earlier to exist in two different conformations in the absence of ligands. In agreement with our earlier findings (Godoy-Ruiz in J Am Chem Soc 133:19578-195781, 2011), no evidence of dimer asymmetry has been observed in either amide- or methyl-detected experiments.

  9. Residual deposits (residual soil)

    International Nuclear Information System (INIS)

    Khasanov, A.Kh.

    1988-01-01

    Residual soil deposits is accumulation of new formate ore minerals on the earth surface, arise as a result of chemical decomposition of rocks. As is well known, at the hyper genes zone under the influence of different factors (water, carbonic acid, organic acids, oxygen, microorganism activity) passes chemical weathering of rocks. Residual soil deposits forming depends from complex of geologic and climatic factors and also from composition and physical and chemical properties of initial rocks

  10. Physicochemical characterisation of rVIII-SingleChain, a novel recombinant single-chain factor VIII.

    Science.gov (United States)

    Schmidbauer, Stefan; Witzel, Reinhild; Robbel, Lars; Sebastian, Petra; Grammel, Nicolas; Metzner, Hubert J; Schulte, Stefan

    2015-08-01

    rVIII-SingleChain is a novel recombinant single-chain factor VIII (FVIII) construct, comprising covalently bonded heavy and light chains. Post-translational modifications of FVIII affect physicochemical parameters, including hydrophobicity and charge. The most relevant post-translational modifications of FVIII products are N-glycosylation of asparagine residues and tyrosine sulphations. Here, the physicochemical properties, thrombin cleavage products and post-translational modifications of rVIII-SingleChain were investigated and compared against commercially available recombinant FVIII (rFVIII) products with a predominant two-chain structure (B-domain deleted rFVIII and full-length rFVIII). rVIII-SingleChain was expressed in Chinese hamster ovary (CHO) cells and purified by chromatographic methods. Physicochemical properties of rVIII-SingleChain or thrombin-derived cleavage products were assessed using size-exclusion chromatography, reversed-phase chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Analysis of the respective carbohydrate structures was performed after release of N-glycans by PNGase F followed by fluorescence labelling and high-performance liquid chromatography. Proteolysis by trypsin generated the corresponding peptides, which were analysed for sulphated tyrosines by liquid chromatography-electrospray ionisation time of flight-mass spectrometry. rVIII-SingleChain was shown to be of high purity and homogeneity, and presented a well-defined single-chain molecule with predominant β-sheet conformation. The coagulation-relevant thrombin-activation products of rVIII-SingleChain were comparable with those obtained by activation of commercially available rFVIII products. rVIII-SingleChain post-translational modifications were similar to other CHO cell-derived rFVIII products for N-glycopattern and tyrosine sulphation. In conclusion, rVIII-SingleChain is of high homogeneity and purity, and provides an expected cleavage pattern on

  11. Characterization of the hypertonically induced tyrosine phosphorylation of erythrocyte band 3.

    Science.gov (United States)

    Minetti, G; Seppi, C; Ciana, A; Balduini, C; Low, P S; Brovelli, A

    1998-01-01

    Human erythrocyte band 3 becomes rapidly phosphorylated on tyrosine residues after exposure of erythrocytes to hypertonic conditions. The driving force for this phosphorylation reaction seems to be a decrease in cell volume, because (1) changes in band 3 phosphotyrosine content accurately track repeated changes in erythrocyte volume through several cycles of swelling and shrinking; (2) the level of band 3 phosphorylation is independent of the osmolyte employed but strongly sensitive to the magnitude of cell shrinkage; and (3) exposure of erythrocytes to hypertonic buffers under conditions in which intracellular osmolarity increases but volume does not change (nystatin-treated cells) does not promote an increase in tyrosine phosphorylation. We hypothesize that shrinkage-induced tyrosine phosphorylation results either from an excluded-volume effect, stemming from an increase in intracellular crowding, or from changes in membrane curvature that accompany the decrease in cell volume. Although the net phosphorylation state of band 3 is shown to be due to a delicate balance between a constitutively active tyrosine phosphatase and constitutively active tyrosine kinase, the increase in phosphorylation during cell shrinkage was demonstrated to derive specifically from an activation of the latter. Further, a peculiar inhibition pattern of the volume-sensitive erythrocyte tyrosine kinase that matched that of p72syk, a tyrosine kinase already known to associate with band 3 in vivo, suggested the involvement of this kinase in the volume-dependent response. PMID:9761728

  12. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  13. Tracer experiment administering L-phenylalanine-U-14C and L-tyrosine-U-14C to the tissue slices of bamboo shoots

    International Nuclear Information System (INIS)

    Kozukue, E.; Mizuno, S.

    1987-01-01

    Uniformly 14 C-labeled L-phenylalanine and L-tyrosine were administered to tissue slices of both top and base sections of bamboo shoots. Alcohol soluble substances were extracted and then separated into organic acid, sugar and amino acid fractions by ion exchange chromatography. The homogentisic acid fraction among the organic acids was collected by high-performance liquid chromatography (HPLC) and its radioactivity was measured, while the alcohol insoluble residue was used for the analysis of lignin aldehyde by the method of alkaline nitrobenzene oxidation. 1. The two labeled amino acids were steadily incorporated into the tissues during incubation and rapidly converted to organic acid, sugar and alcohol insoluble residue, especially the latter. 2. On determining the amount of phenylalanine converted to tyrosine, it was found that this was extremely small. 3. The incorporation of phenylalanine-U- 14 C into alcohol insoluble residue was higher than that of tyrosine in both sections. 4. Although the conversion into lignin aldehyde from phenylalanine-U- 14 C was higher than that from tyrosine-U- 14 C, it was found that tyrosine incorporated into the shoots was converted to a remarkable extent for formation of lignin aldehyde. 5. The incorporation of phenylalanine and tyrosine into homogentisic acid was very low. From these results, we assume that the conversion of phenylalanine to tyrosine or of tyrosine to homogentisic acid is very small, and that a part of the high amount of tyrosine in the shoots may be used for formation of lignin

  14. A curvature-dependent membrane binding by tyrosine kinase Fer involves an intrinsically disordered region.

    Science.gov (United States)

    Yamamoto, Hikaru; Kondo, Akihiro; Itoh, Toshiki

    2018-01-01

    Tyrosine kinases are important enzymes that mediate signal transduction at the plasma membrane. While the significance of membrane localization of tyrosine kinases has been well evaluated, the role of membrane curvature in their regulation is unknown. Here, we demonstrate that an intrinsically disordered region in the tyrosine kinase Fer acts as a membrane curvature sensor that preferentially binds to highly curved membranes in vitro. This region forms an amphipathic α-helix upon interaction with curved membranes, aligning hydrophobic residues on one side of the helical structure. Further, the tyrosine kinase activity of Fer is significantly enhanced by the membrane in a manner dependent on curvature. We propose a model for the regulation of Fer based on an intramolecular interaction and the curvature-dependent membrane binding mediated by its intrinsically disordered region. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  16. Single-particle characterization of ice-nucleating particles and ice particles residuals sampled by three different techniques

    Science.gov (United States)

    Kandler, Konrad; Worringen, Annette; Benker, Nathalie; Dirsch, Thomas; Mertes, Stephan; Schenk, Ludwig; Kästner, Udo; Frank, Fabian; Nillius, Björn; Bundke, Ulrich; Rose, Diana; Curtius, Joachim; Kupiszewski, Piotr; Weingartner, Ernest; Vochezer, Paul; Schneider, Johannes; Schmidt, Susan; Weinbruch, Stephan; Ebert, Martin

    2015-04-01

    During January/February 2013, at the High Alpine Research Station Jungfraujoch a measurement campaign was carried out, which was centered on atmospheric ice-nucleating particles (INP) and ice particle residuals (IPR). Three different techniques for separation of INP and IPR from the non-ice-active particles are compared. The Ice Selective Inlet (ISI) and the Ice Counterflow Virtual Impactor (Ice-CVI) sample ice particles from mixed phase clouds and allow for the analysis of the residuals. The combination of the Fast Ice Nucleus Chamber (FINCH) and the Ice Nuclei Pumped Counterflow Virtual Impactor (IN-PCVI) provides ice-activating conditions to aerosol particles and extracts the activated INP for analysis. Collected particles were analyzed by scanning electron microscopy and energy-dispersive X-ray microanalysis to determine size, chemical composition and mixing state. All INP/IPR-separating techniques had considerable abundances (median 20 - 70 %) of instrumental contamination artifacts (ISI: Si-O spheres, probably calibration aerosol; Ice-CVI: Al-O particles; FINCH+IN-PCVI: steel particles). Also, potential sampling artifacts (e.g., pure soluble material) occurred with a median abundance of metal oxides were the major INP/IPR particle types separated by all three techniques. Soot was a minor contributor. Lead was detected in less than 10 % of the particles, of which the majority were internal mixtures with other particle types. Sea-salt and sulfates were identified by all three methods as INP/IPR. Most samples showed a maximum of the INP/IPR size distribution at 400 nm geometric diameter. In a few cases, a second super-micron maximum was identified. Soot/carbonaceous material and metal oxides were present mainly in the submicron range. ISI and FINCH yielded silicates and Ca-rich particles mainly with diameters above 1 µm, while the Ice-CVI also separated many submicron IPR. As strictly parallel sampling could not be performed, a part of the discrepancies between

  17. Chimeras Reveal a Single Lipid-Interface Residue that Controls MscL Channel Kinetics as well as Mechanosensitivity

    Directory of Open Access Journals (Sweden)

    Li-Min Yang

    2013-02-01

    Full Text Available MscL, the highly conserved bacterial mechanosensitive channel of large conductance, serves as an osmotic “emergency release valve,” is among the best-studied mechanosensors, and is a paradigm of how a channel senses and responds to membrane tension. Although all homologs tested thus far encode channel activity, many show functional differences. We tested Escherichia coli and Staphylococcus aureus chimeras and found that the periplasmic region of the protein, particularly E. coli I49 and the equivalent S. aureus F47 at the periplasmic lipid-aqueous interface of the first transmembrane domain, drastically influences both the open dwell time and the threshold of channel opening. One mutant shows a severe hysteresis, confirming the importance of this residue in determining the energy barriers for channel gating. We propose that this site acts similarly to a spring for a clasp knife, adjusting the resistance for obtaining and stabilizing an open or closed channel structure.

  18. Dynamics of allostery in hemoglobin: roles of the penultimate tyrosine H bonds.

    Science.gov (United States)

    Kneipp, Janina; Balakrishnan, Gurusamy; Chen, Ruopian; Shen, Tong-Jian; Sahu, Sarata C; Ho, Nancy T; Giovannelli, Janel L; Simplaceanu, Virgil; Ho, Chien; Spiro, Thomas G

    2006-02-17

    The tyrosine residues adjacent to the C termini of the hemoglobin (Hb) subunits, alphaY140 and betaY145, are expected to play important structural roles, because the C termini are the loci of T-state quaternary salt-bridges, and because the tyrosine side-chains bridge the H and F helices via H bonds to the alphaV93 and betaV98 carbonyl groups. These roles have been investigated via measurements of oxygen binding, (1)H NMR spectra, resonance Raman (RR) spectra, and time-resolved resonance Raman (TR(3)) spectra on site mutants in which the Hcdots, three dots, centeredF H bonds are eliminated by replacing the tyrosine residues with phenylalanine. The TR(3) spectra confirm the hypothesis, based on TR(3) studies of wild-type Hb, that the Hcdots, three dots, centeredF H bonds break and then re-form during the sub-microsecond phase of the R-T quaternary transition. The TR(3) spectra support the inference from other mutational studies that the alphabeta dimers act as single dynamic units in this early phase, motions of the E and F helices being coupled tightly across the dimer interface. Formation of T quaternary contacts occurs at about the same rate in the mutants as in HbA. However, these contacts are weakened substantially by the Y/F substitutions. Equilibrium perturbations are apparent also, especially for the alpha-subunits, in which relaxation of the Fe-His bond, strengthening of the Acdots, three dots, centeredE interhelical H bond, and weakening of the "switch" quaternary contact in deoxyHb are all apparent. Structural effects are less marked for the beta-chain Y/F replacement, but the Bohr effect is reduced by 25%, indicating that the salt-bridge and H bond interactions of the adjacent C terminus are loosened. The alpha-chain replacement reduces the Bohr effect much more, consistent with the global perturbations detected by the structure probes.

  19. Single-particle characterization of ice-nucleating particles and ice particle residuals sampled by three different techniques

    Science.gov (United States)

    Worringen, A.; Kandler, K.; Benker, N.; Dirsch, T.; Mertes, S.; Schenk, L.; Kästner, U.; Frank, F.; Nillius, B.; Bundke, U.; Rose, D.; Curtius, J.; Kupiszewski, P.; Weingartner, E.; Vochezer, P.; Schneider, J.; Schmidt, S.; Weinbruch, S.; Ebert, M.

    2015-04-01

    In the present work, three different techniques to separate ice-nucleating particles (INPs) as well as ice particle residuals (IPRs) from non-ice-active particles are compared. The Ice Selective Inlet (ISI) and the Ice Counterflow Virtual Impactor (Ice-CVI) sample ice particles from mixed-phase clouds and allow after evaporation in the instrument for the analysis of the residuals. The Fast Ice Nucleus Chamber (FINCH) coupled with the Ice Nuclei Pumped Counterflow Virtual Impactor (IN-PCVI) provides ice-activating conditions to aerosol particles and extracts the activated particles for analysis. The instruments were run during a joint field campaign which took place in January and February 2013 at the High Alpine Research Station Jungfraujoch (Switzerland). INPs and IPRs were analyzed offline by scanning electron microscopy and energy-dispersive X-ray microanalysis to determine their size, chemical composition and mixing state. Online analysis of the size and chemical composition of INP activated in FINCH was performed by laser ablation mass spectrometry. With all three INP/IPR separation techniques high abundances (median 20-70%) of instrumental contamination artifacts were observed (ISI: Si-O spheres, probably calibration aerosol; Ice-CVI: Al-O particles; FINCH + IN-PCVI: steel particles). After removal of the instrumental contamination particles, silicates, Ca-rich particles, carbonaceous material and metal oxides were the major INP/IPR particle types obtained by all three techniques. In addition, considerable amounts (median abundance mostly a few percent) of soluble material (e.g., sea salt, sulfates) were observed. As these soluble particles are often not expected to act as INP/IPR, we consider them as potential measurement artifacts. Minor types of INP/IPR include soot and Pb-bearing particles. The Pb-bearing particles are mainly present as an internal mixture with other particle types. Most samples showed a maximum of the INP/IPR size distribution at 200

  20. Analysis of tomato matrix effect in pesticide residue quantification through QuEChERS and single quadrupole GC/MS

    Directory of Open Access Journals (Sweden)

    Ana M Domínguez

    2014-04-01

    Full Text Available The detection of pesticide in tomato (Solanum lycopersicum L. matrix using Gas Chromatography hyphenated to Mass Spectrometry detector (GC/MS can affect the sensitivity of the analysis by enhancement or suppression of their chromatography response, the percentages of recoveries and leading to errors in the quantification of the pesticides. In this study, the matrix effect was investigated using nine pesticides, and "Quick, Easy, Cheap, Effective, Rugged and Safe" (QuEChERS-GC/MS analytical technique was validated for pesticides multiresidue analysis. The matrix effect was determined using not statistical and statistical procedures including ANOVA, with similar results. Strong negative matrix effect was found for the pesticides trifluralin, 4,4'-DDT, and permethrin, resulting in the increment of the chromatogram background and a decrease in their detection responses. Contrary, an enhancement induced by the matrix presence was obtained for carbaryl and azinphos methyl, showing a positive medium matrix effect. While, dimethoate, simazine, 4,4'-DDE, and iprodione did not exhibit matrix effect. The detection limits (LOD obtained, varied from 0.003 to 0.1 mg kg-1. Reproducibility was less than 20% for each pesticide. Recoveries were found to be between 71% and 121%, except for dimethoate, carbaryl, and azinphos methyl which reached values lower than 70%. Recoveries relative standard deviations were less than 22%. QuEChERS-GC/MS technique was used for evaluation of fresh commercial tomatoes samples, detecting carbaryl in all of them, but in concentration levels lower than the maximum residue limits according to regulations of Codex.

  1. Detecting Pesticide Residue by Using Modulating Temperature Over a Single SnO2-Based Gas Sensor

    Directory of Open Access Journals (Sweden)

    Zengliang Yu

    2003-09-01

    Full Text Available A new rapid detecting method (called dynamic measurements was reported to detect and distinguish the presence of two pesticide gases in the ambient atmosphere. The method employed only a single SnO2-based gas sensor in a rectangular temperature mode to perform the qualitative analysis of a binary gas mixture (acephate and trichlorphon in air. Polar plots was used for quantitative analysis which the feature extraction was performed by FFT. Experimental results showed that high selectivity of the sensor achieved in the range of 250~3000C and modulating frequency 20mHz, one can easily observe the qualitative difference among the response to pure acephate and trichlorphon gases of the same concentration and to the mixture, and the concentration of pesticide gases can be obtained based on the changes of polar plots.

  2. In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Musumeci, Lucia; Tautz, Lutz

    2005-01-01

    Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains...

  3. Multiple forms of the human tyrosine phosphatase RPTP alpha. Isozymes and differences in glycosylation

    DEFF Research Database (Denmark)

    Daum, G; Regenass, S; Sap, J

    1994-01-01

    Among all the receptor-linked protein-tyrosine-phosphatase RPTP alpha clones described from mammalian tissues, one differed in that it encoded a 9-amino-acid insert 3 residues upstream from the transmembrane segment (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R. Ravera, M., Ricca, G...

  4. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

    2005-01-01

    of protein-tyrosine phosphorylation. We discuss the approaches currently used to chart this network: ranging from studies of substrate specifi city and the physiological role of tyrosine phosphorylation of individual enzymes to the global approaches at the level of systems biology....... on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

  5. Protein tyrosine nitration in plants: Present knowledge, computational prediction and future perspectives.

    Science.gov (United States)

    Kolbert, Zsuzsanna; Feigl, Gábor; Bordé, Ádám; Molnár, Árpád; Erdei, László

    2017-04-01

    Nitric oxide (NO) and related molecules (reactive nitrogen species) regulate diverse physiological processes mainly through posttranslational modifications such as protein tyrosine nitration (PTN). PTN is a covalent and specific modification of tyrosine (Tyr) residues resulting in altered protein structure and function. In the last decade, great efforts have been made to reveal candidate proteins, target Tyr residues and functional consequences of nitration in plants. This review intends to evaluate the accumulated knowledge about the biochemical mechanism, the structural and functional consequences and the selectivity of plants' protein nitration and also about the decomposition or conversion of nitrated proteins. At the same time, this review emphasizes yet unanswered or uncertain questions such as the reversibility/irreversibility of tyrosine nitration, the involvement of proteasomes in the removal of nitrated proteins or the effect of nitration on Tyr phosphorylation. The different NO producing systems of algae and higher plants raise the possibility of diversely regulated protein nitration. Therefore studying PTN from an evolutionary point of view would enrich our present understanding with novel aspects. Plant proteomic research can be promoted by the application of computational prediction tools such as GPS-YNO 2 and iNitro-Tyr software. Using the reference Arabidopsis proteome, Authors performed in silico analysis of tyrosine nitration in order to characterize plant tyrosine nitroproteome. Nevertheless, based on the common results of the present prediction and previous experiments the most likely nitrated proteins were selected thus recommending candidates for detailed future research. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  6. The oncogenic tyrosine kinase Lyn impairs the pro-apoptotic function of Bim.

    Science.gov (United States)

    Aira, Lazaro E; Villa, Elodie; Colosetti, Pascal; Gamas, Parvati; Signetti, Laurie; Obba, Sandrine; Proics, Emma; Gautier, Fabien; Bailly-Maitre, Béatrice; Jacquel, Arnaud; Robert, Guillaume; Luciano, Frédéric; Juin, Philippe P; Ricci, Jean-Ehrland; Auberger, Patrick; Marchetti, Sandrine

    2018-02-02

    Phosphorylation of Ser/Thr residues is a well-established modulating mechanism of the pro-apoptotic function of the BH3-only protein Bim. However, nothing is known about the putative tyrosine phosphorylation of this Bcl-2 family member and its potential impact on Bim function and subsequent Bax/Bak-mediated cytochrome c release and apoptosis. As we have previously shown that the tyrosine kinase Lyn could behave as an anti-apoptotic molecule, we investigated whether this Src family member could directly regulate the pro-apoptotic function of Bim. In the present study, we show that Bim is phosphorylated onto tyrosine residues 92 and 161 by Lyn, which results in an inhibition of its pro-apoptotic function. Mechanistically, we show that Lyn-dependent tyrosine phosphorylation of Bim increases its interaction with anti-apoptotic members such as Bcl-xL, therefore limiting mitochondrial outer membrane permeabilization and subsequent apoptosis. Collectively, our data uncover one molecular mechanism through which the oncogenic tyrosine kinase Lyn negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or the chemotherapeutic resistance of cancer cells.

  7. Tyrosine dephosphorylation regulates AMPAR internalisation in mGluR-LTD.

    Science.gov (United States)

    Gladding, Clare M; Collett, Valerie J; Jia, Zhengping; Bashir, Zafar I; Collingridge, Graham L; Molnár, Elek

    2009-02-01

    Long-term depression (LTD) can be induced at hippocampal CA1 synapses by activation of either NMDA receptors (NMDARs) or group I metabotropic glutamate receptors (mGluRs), using their selective agonists NMDA and (RS)-3,5-dihydroxyphenylglycine (DHPG), respectively. Recent studies revealed that DHPG-LTD is dependent on activation of postsynaptic protein tyrosine phosphatases (PTPs), which transiently dephosphorylate tyrosine residues in AMPA receptors (AMPARs). Here we show that while both endogenous GluR2 and GluR3 AMPAR subunits are tyrosine phosphorylated at basal activity, only GluR2 is dephosphorylated in DHPG-LTD. The tyrosine dephosphorylation of GluR2 does not occur in NMDA-LTD. Conversely, while NMDA-LTD is associated with the dephosphorylation of GluR1-serine-845, DHPG-LTD does not alter the phosphorylation of this site. The increased AMPAR endocytosis in DHPG-LTD is PTP-dependent and involves tyrosine dephosphorylation of cell surface AMPARs. Together, these results indicate that the subunit selective tyrosine dephosphorylation of surface GluR2 regulates AMPAR internalisation in DHPG-LTD but not in NMDA-LTD in the hippocampus.

  8. Nonreceptor Tyrosine Kinases in Prostate

    Directory of Open Access Journals (Sweden)

    Cancer Yu-Ming Chang

    2007-02-01

    Full Text Available BACKGROUND: Carcinoma of the prostate (CaP is the most commonly diagnosed cancer in men in the United States. Signal transduction molecules such as tyrosine kinases play important roles in CaP. Src, a nonreceptor tyrosine kinase (NRTK and the first proto-oncogene discovered is shown to participate in processes such as cell proliferation and migration in CaP. Underscoring NRTK's and, specifically, Src's importance in cancer is the recent approval by the US Food and Drug Administration of dasatinib, the first commercial Src inhibitor for clinical use in chronic myelogenous leukemia (CML. In this review we will focus on NRTKs and their roles in the biology of CaP. MATERIALS AND METHODS: Publicly available literature from PubMed regarding the topic of members of NRTKs in CaP was searched and reviewed. RESULTS: Src, FAK, JaK1/2, and ETK are involved in processes indispensable to the biology of CaP: cell growth, migration, invasion, angiogenesis, and apoptosis. CONCLUSIONS: Src emerges as a common signaling and regulatory molecule in multiple biological processes in CaP. Src's relative importance in particular stages of CaP, however, required further definition. Continued investigation of NRTKs will increase our understanding of their biological function and potential role as new therapeutic targets.

  9. An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

    Science.gov (United States)

    Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

    2016-01-01

    Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells. PMID:27028870

  10. Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung

    Directory of Open Access Journals (Sweden)

    Sabrina V. Martini

    2016-07-01

    Full Text Available Background/Aims: Adeno-associated virus (AAV vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Eighteen C57BL/6 mice were randomly assigned into three groups: (1 a control group (CTRL animals underwent intratracheal (i.t. instillation of saline, (2 the wild-type AAV9 group (WT-AAV9, 1010 vg, and (3 the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg, which received (i.t. self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP. Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. Results: No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30% compared with their wild-type counterparts, without eliciting an inflammatory response. Conclusion: Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.

  11. Eficiência dos testes COPAN (Microplate e Single na detecção de resíduos de antimicrobianos no leite Efficacy of COPAN (Microplate and Single kits in detection of antimicrobials residues in milk

    Directory of Open Access Journals (Sweden)

    C.G.M.S.C. Tenório

    2009-04-01

    Full Text Available Avaliou-se a eficiência dos testes microbiológicos COPAN (Microplate e Single para detecção de resíduos de 13 antimicrobianos inoculados em leite isento de resíduos. Foram utilizadas quatro concentrações, sendo a primeira equivalente à metade do limite de detecção declarado pelo fabricante (C1; a segunda equivalente ao limite máximo de resíduos (LMR estabelecido pela legislação brasileira (C2; a terceira equivalente ao limite de detecção declarado pelo fabricante (C3; e a quarta equivalente ao dobro do limite de detecção declarado pelo fabricante (C4. Os testes apresentaram 100% de sensibilidade na detecção de amoxicilina, ampicilina, cloxacilina, penicilina, tilosina, sulfadiazina, sulfametoxazol, e 96,7% na detecção de oxacilina para as concentrações informadas pelo fabricante como limite de detecção. Em relação às concentrações estabelecidas pela legislação brasileira, verificou-se que os testes foram capazes de detectá-las para a maioria dos antimicrobianos avaliados. Os testes não se mostraram eficientes na detecção das concentrações estabelecidas pela legislação para eritromicina, oxitetraciclina, tetraciclina, trimetoprim e gentamicina (Microplate.The efficacy of the microbiological kits COPAN (Microplate and Single for detection of antimicrobials residues was tested. Thirteen drugs were inoculated into milk without residues at four concentration levels: C1 - half of the detection threshold declared by the manufacturer, C2 - the maximum residues threshold established by the Brazilian legislation, C3 - the detection threshold declared by the manufacturer, and C4 - two fold the detection threshold declared by the manufacturer. The tests showed 100% efficient in detection of amoxicillin, cloxacillin, penicillin, tilosin, sulfadiazine, and sulfa methoxazol; and 96.7% for oxacillin considering the concentration stated by the manufacturer as threshold level. Regarding the concentrations established by

  12. Dietary Tyrosine Benefits Cognitive and Psychomotor Performance During Body Cooling

    National Research Council Canada - National Science Library

    O'Brien, Catherine; Mahoney, Caroline; Tharion, William J; Sils, Ingrid V; Castellani, John W

    2007-01-01

    Supplemental tyrosine is effective at limiting cold-induced decreases in working memory, presumably by augmenting brain catecholamine levels, since tyrosine is a precursor for catecholamine synthesis...

  13. Coumarins as turn on/off fluorescent probes for detection of residual acetone in cosmetics following headspace single-drop microextraction.

    Science.gov (United States)

    Cabaleiro, N; de la Calle, I; Bendicho, C; Lavilla, I

    2014-11-01

    In this work, a new method based on headspace-single drop microextraction for the determination of residual acetone in cosmetics by microfluorospectrometry is proposed. Acetone causes fluorescence changes in a 2.5 µL-ethanolic drop (40% v/v) containing 3.10(-4) mol L(-1) 7-hydroxy-4-methylcoumarin ('turn off') or 6.10(-6) mol L(-1) 7-diethylamino-4-methylcoumarin ('turn on'). Polarity and ability to form hydrogen bonds of short chain alcohols (polar protic solvents) were crucial in order to observe these changes in the presence of acetone (polar aprotic solvent). Parameters related with the HS-SDME procedure were studied, namely headspace volume, composition, volume and temperature of drop, microextraction time, stirring rate, mass and temperature of sample, as well as the effect of potential interferents (alcohols and fragrances). The high volatility of acetone allows its extraction from an untreated cosmetic sample within 3 min. A detection limit of 0.26 µg g(-1) and repeatability, expressed as relative standard deviation, around 5% were reached. Accuracy of the proposed methodology was evaluated by means of recovery studies. The method was successfully used to analyze different cosmetics. Simplicity and high sample throughput can be highlighted. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Internet-Based Cognitive Behavioral Therapy for Residual Symptoms in Bipolar Disorder Type II: A Single-Subject Design Pilot Study.

    Science.gov (United States)

    Holländare, Fredrik; Eriksson, Annsofi; Lövgren, Lisa; Humble, Mats B; Boersma, Katja

    2015-04-23

    Bipolar disorder is a chronic condition with recurring episodes that often lead to suffering, decreased functioning, and sick leave. Pharmacotherapy in the form of mood stabilizers is widely available, but does not eliminate the risk of a new depressive or (hypo)manic episode. One way to reduce the risk of future episodes is to combine pharmacological treatment with individual or group psychological interventions. However, access to such interventions is often limited due to a shortage of trained therapists. In unipolar depression there is now robust evidence of the effectiveness of Internet-based psychological interventions, usually comprising psychoeducation and cognitive behavioral therapy (CBT). Internet-based interventions for persons suffering from bipolar disorder could increase access to psychological treatment. The aim of this study was to investigate the feasibility of an Internet-based intervention, as well as its effect on residual depressive symptoms in persons diagnosed with bipolar disorder type II (BP-II). The most important outcomes were depressive symptoms, treatment adherence, and whether the patient perceived the intervention as helpful. A total of 7 patients diagnosed with bipolar disorder type II at a Swedish psychiatric outpatient clinic were offered the opportunity to participate. Of the 7 patients, 3 (43%) dropped out before treatment began, and 4 (57%) were treated by means of an online, Internet-based intervention based on CBT (iCBT). The intervention was primarily aimed at psychoeducation, treatment of residual depressive symptoms, emotion regulation, and improved sleep. All patients had ongoing pharmacological treatment at recruitment and established contact with a psychiatrist. The duration of BP-II among the treated patients was between 6 and 31 years. A single-subject design was used and the results of the 4 participating patients were presented individually. Initiating treatment was perceived as too demanding under current life

  15. Tyrosine kinase fusion genes in pediatric BCR-ABL1-like acute lymphoblastic leukemia.

    Science.gov (United States)

    Boer, Judith M; Steeghs, Elisabeth M P; Marchante, João R M; Boeree, Aurélie; Beaudoin, James J; Beverloo, H Berna; Kuiper, Roland P; Escherich, Gabriele; van der Velden, Vincent H J; van der Schoot, C Ellen; de Groot-Kruseman, Hester A; Pieters, Rob; den Boer, Monique L

    2017-01-17

    Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (pfusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome.

  16. Nicotinic stimulation of catecholamine synthesis and tyrosine hydroxylase phosphorylation in cervine adrenal medullary chromaffin cells.

    Science.gov (United States)

    Knowles, P J; Douglas, S A; Bunn, S J

    2011-03-01

    The synthesis and secretion of catecholamines by the adrenal medulla is of major importance in the stress response. Tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, has been extensively studied in adrenal medullary chromaffin cells from a number of species. Cervine chromaffin cells are of interest because the deer is known to be a relatively stress-prone reactive species. We report the first characterisation of tyrosine hydroxylase regulation in cervine chromaffin cells. Nicotinic receptor activation resulted in a time- and concentration-dependent increase in catecholamine synthesis, which was significantly reduced by the extracellular signal-regulated kinase (ERK)1/2 signalling pathway inhibitor PD98059 and the calcium/calmodulin protein kinase II inhibitor KN-93, but not by H89 or bisindolylmaleimide I, inhibitors of protein kinase A and C, respectively. Nicotinic stimulation also increased the phosphorylation of ERK1/2 and tyrosine hydroxylase. This latter response occurred on serine residues 19, 31 and 40 of the enzyme. The nicotinic-induced phosphorylation of ERK1/2 and serine 31 of tyrosine hydroxylase was suppressed by PD98059 but not bisindolylmaleimide I. These data indicate that nicotinic stimulation of tyrosine hydroxylase involves the phosphorylation of serine 31 via an ERK1/2-dependent, protein kinase C-independent pathway. Protein kinase C activation by phorbol 12-myristate 13-acetate also caused an ERK1/2-dependent increase in the serine 31 phosphorylation of tyrosine hydroxylase but, in contrast to the nicotinic response, was not accompanied by an increase in enzyme activity. Thus, ERK1/2-mediated serine 31 phosphorylation of tyrosine hydroxylase appears necessary but not sufficient for nicotinic activation of catecholamine synthesis in cervine chromaffin cells. These data present potentially important similarities and differences between the regulation of catecholamine synthesis in cervine and the more widely studied

  17. Environmental Stress Testing of the Single Sample Cylinder: A Proven Consensus Standard for Internal Gas Analysis (IGA) or Residual Gas Analysis (RGA)

    Science.gov (United States)

    Schuessler, Philipp WH

    2010-01-01

    In August 2008, Schuessler Consulting was contracted by NASA GSFC in support of the NASA Electronic Parts and Packaging (NEPP) program to perform two separate studies on moisture laden air in a stainless steel cylinder that had been designed to become a consensus standard for Test Method 1018. This Test Method was originally released for hybrids under Mil. Std. 883 but was quickly utilized on other microelectronic devices under the auspice of Mil. Std. 750. The cylinder had subsequently been fabricated for the 750 community. It was back-filled with moist air and subsequently analyzed over a period of time under a previous NASA contract. It had been shown that moisture in the 4000 - 5000 ppm range could be analyzed rather precisely with a mass spectrometer, commonly referred to as a Residual Gas Analyzer (RGA). The scope of this study was to ascertain if the composition and precision varied as a function of thermal shock at sub-zero temperatures and whether there was consensus when the standard was submitted to other RGA units. It was demonstrated and published that the consensus standard would yield precise RGA data for moisture within +/- 1% when optimized for a given RGA unit. It has been subsequently shown in this study at Oneida Research Services, that sub-zero storage did not affect that precision when a well-defined protocol for the analysis was followed. The consensus standard was taken to a second facility for analysis where it was found that moisture adsorption on the transfer lines caused precision to drop to +/- 12%. The Single Sample Cylinder (SSC) is a one liter stainless steel cylinder with associated sampling valves and has considerable weight and volume. But this considerable size allows for approximately 300 gas samples of the same composition to be delivered to any RGA unit. Lastly, a smaller cylinder, approximately 75 cc, of a second consensus standard was fabricated and tested with a different mix of fixed gases where moisture was kept in the

  18. Online single particle analysis of ice particle residuals from mountain-top mixed-phase clouds using laboratory derived particle type assignment

    Science.gov (United States)

    Schmidt, Susan; Schneider, Johannes; Klimach, Thomas; Mertes, Stephan; Schenk, Ludwig Paul; Kupiszewski, Piotr; Curtius, Joachim; Borrmann, Stephan

    2017-01-01

    In situ single particle analysis of ice particle residuals (IPRs) and out-of-cloud aerosol particles was conducted by means of laser ablation mass spectrometry during the intensive INUIT-JFJ/CLACE campaign at the high alpine research station Jungfraujoch (3580 m a.s.l.) in January-February 2013. During the 4-week campaign more than 70 000 out-of-cloud aerosol particles and 595 IPRs were analyzed covering a particle size diameter range from 100 nm to 3 µm. The IPRs were sampled during 273 h while the station was covered by mixed-phase clouds at ambient temperatures between -27 and -6 °C. The identification of particle types is based on laboratory studies of different types of biological, mineral and anthropogenic aerosol particles. The outcome of these laboratory studies was characteristic marker peaks for each investigated particle type. These marker peaks were applied to the field data. In the sampled IPRs we identified a larger number fraction of primary aerosol particles, like soil dust (13 ± 5 %) and minerals (11 ± 5 %), in comparison to out-of-cloud aerosol particles (2.4 ± 0.4 and 0.4 ± 0.1 %, respectively). Additionally, anthropogenic aerosol particles, such as particles from industrial emissions and lead-containing particles, were found to be more abundant in the IPRs than in the out-of-cloud aerosol. In the out-of-cloud aerosol we identified a large fraction of aged particles (31 ± 5 %), including organic material and secondary inorganics, whereas this particle type was much less abundant (2.7 ± 1.3 %) in the IPRs. In a selected subset of the data where a direct comparison between out-of-cloud aerosol particles and IPRs in air masses with similar origin was possible, a pronounced enhancement of biological particles was found in the IPRs.

  19. Structure-function paradigm in human myoglobin: how a single-residue substitution affects NO reactivity at low pO2.

    Science.gov (United States)

    Scorciapino, Mariano Andrea; Spiga, Enrico; Vezzoli, Alessandra; Mrakic-Sposta, Simona; Russo, Rosaria; Fink, Bruno; Casu, Mariano; Gussoni, Maristella; Ceccarelli, Matteo

    2013-05-22

    This work is focused on the two more expressed human myoglobin isoforms. In the literature, their different overexpression in high-altitude natives was proposed to be related to alternative/complementary functions in hypoxia. Interestingly, they differ only at residue-54, lysine or glutamate, which is external and far from the main binding site. In order to ascertain whether these two almost identical myoglobins might exert different functions and to contribute to a deeper understanding about myoglobin's oxygen-level dependent functioning, they have been compared with respect to dynamics, heme electronic structure, and NO reactivity at different O2 levels. Electron paramagnetic resonance (EPR) spectroscopy was employed to investigate the electronic structure of the nitrosyl-form, obtaining fundamental clues about a different bond interaction between the heme-iron and the proximal histidine and highlighting striking differences in NO reactivity, especially at a very low pO2. The experimental results well matched with the information provided by molecular dynamics simulations, which showed a significantly different dynamics for the two proteins only in the absence of O2. The single mutation differentiating the two myoglobins resulted in strongly affecting the plasticity of the CD-region (C-helix-loop-D-helix), whose fluctuations, being coupled to the solvent, were found to be correlated with the dynamics of the distal binding site. In the absence of O2, on the one hand a significantly different probability for the histidine-gate opening has been shown by MD simulations, and on the other a different yield of myoglobin-NO formation was experimentally observed through EPR.

  20. Organization and evolution of the rat tyrosine hydroxylase gene

    International Nuclear Information System (INIS)

    Brown, E.R.; Coker, G.T. III; O'Malley, K.L.

    1987-01-01

    This report describes the organization of the rat tyrosine hydroxylase (TH) gene and compares its structure with the human phenylalanine hydroxylase gene. Both genes are single copy and contain 13 exons separated by 12 introns. Remarkably, the positions of 10 out 12 intron/exon boundaries are identical for the two genes. These results support the idea that these hydroxylases genes are members of a gene family which has a common evolutionary origin. The authors predict that this ancestral gene would have encoded exons similar to those of TH prior to evolutionary drift to other members of this gene family

  1. No effect of oral tyrosine on total tyrosine levels in breast milk: implications for dietary supplementation in early postpartum.

    Science.gov (United States)

    Dowlati, Yekta; Ravindran, Arun V; Maheux, Maxim; Steiner, Meir; Stewart, Donna E; Meyer, Jeffrey H

    2014-12-01

    Postpartum depression (PPD) is the most common complication of childbearing with a 13 % prevalence rate, and there is no widespread approach for prevention. There is an appealing theoretical rationale for oral tyrosine to help prevent PPD. However, the effect of oral tyrosine on its total and free concentrations in breast milk and plasma of breastfeeding mothers is not known. Twenty-four healthy breastfeeding women were randomly assigned to 0, 2, 5, or 10 g of oral tyrosine. Free and total tyrosine in breast milk and free tyrosine in plasma were measured. Free tyrosine was also measured in 12 different infant formulas. Total tyrosine in breast milk did not rise, but there was a slight tendency towards a reduction (up to −12 %; repeated measures ANOVA (RMANOVA): p = 0.074). Maternal plasma tyrosine rose (RMANOVA: p oral tyrosine on its concentration in breast milk supports further development of oral tyrosine as part of a prevention strategy for PPD.

  2. Residuation theory

    CERN Document Server

    Blyth, T S; Sneddon, I N; Stark, M

    1972-01-01

    Residuation Theory aims to contribute to literature in the field of ordered algebraic structures, especially on the subject of residual mappings. The book is divided into three chapters. Chapter 1 focuses on ordered sets; directed sets; semilattices; lattices; and complete lattices. Chapter 2 tackles Baer rings; Baer semigroups; Foulis semigroups; residual mappings; the notion of involution; and Boolean algebras. Chapter 3 covers residuated groupoids and semigroups; group homomorphic and isotone homomorphic Boolean images of ordered semigroups; Dubreil-Jacotin and Brouwer semigroups; and loli

  3. UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 2: selected applications.

    Science.gov (United States)

    Antosiewicz, Jan M; Shugar, David

    2016-06-01

    In Part 2 we discuss application of several different types of UV-Vis spectroscopy, such as normal, difference, and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, of the side-chain of tyrosine residues in different molecular environments. We review the ways these spectroscopies can be used to probe complex protein structures.

  4. UV?Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 2: selected applications

    OpenAIRE

    Antosiewicz, Jan M.; Shugar, David

    2016-01-01

    In Part 2 we discuss application of several different types of UV?Vis spectroscopy, such as normal, difference, and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, of the side-chain of tyrosine residues in different molecular environments. We review the ways these spectroscopies can be used to probe complex protein structures.

  5. Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism

    Science.gov (United States)

    Yuta Miki; Rebecca Pogni; Sandra Acebes; Fatima Lucas; Elena Fernandez-Fueyo; Maria Camilla Baratto; Maria I. Fernandez; Vivian De Los Rios; Francisco J. Ruiz-duenas; Adalgisa Sinicropi; Riccardo Basosi; Kenneth E. Hammel; Victor Guallar; Angel T. Martinez

    2013-01-01

    LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr181) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H2O2...

  6. Conformational basis for substrate recruitment in Protein Tyrosine Phosphatase 10D†

    OpenAIRE

    Madan, Lalima L.; Gopal, B.

    2011-01-01

    The coordinated activity of Protein Tyrosine Phosphatases (PTP) is crucial to initiate, modulate and terminate diverse cellular processes. The catalytic activity of this protein depends on a nucleophilic cysteine at the active site that mediates the hydrolysis of the incoming phosphotyrosine substrate. While the role of conserved residues in the catalytic mechanism of PTPs has been extensively examined, the diversity in the mechanisms of substrate recognition and modulation of catalytic activ...

  7. [Residual neuromuscular blockade].

    Science.gov (United States)

    Fuchs-Buder, T; Schmartz, D

    2017-06-01

    Even small degrees of residual neuromuscular blockade, i. e. a train-of-four (TOF) ratio >0.6, may lead to clinically relevant consequences for the patient. Especially upper airway integrity and the ability to swallow may still be markedly impaired. Moreover, increasing evidence suggests that residual neuromuscular blockade may affect postoperative outcome of patients. The incidence of these small degrees of residual blockade is relatively high and may persist for more than 90 min after a single intubating dose of an intermediately acting neuromuscular blocking agent, such as rocuronium and atracurium. Both neuromuscular monitoring and pharmacological reversal are key elements for the prevention of postoperative residual blockade.

  8. Mechanisms of Peroxynitrite Mediated Nitration of Tyrosine

    Science.gov (United States)

    Gunaydin, Hakan; Houk, K. N.

    2009-01-01

    The mechanisms of tyrosine nitration by peroxynitrous acid or nitrosoperoxycarbonate were investigated with the CBS-QB3 method. Either the protonation of peroxynitrite, or a reaction with carbon dioxide gives a reactive peroxide intermediate. Peroxynitrous acid mediated nitration of phenol occurs via the unimolecular decomposition to give nitrogen dioxide and hydroxyl radicals. Nitrosoperoxycarbonate also undergoes unimolecular decomposition to give carbonate and nitrogen dioxide radicals. The reactions of tyrosine with the hydroxyl or carbonate radicals give a phenoxy radical intermediate. The reaction of the nitrogen dioxide with this radical intermediate followed by tautomerization gives nitrated tyrosine in both cases. According to CBS-QB3 calculations, the rate-limiting step for the nitration of phenol is the decomposition of peroxynitrous acid or of nitrosoperoxycarbonate. PMID:19374346

  9. Residue processing

    Energy Technology Data Exchange (ETDEWEB)

    Gieg, W.; Rank, V.

    1942-10-15

    In the first stage of coal hydrogenation, the liquid phase, light and heavy oils were produced; the latter containing the nonliquefied parts of the coal, the coal ash, and the catalyst substances. It was the problem of residue processing to extract from these so-called let-down oils that which could be used as pasting oils for the coal. The object was to obtain a maximum oil extraction and a complete removal of the solids, because of the latter were returned to the process they would needlessly burden the reaction space. Separation of solids in residue processing could be accomplished by filtration, centrifugation, extraction, distillation, or low-temperature carbonization (L.T.C.). Filtration or centrifugation was most suitable since a maximum oil yield could be expected from it, since only a small portion of the let-down oil contained in the filtration or centrifugation residue had to be thermally treated. The most satisfactory centrifuge at this time was the Laval, which delivered liquid centrifuge residue and centrifuge oil continuously. By comparison, the semi-continuous centrifuges delivered plastic residues which were difficult to handle. Various apparatus such as the spiral screw kiln and the ball kiln were used for low-temperature carbonization of centrifuge residues. Both were based on the idea of carbonization in thin layers. Efforts were also being made to produce electrode carbon and briquette binder as by-products of the liquid coal phase.

  10. Phenylketonuria : tyrosine supplementation in phenylalanine-restricted diets

    NARCIS (Netherlands)

    van Spronsen, FJ; van Rijn, M; Bekhof, J; Koch, R; Smit, PGA

    Treatment of phenylketonuria (PKU) consists of restriction of natural protein and provision of a protein substitute that lacks phenylalanine but is enriched in tyrosine. Large and unexplained differences exist, however, in the tyrosine enrichment of the protein substitutes. Furthermore, some

  11. Chlorinated tyrosine derivatives in insect cuticle

    DEFF Research Database (Denmark)

    Andersen, Svend Olav

    2004-01-01

    , not-yet sclerotized cuticle of adult femur and tibia, the amounts increased rapidly during the first 24 h after ecdysis and more slowly during the next two weeks. Control analyses using stable isotope dilution mass spectrometry have confirmed that the chlorinated tyrosines are not artifacts formed...

  12. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.

    2003-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  13. Morphological Features of Tyrosine Hydroxylase Immunoreactive ...

    African Journals Online (AJOL)

    The current immunohistochemical study used the antibody against tyrosine hydroxylase (TH) to observe the immunoreactive elements in the mouse pancreas. The results indicated the presence of immunoreactive nerve fibers and endocrine cells. The immunopositive nerve fibers appeared as thick and thin bundles; thick ...

  14. Research of nitroxynil residues in bovine milk following a single administration in the dry period by ultra-performance liquid chromatography tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Claudia Chirollo

    2013-10-01

    Full Text Available Nitroxynil (NIT is a halogenated phenol used to control fascioliasis in cattle and sheep. The Commission Regulation EU No 37/2010 has established maximum residue limits for NIT in bovine and ovine muscle (400 μg kg−1, fat (200 μg kg−1, liver (20 μg kg−1 and kidney (400 μg kg−1, and more recently in bovine and ovine milk (20 μg kg−1. Thirty-five pregnant dairy cows were treated in this study with nitroxynil (340 mg/mL solution for injection at the recommended dose of 10 mg/kg body weight at the start of the dry period, i.e. 53 to 74 days before the expected calving. Calving occurred between 43 days and 79 days after treatment. The concentrations of NIT in the milk were monitored for up to 120 days after calving. NIT residues were extracted using acetonitrile; magnesium sulfate and sodium chloride were added to induce liquid-liquid partitioning and purified by dispersive solid phase extraction for clean-up. NIT was detected by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS in negative ionization mode. The highest concentrations of this drug were found in two animals at the first milking, 48 and 53 day post treatment with levels of 362 and 657 μg kg–1, respectively. NIT residues were below the limit of detection of the method (0.24 μg/kg–1 between 67 and 106 day post-treatment. Following calving, residues rapidly depleted in animals and were non-detectable from 10 to 38 days post-calving. In particular, in all animals milk resulted compliant (<20 μg/kg−1 three days post partum.

  15. Mutational properties of amino acid residues: implications for evolvability of phosphorylatable residues

    DEFF Research Database (Denmark)

    Creixell, Pau; Schoof, Erwin M.; Tan, Chris Soon Heng

    2012-01-01

    in terms of their mutational activity. Moreover, we highlight the importance of the genetic code and physico-chemical properties of the amino acid residues as likely causes of these inequalities and uncover serine as a mutational hot spot. Finally, we explore the consequences that these different......; it is typically assumed that all amino acid residues are equally likely to mutate or to result from a mutation. Here, by reconstructing ancestral sequences and computing mutational probabilities for all the amino acid residues, we refute this assumption and show extensive inequalities between different residues...... mutational properties have on phosphorylation site evolution, showing that a higher degree of evolvability exists for phosphorylated threonine and, to a lesser extent, serine in comparison with tyrosine residues. As exemplified by the suppression of serine's mutational activity in phosphorylation sites, our...

  16. Phenylketonuria : Tyrosine beyond the phenylalanine-restricted diet

    NARCIS (Netherlands)

    van Spronsen, FJ; Smit, PGA; Koch, R

    Controversies exist on the role of tyrosine in the pathogenesis of phenylketonuria (PKU) and, consequently, on the therapeutic role of tyrosine. This review examines data and theoretical considerations on the role of tyrosine in the pathogenesis and treatment of PKU. It is concluded that treatment

  17. Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides

    DEFF Research Database (Denmark)

    Winterbourn, Christine C; Parsons-Mair, Helena N; Gebicki, Silvia

    2004-01-01

    requirements for hydroperoxide formation using tyrosine analogues and di- and tri-peptides. Superoxide and phenoxyl radicals were generated using xanthine oxidase, peroxidase and the respective tyrosine derivative, or by gamma-radiation. Peroxides were measured using FeSO4/Xylenol Orange. Tyrosine and tyramine...

  18. Artificial Neural Network for the Prediction of Tyrosine-Based Sorting Signal Recognition by Adaptor Complexes

    Directory of Open Access Journals (Sweden)

    Debarati Mukherjee

    2012-01-01

    Full Text Available Sorting of transmembrane proteins to various intracellular compartments depends on specific signals present within their cytosolic domains. Among these sorting signals, the tyrosine-based motif (YXXØ is one of the best characterized and is recognized by μ-subunits of the four clathrin-associated adaptor complexes (AP-1 to AP-4. Despite their overlap in specificity, each μ-subunit has a distinct sequence preference dependent on the nature of the X-residues. Moreover, combinations of these residues exert cooperative or inhibitory effects towards interaction with the various APs. This complexity makes it impossible to predict a priori, the specificity of a given tyrosine-signal for a particular μ-subunit. Here, we describe the results obtained with a computational approach based on the Artificial Neural Network (ANN paradigm that addresses the issue of tyrosine-signal specificity, enabling the prediction of YXXØ-μ interactions with accuracies over 90%. Therefore, this approach constitutes a powerful tool to help predict mechanisms of intracellular protein sorting.

  19. Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

    DEFF Research Database (Denmark)

    Su, J; Muranjan, M; Sap, J

    1999-01-01

    BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family...... of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well...... established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from...

  20. Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

    Directory of Open Access Journals (Sweden)

    Thomas H Sanderson

    Full Text Available Recent advancements in isolation techniques for cytochrome c (Cytc have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

  1. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine-phos...

  2. Determination of low-level agricultural residues in soft drinks and sports drinks by liquid chromatography/tandem mass spectrometry: single-laboratory validation.

    Science.gov (United States)

    Paske, Nathan; Berry, Bryan; Schmitz, John; Sullivan, Darryl

    2007-01-01

    In this study, sponsored by PepsiCo Inc., a method was validated for measurement of 11 pesticide residues in soft drinks and sports drinks. The pesticide residues determined in this validation were alachlor, atrazine, butachlor, isoproturon, malaoxon, monocrotophos, paraoxon-methyl, phorate, phorate sulfone, phorate sulfoxide, and 2,4-dichlorophenoxyacetic acid (2,4-D) when spiked at 0.100 microg/L (1.00 microg/L for phorate). Samples were filtered (if particulate matter was present), degassed (if carbonated), and analyzed using liquid chromatography with tandem mass spectrometry. Quantitation was performed with matrix-matched external standard calibration solutions. The standard curve range for this assay was 0.0750 to 10.0 microg/L. The calibration curves for all agricultural residues had coefficient of determination (r2) values greater than or equal to 0.9900 with the exception of 2 values that were 0.9285 and 0.8514. Fortification spikes at 0.100 microg/L (1.00 microg/L for phorate) over the course of 2 days (n=8 each day) for 3 matrixes (7UP, Gatorade, and Diet Pepsi) yielded average percent recoveries (and percent relative standard deviations) as follows (n=48): 94.4 (15.2) for alachlor, 98.2 (13.5) for atrazine, 83.1 (41.6) for butachlor, 89.6 (24.5) for isoproturon, 87.9 (24.4) for malaoxon, 96.1 (9.26) for monocrotophos, 101 (25.7) for paraoxon-methyl, 86.6 (20.4) for phorate, 101 (16.5) for phorate sulfone, 93.6 (25.5) for phorate sulfoxide, and 98.2 (6.02) for 2,4-D.

  3. Classification of binding site conformations of protein tyrosine phosphatase 1B.

    Science.gov (United States)

    Tanchuk, Vsevolod Yu; Tanin, Volodymyr O; Vovk, Andriy I

    2012-07-01

    Hundred and two binding sites from 91 Protein Data Bank files for protein tyrosine phosphatase 1B with different ligands have been compared. It was found that they can be divided into five clusters. Additional clusters were formed by the unliganded and oxidized enzyme. The centroids of the clusters can be used as starting points for further studies of enzyme-inhibitor interaction by computer simulations. A special software tool has been created for the investigation of protein tyrosine phosphatase 1B and other enzymes. It performs multiple comparisons of selected parts of Protein Data Bank files, as well as further clustering, and determines mobility of separate residues. © 2012 John Wiley & Sons A/S.

  4. Residual risk

    African Journals Online (AJOL)

    ing the residual risk of transmission of HIV by blood transfusion. An epidemiological approach assumed that all HIV infections detected serologically in first-time donors were pre-existing or prevalent infections, and that all infections detected in repeat blood donors were new or incident infections. During 1986 - 1987,0,012%.

  5. Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response.

    Science.gov (United States)

    Zhang, Xu; Maity, Tapan; Kashyap, Manoj K; Bansal, Mukesh; Venugopalan, Abhilash; Singh, Sahib; Awasthi, Shivangi; Marimuthu, Arivusudar; Charles Jacob, Harrys Kishore; Belkina, Natalya; Pitts, Stephanie; Cultraro, Constance M; Gao, Shaojian; Kirkali, Guldal; Biswas, Romi; Chaerkady, Raghothama; Califano, Andrea; Pandey, Akhilesh; Guha, Udayan

    2017-05-01

    Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747 LREA 750 , are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238

  6. Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase*

    Science.gov (United States)

    Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F.

    2010-01-01

    The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr687 in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr687 and association with components of the Tyr1062 signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser696, a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr687 as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions. PMID:20682772

  7. Protein-tyrosine phosphatase SHP2 contributes to GDNF neurotrophic activity through direct binding to phospho-Tyr687 in the RET receptor tyrosine kinase.

    Science.gov (United States)

    Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F

    2010-10-08

    The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

  8. Ror receptor tyrosine kinases: orphans no more.

    Science.gov (United States)

    Green, Jennifer L; Kuntz, Steven G; Sternberg, Paul W

    2008-11-01

    Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.

  9. Analysis of tyrosine-O-sulfation

    DEFF Research Database (Denmark)

    Bundgaard, J.R.; Sen, J.W.; Johnsen, A.H.

    2008-01-01

    Tyrosine O-sulfation was first described about 50 years ago as a post-translational modification of fibrinogen. In the following 30 years it was considered to be a rare modification affecting only a few proteins and peptides. However, in the beginning of the 1980s tyrosine (Tyr) sulfation was shown...... to be a common modification and since then an increasing number of proteins have been identified as sulfated. The target proteins belong to the classes of secretory, plasma membrane, and lysosomal proteins, which reflects the intracellular localization of the enzymes catalyzing Tyr sulfation, the tyrosylprotein...... sulfotransferases (TPSTs).Traditionally, Tyr sulfation has been analyzed by incorporation of radiolabeled sulfate into target cells followed by purification of the target protein. Subsequently, the protein is degraded enzymatically or by alkaline hydrolysis followed by thin-layer electrophoresis to demonstrate...

  10. Analysis of tyrosine-O-sulfation

    DEFF Research Database (Denmark)

    Bundgaard, J.R.; Sen, J.W.; Johnsen, A.H.

    2008-01-01

    to be a common modification and since then an increasing number of proteins have been identified as sulfated. The target proteins belong to the classes of secretory, plasma membrane, and lysosomal proteins, which reflects the intracellular localization of the enzymes catalyzing Tyr sulfation, the tyrosylprotein......Tyrosine O-sulfation was first described about 50 years ago as a post-translational modification of fibrinogen. In the following 30 years it was considered to be a rare modification affecting only a few proteins and peptides. However, in the beginning of the 1980s tyrosine (Tyr) sulfation was shown...... sulfotransferases (TPSTs).Traditionally, Tyr sulfation has been analyzed by incorporation of radiolabeled sulfate into target cells followed by purification of the target protein. Subsequently, the protein is degraded enzymatically or by alkaline hydrolysis followed by thin-layer electrophoresis to demonstrate...

  11. Receptor tyrosine kinase structure and function in health and disease

    Directory of Open Access Journals (Sweden)

    Oleg A. Karpov

    2015-09-01

    Full Text Available Receptor tyrosine kinases (RTKs are membrane proteins that control the flow of information through signal transduction pathways, impacting on different aspects of cell function. RTKs are characterized by a ligand-binding ectodomain, a single transmembrane α-helix, a cytosolic region comprising juxtamembrane and kinase domains followed by a flexible C-terminal tail. Somatic and germline RTK mutations can induce aberrant signal transduction to give rise to cardiovascular, developmental and oncogenic abnormalities. RTK overexpression occurs in certain cancers, correlating signal strength and disease incidence. Diverse RTK activation and signal transduction mechanisms are employed by cells during commitment to health or disease. Small molecule inhibitors are one means to target RTK function in disease initiation and progression. This review considers RTK structure, activation, and signal transduction and evaluates biological relevance to therapeutics and clinical outcomes.

  12. SRC Residual fuel oils

    Science.gov (United States)

    Tewari, Krishna C.; Foster, Edward P.

    1985-01-01

    Coal solids (SRC) and distillate oils are combined to afford single-phase blends of residual oils which have utility as fuel oils substitutes. The components are combined on the basis of their respective polarities, that is, on the basis of their heteroatom content, to assure complete solubilization of SRC. The resulting composition is a fuel oil blend which retains its stability and homogeneity over the long term.

  13. A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

    DEFF Research Database (Denmark)

    Carlsson, A.S.; LaBrie, S.T.; Kinney, A.J.

    2002-01-01

    in Arabidopsis KAS2 that results in a Leu337Phe substitution. The Leu337 residue is conserved among plant and bacterial KAS proteins, and in the crystal structures of E. coli KAS I and KAS II, this leucine abuts a phenylalanine whose imidazole ring extends into the substrate binding cavity causing the fatty acid...... chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size...

  14. A single amino acid residue, Ala 105, confers 16alpha-hydroxylase activity to human cytochrome P450 17alpha-hydroxylase/17,20 lyase.

    Science.gov (United States)

    Swart, Amanda C; Storbeck, Karl-Heinz; Swart, Pieter

    2010-04-01

    In adrenal steroidogenesis, CYP17 catalyses the 17alpha-hydroxylation of pregnenolone and progesterone and the subsequent 17,20-lyase reaction, yielding adrenal androgens. The enzyme exhibits distinctly different selectivities towards these substrates in various species. CYP17 has also been shown to exhibit 16alpha-hydroxylase activity towards progesterone in some species, with only human and chimp CYP17 catalysing the biosynthesis of substantial amounts of 16-OHprogesterone. The 16alpha-hydroxylase activity was investigated by introducing an Ala105Leu substitution into human CYP17. The converse mutation, Leu105Ala was introduced into the baboon, goat and pig enzymes. Wt human CYP17 converted approximately 30% progesterone to 16-OHprogesterone while the Ala105Leu mutant converted negligible amounts to 16-OHprogesterone ( approximately 9%), comparable to wt CYP17 of the other three species when expressed in COS-1 cells. The ratio of 17-hydroxylated products to 16-OHprogesterone of human CYP17 was 2.7 and that of the mutant human construct 10.5. Similar ratios were observed for human and goat CYP17 with the corresponding Ala or Leu residues. Although the Leu105Ala mutation of both baboon and pig CYP17 exhibited the same trend regarding the ratios, the rate of progesterone conversion was reduced. Coexpression with cytochrome b(5) significantly decreased the ratio of 17-hydroxylated products to 16-OHprogesterone in the Leu105 constructs, while effects were negligible with Ala at this position. Homology models show that Ala105 faces towards the active pocket in the predicted B'-C domain of CYP17. The smaller residue allows more flexibility of movement in the active pocket than Leu, presenting both the C16 and C17 of progesterone to the iron-oxy complex. 2010 Elsevier Ltd. All rights reserved.

  15. Residual basins

    International Nuclear Information System (INIS)

    D'Elboux, C.V.; Paiva, I.B.

    1980-01-01

    Exploration for uranium carried out over a major portion of the Rio Grande do Sul Shield has revealed a number of small residual basins developed along glacially eroded channels of pre-Permian age. Mineralization of uranium occurs in two distinct sedimentary units. The lower unit consists of rhythmites overlain by a sequence of black shales, siltstones and coal seams, while the upper one is dominated by sandstones of probable fluvial origin. (Author) [pt

  16. Determination of low-level agricultural residues in soft drinks and sports drinks by gas chromatography with mass-selective detection: single-laboratory validation.

    Science.gov (United States)

    Paske, Nathan; Berry, Bryan; Schmitz, John; Sullivan, Darryl

    2007-01-01

    In this study, sponsored by PepsiCo Inc., a method was validated for measurement of 19 pesticide residues in soft drinks and sports drinks by gas chromatography/mass spectrometry (GC/MS) with mass selective detection The pesticide residues determined in this validation were alpha-benzenehexachloride (BHC); beta-BHC; gamma-BHC; delta-BHC; methyl parathion; malathion; chlorpyrifos; aldrin; 2,4-dichlorodiphenyldichloroethylene (DDE); alpha-endosulfan; 4,4-DDE; 2,4-dichlorodiphenyldichloroethane (DDD); dieldrin; ethion; 4,4-DDD; 2,4-dichlorodiphenyltrichloroethylene (DDT); beta-endosulfan; 4,4-DDT; and endosulfan sulfate when spiked into a 200 mL matrix sample at 0.50 microg/L. The samples were diluted with acetonitrile and water, then liquid-liquid phase extracted into petroleum ether. The resulting extract was concentrated to near dryness and diluted with hexane:dichloromethane (50:50). The concentrated samples were purified by gel permeation chromatography. The resulting solution was concentrated and separated on a Florisil substrate. The eluent was concentrated to near dryness, reconstituted to produce a 200-fold concentration, and analyzed using a GC/MS instrument operated in the selective ion monitoring mode. The GC/MS instrument was equipped with a large volume injector capable of injecting 25 microL. External standards prepared in dichloromethane were used for quantification without the need for matrix-matched calibration because the extraction step minimized the matrix effects. The calibration curves for all agricultural residues had coefficients of determination (r2) of greater than or equal to 0.9900, with the exception of one value that was 0.988. Fortification spikes at 0.50 microg/L in 3 matrixes (7UP, Gatorade, and Diet Pepsi) over the course of 2 days (4 days for Gatorade), where n=8 each day, yielded average percent recoveries (and percent relative standard deviations) as follows (n=64): 95.6 (24.8) for alpha-BHC; 91.9 (23.6) for beta-BHC; 89.1 (21

  17. Ultrafast Spectroscopic and AB Initio Computational Investigations on Solvation Dynamics of Neutral and Deprotonated Tyrosine.

    Science.gov (United States)

    Fujiwara, Takashige; Zgierski, Marek Z.

    2015-06-01

    We have studied one of the aromatic amino acids, tyrosine, regarding its photophysical properties in various solvent conditions by using a femtosecond fluorescence up-conversion technique and high-level TDDFT and CC2 computations. In this talk, profound details not only on ultrafast solvation dynamics on a neutral tyrosine in various solvents, but also on the excited-state dynamics for a single- (or doubly-) deprotonated tyrosine under various pH solutions will be presented. In high basicity, a tyrosine shows different absorption/emission spectra, and a total spectrum consists of a combination of these individual spectra that depend on the pH of the solution. The time scale of acid--base equilibrium is essential in solvation dynamics; whereas the protonation is simply controlled by diffusion, the de-protonation is considered to be slow process such that acid--base equilibrium may not be reached in the short-lived excited state after photo-excitation. Experimental and computational approaches taken and insights obtained in this concerted work will be described.

  18. Rapid Phospho-Turnover by Receptor Tyrosine Kinases Impacts Downstream Signaling and Drug Binding

    OpenAIRE

    Kleiman, Laura B.; Maiwald, Thomas; Conzelmann, Holger; Lauffenburger, Douglas A.; Sorger, Peter K.

    2011-01-01

    Epidermal growth factor receptors (ErbB1–4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100–1000 times in the course of a typical immediate-early response t...

  19. Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?

    DEFF Research Database (Denmark)

    Petrone, A; Sap, J

    2000-01-01

    Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions...... with signaling pathways involving SRC family kinases, which result from their ability to control phosphorylation of both activating and inhibitory sites in these kinases and possibly also their substrates. Similarly, integrin signaling illustrates how phosphorylation of a single protein, or the activity...

  20. Sensitive determination of mixtures of neonicotinoid and fungicide residues in pollen and single bumblebees using a scaled down QuEChERS method for exposure assessment.

    Science.gov (United States)

    David, Arthur; Botías, Cristina; Abdul-Sada, Alaa; Goulson, Dave; Hill, Elizabeth M

    2015-10-01

    To accurately estimate exposure of bees to pesticides, analytical methods are needed to enable quantification of nanogram/gram (ng/g) levels of contaminants in small samples of pollen or the individual insects. A modified QuEChERS extraction method coupled with ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis was tested to quantify residues of 19 commonly used neonicotinoids and fungicides and the synergist, piperonyl butoxide, in 100 mg samples of pollen and in samples of individual bumblebees (Bombus terrestris). Final recoveries ranged from 71 to 102 % for most compounds with a repeatability of below 20 % for both pollen and bumblebee extracts spiked at 5 and 40 ng/g. The method enables the detection of all compounds at sub-ng/g levels in both matrices and the method detection limits (MDL) ranged from 0.01 to 0.84 ng/g in pollen and 0.01 to 0.96 ng/g in individual bumblebees. Using this method, mixtures of neonicotinoids (thiamethoxam, clothianidin, imidacloprid and thiacloprid) and fungicides (carbendazim, spiroxamine, boscalid, tebuconazole, prochloraz, metconazole, fluoxastrobin, pyraclostrobin and trifloxystrobin) were detected in pollens of field bean, strawberry and raspberry at concentrations ranging from neonicotinoids and from MDL, and in some bees, the fungicides carbendazim, boscalid, tebuconazole, flusilazole and metconazole were present at concentrations between 0.80 to 30 ng/g. This new method allows the analysis of mixtures of neonicotinoids and fungicides at trace levels in small quantities of pollen and individual bumblebees and thus will facilitate exposure assessment studies.

  1. Entry of a Six-Residue Antimicrobial Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging their Membranes.

    Science.gov (United States)

    Moniruzzaman, Md; Islam, Md Zahidul; Sharmin, Sabrina; Dohra, Hideo; Yamazaki, Masahito

    2017-08-22

    Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4-9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4-9) and lissamine rhodamine B red-labeled LfcinB (4-9) (Rh-LfcinB (4-9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4-9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4-9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4-9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4-9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4-9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4-9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4-9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.

  2. Src Inhibits the Hippo Tumor Suppressor Pathway through Tyrosine Phosphorylation of Lats1.

    Science.gov (United States)

    Si, Yuan; Ji, Xinyan; Cao, Xiaolei; Dai, Xiaoming; Xu, Lingyi; Zhao, Hongxia; Guo, Xiaocan; Yan, Huan; Zhang, Haitao; Zhu, Chu; Zhou, Qi; Tang, Mei; Xia, Zongping; Li, Li; Cong, Yu-Sheng; Ye, Sheng; Liang, Tingbo; Feng, Xin-Hua; Zhao, Bin

    2017-09-15

    The Hippo pathway regulates cell proliferation, apoptosis, and stem cell self-renewal, and its inactivation in animal models causes organ enlargement followed by tumorigenesis. Hippo pathway deregulation occurs in many human cancers, but the underlying mechanisms are not fully understood. Here, we report tyrosine phosphorylation of the Hippo pathway tumor suppressor LATS1 as a mechanism underlying its regulation by cell adhesion. A tyrosine kinase library screen identified Src as the kinase to directly phosphorylate LATS1 on multiple residues, causing attenuated Mob kinase activator binding and structural alteration of the substrate-binding pocket in the kinase domain. Cell matrix adhesion activated the Hippo pathway effector transcription coactivator YAP partially through Src-mediated phosphorylation and inhibition of LATS1. Aberrant Src activation abolished the tumor suppressor activity of LATS1 and induced tumorigenesis in a YAP-dependent manner. Protein levels of Src in human breast cancer tissues correlated with accumulation of active YAP dephosphorylated on the LATS1 target site. These findings reveal tyrosine phosphorylation of LATS1 by Src as a novel mechanism of Hippo pathway regulation by cell adhesion and suggest Src activation as an underlying reason for YAP deregulation in tumorigenesis. Cancer Res; 77(18); 4868-80. ©2017 AACR . ©2017 American Association for Cancer Research.

  3. Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

    Directory of Open Access Journals (Sweden)

    Alessandra Pasquo

    Full Text Available Protein tyrosine phosphatase ρ (PTPρ belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

  4. Euglena mitochondria and chloroplasts form tyrosine-O-sulfate

    Energy Technology Data Exchange (ETDEWEB)

    Saidha, T.; Hanfstingl, U.; Schiff, J.A. (Brandeis Univ., Waltham, MA (USA))

    1989-04-01

    Mitochondria from light-grown wild-type Euglena gracilis var. bacillaris Cori or dark-grown mutant W{sub 10}BSmL incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, or with {sup 14}C-tyrosine, non-radioactive sulfate and ATP accumulate a labeled compound in the medium. Since this compound shows exact coelectrophoresis with tyrosine-O-sulfate (TOS) at pH 2.0, 5.8 or 8.0., yields sulfate and tyrosine on acid hydrolysis, and treatment with aryl sulfatase from Aerobacter aerogenes yields sulfate and tyrosine but no tyrosine methyl ester, it is identified as TOS. No TOS is found outside purified developing chloroplasts incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, but both chloroplasts and mitochondria form to {sup 35}S externally when incubated with adenosine 3{prime} phosphate 5{prime}phospho({sup 35}S) sulfate (PAP{sup 35}S). Since no tyrosine need be added, tyrosine is provided from endogenous sources. Although TOS is found in the free pool of Euglena cells it cannot be detected in proteins of cells or mucus ruling our sulfation of tyrosine of protein or incorporation of TOS into proteins. The system forming TOS is membrane-bound and may be involved in tyrosine transport.

  5. Tyrosine metabolic enzymes from insects and mammals: a comparative perspective.

    Science.gov (United States)

    Vavricka, Christopher John; Han, Qian; Mehere, Prajwalini; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2014-02-01

    Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess fine-tuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modern genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precursor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mammalian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  6. Dietary Tyrosine Benefits Cognitive and Psychomotor Performance During Body Cooling

    National Research Council Canada - National Science Library

    O'Brien, Catherine; Mahoney, Caroline; Tharion, William J; Sils, Ingrid V; Castellani, John W

    2007-01-01

    ... examined. This study evaluated the effect of tyrosine supplementation on cognitive, psychomotor, and physical performance following a cold water immersion protocol that lowered body core temperature...

  7. SH3 domain tyrosine phosphorylation--sites, role and evolution.

    Directory of Open Access Journals (Sweden)

    Zuzana Tatárová

    Full Text Available BACKGROUND: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. RESULTS: To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F. This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. CONCLUSIONS: While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.

  8. RESIDUAL RISK ASSESSMENTS - RESIDUAL RISK ...

    Science.gov (United States)

    This source category previously subjected to a technology-based standard will be examined to determine if health or ecological risks are significant enough to warrant further regulation for Coke Ovens. These assesments utilize existing models and data bases to examine the multi-media and multi-pollutant impacts of air toxics emissions on human health and the environment. Details on the assessment process and methodologies can be found in EPA's Residual Risk Report to Congress issued in March of 1999 (see web site). To assess the health risks imposed by air toxics emissions from Coke Ovens to determine if control technology standards previously established are adequately protecting public health.

  9. Large daily fluctuations in plasma tyrosine in treated patients with phenylketonuria

    NARCIS (Netherlands)

    vanSpronsen, FJ; vanDijk, T; Smit, GPA; vanRijn, M; Reijngoud, DJ; Berger, Ruud; Heymans, HSA

    1996-01-01

    In patients with phenylketonuria (PKU), extra tyrosine supplementation is advocated in addition to tyrosine-enriched amino acid mixtures. PKU patients have low fasting plasma tyrosine concentrations, but little is known about tyrosine fluctuations during the day. Plasma tyrosine concentrations were

  10. ROR-Family Receptor Tyrosine Kinases.

    Science.gov (United States)

    Stricker, Sigmar; Rauschenberger, Verena; Schambony, Alexandra

    2017-01-01

    ROR-family receptor tyrosine kinases form a small subfamily of receptor tyrosine kinases (RTKs), characterized by a conserved, unique domain architecture. ROR RTKs are evolutionary conserved throughout the animal kingdom and act as alternative receptors and coreceptors of WNT ligands. The intracellular signaling cascades activated downstream of ROR receptors are diverse, including but not limited to ROR-Frizzled-mediated activation of planar cell polarity signaling, RTK-like signaling, and antagonistic regulation of WNT/β-Catenin signaling. In line with their diverse repertoire of signaling functions, ROR receptors are involved in the regulation of multiple processes in embryonic development such as development of the axial and paraxial mesoderm, the nervous system and the neural crest, the axial and appendicular skeleton, and the kidney. In humans, mutations in the ROR2 gene cause two distinct developmental syndromes, recessive Robinow syndrome (RRS; MIM 268310) and dominant brachydactyly type B1 (BDB1; MIM 113000). In Robinow syndrome patients and animal models, the development of multiple organs is affected, whereas BDB1 results only in shortening of the distal phalanges of fingers and toes, reflecting the diversity of functions and signaling activities of ROR-family RTKs. In this chapter, we give an overview on ROR receptor structure and function. We discuss their signaling functions and role in vertebrate embryonic development with a focus on those developmental processes that are affected by mutations in the ROR2 gene in human patients. © 2017 Elsevier Inc. All rights reserved.

  11. Residual nilpotence and residual solubility of groups

    International Nuclear Information System (INIS)

    Mikhailov, R V

    2005-01-01

    The properties of the residual nilpotence and the residual solubility of groups are studied. The main objects under investigation are the class of residually nilpotent groups such that each central extension of these groups is also residually nilpotent and the class of residually soluble groups such that each Abelian extension of these groups is residually soluble. Various examples of groups not belonging to these classes are constructed by homological methods and methods of the theory of modules over group rings. Several applications of the theory under consideration are presented and problems concerning the residual nilpotence of one-relator groups are considered.

  12. Analysis of conformational flexibility of loop 110-120 of protein tyrosine phosphatase 1B.

    Science.gov (United States)

    Tanchuk, V Yu; Tanin, V O; Vovk, A I

    2013-01-01

    Conformations of the catalytic center of protein tyrosine phosphatase 1B (PTP1B) and surrounding loops are known to be important in catalysis and inhibition of the enzyme. There were 98 conformations from 88 PDB files representing PTP1B with different ligands which were analyzed to investigate the details of loop 110-120 movement and mobility of separate residues. The differences were identified by a special software tool which performs multiple comparisons of selected parts of PDB files. The conformations were divided into 6 clusters. It was found that the loop formed by residues 110-120 can be characterized by four main conformations. Predominantly, the loop 110-120 adopts the main conformation and keeps it during WPD loop movement. Three other conformations appear to be stabilized in case of closed WPD loop and seem to be favorable for PTP1B with subunit structure.

  13. Deuterium Labelling of L-Tyrosine with Raney Alloys in Alkaline Deuterium Oxide Solutions

    OpenAIRE

    Tsuzuki, Hirohisa; Mukumoto, Mamoru; Udagawa, Jun; Mataka, Shuntaro; Tashiro, Masashi

    1997-01-01

    The synthesis of deuteriated L-tyrosines with Raney alloys in alkaline deuterium oxide solutions, involving reductive debromination of brominated L-tyrosines and hydrogen-deuterium (H-D) exchange of L-tyrosines, without causing racemization, is presented.

  14. Substrate specificities of tyrosine-specific protein kinases toward cytoskeletal proteins in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Akiyama, T.; Kadowaki, T.; Nishida, E.; Kadooka, T.; Ogawara, H.; Fukami, Y.; Sakai, H.; Takaku, F.; Kasuga, M.

    1986-11-05

    The authors have previously reported that fodrin (..beta.. subunit), tubulin (..cap alpha.. subunit) and microtubule-associated proteins (MAPs; MAP2 and tau) are good substrates for the purified insulin receptor kinase. In this study, to investigate the substrate specificities of tyrosine kinases, they have examined the actions of the purified epidermal growth factor (EGF) receptor kinase and Rous sarcoma virus src kinase on purified microfilament- and microtubule-related proteins. Among microfilament-related proteins examined, the purified EGF receptor kinase phosphorylated the ..beta.. subunit, but not the ..cap alpha.. subunit, of fodrin on tyrosine residues with a K/sub m/ below the micromolar range. The fodrin phosphorylation by the EGF receptor kinase was markedly inhibited by F-actin. In contrast, the purified src kinase preferentially phosphorylated the ..cap alpha.. subunit of fodrin on tyrosine residues. Fodrin phosphorylation by the src kinase was not inhibited by F-actin. Among microtubule proteins examined, MAP-2 was the best substrate for the EGF receptor kinase. The peptide mapping of MAP2 phosphorylated by the EGF receptor kinase and by the insulin receptor kinase produced very similar patterns of phosphopeptides, while that of MAP2 phosphorylated by the src kinase gave a distinctly different pattern. When the phosphorylation of the tubulin subunits was examined, the EGF receptor kinase preferred ..beta.. subunit to ..cap alpha.. subunit, but the src kinase phosphorylated both ..cap alpha.. and ..beta.. subunits to a similar extent. These results, together with our previous results, indicate that the substrate specificities of the EGF receptor kinase and the insulin receptor kinase are very similar, but not identical, while that of the src kinase is distinctly different from that of these growth factor receptor kinases.

  15. Determination of residual 1,4-dioxane in surfactants and cleaning agents using headspace single-drop microextraction followed by gas chromatography-flame ionization detection.

    Science.gov (United States)

    Saraji, M; Shirvani, N

    2017-02-01

    Polyethoxylated surfactants are widely used in the formulation of different cleaning agents such as shampoo, dish washing and hand washing products and lotion formulation. During the production of polyethoxylated surfactants, 1,4-dioxane as a toxic and carcinogenic by-product is formed. A simple low-cost method based on headspace single-drop microextraction combined with gas chromatography-flame ionization detection was developed for the determination of 1,4-dioxane in surfactants and cleaning agents. In this method, 1,4-dioxane was extracted from 8.0 mL sample solution into a microdrop of an organic solvent, and then, it was injected to gas chromatography. The effects of such parameters as the solvent type, salt addition, microdrop volume, stirring rate, equilibrium time, extraction time and the temperature of sample solution on the extraction performance were studied and optimized. An ethoxylated surfactant containing 1,4-dioxane was used as the sample for the optimization of the extraction parameters. The linear range, determination coefficient, limit of detection and relative standard deviation of the method were 0.5-100 μg g -1 , 0.9977, 0.4 μg g -1 and 7.2% (n = 5), respectively. Different real samples including sodium lauryl ether sulphate, sodium lauryl sulphate (SLS), four brands of shampoo, and hand washing and dish washing liquids were analysed by the method. 1,4-Dioxane was detected at the concentration range of 2.4-201 μg g -1 in the samples, except dish washing liquid and SLS. A new method with the merits of simplicity, low cost, low organic solvent consumption, short analysis time, good repeatability and suitable detection limit was developed for the analysis of 1,4-dioxane in surfactants and cleaning agents. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  16. Determination of o-tyrosine in irradiated chicken

    International Nuclear Information System (INIS)

    Zoller, O.; Schoeni, D.; Zimmerli, B.

    1991-01-01

    The author explains his method to determine O-Tyrosine in irradiated chickens with a high-performance liquid chromatography. The method is simple and fast, but a proper chromatographic separation is difficult. The detection limit with a high sensitive detector is about 0.05-0.1 mg O-Tyrosine/kg meat (9 refs)

  17. Tyrosine improves working memory in a multitasking environment.

    Science.gov (United States)

    Thomas, J R; Lockwood, P A; Singh, A; Deuster, P A

    1999-11-01

    Previous studies indicate that tyrosine may prove useful in promoting improved performance in situations in which performance is compromised by stress. To extend the generality of previous tyrosine findings, the present study examined the effects of tyrosine ingestion on performance during both a Multiple Task and a Simple Task battery. The multiple task battery was designed to measure working memory, arithmetic skills, and visual and auditory monitoring simultaneously, whereas the simple task battery measured only working memory and visual monitoring. Ten men and 10 women subjects underwent these batteries 1 h after ingesting 150 mg/kg of l-tyrosine or placebo. Administration of tyrosine significantly enhanced accuracy and decreased frequency of list retrieval on the working memory task during the multiple task battery compared with placebo. However, tyrosine induced no significant changes in performance on the arithmetic, visual, or auditory tasks during the Multiple Task, or modified any performance measures during the Simple Task battery. Blood levels of ACTH and cortisol were not, but heart rate and blood pressure were significantly increased during the performance tasks. The present results indicate that tyrosine may sustain working memory when competing requirements to perform other tasks simultaneously degrade performance, and that supplemental tyrosine may be appropriate for maintaining performance when mild to severe decrements are anticipated.

  18. Protein phosphorylation on tyrosine restores expression and glycosylation of cyclooxygenase-2 by 2-deoxy-D-glucose-caused endoplasmic reticulum stress in rabbit articular chondrocyte

    Directory of Open Access Journals (Sweden)

    Seon-Mi Yu

    2012-05-01

    Full Text Available 2-deoxy-D-glucose(2DG-caused endoplasmic reticulum (ERstress inhibits protein phosphorylation at tyrosine residues.However, the accurate regulatory mechanisms, which determinethe inflammatory response of chondrocytes to ER stress via proteintyrosine phosphorylation, have not been systematicallyevaluated. Thus, in this study, we examined whether proteinphosphorylation at tyrosine residues can modulate the expressionand glycosylation of COX-2, which is reduced by 2DG-inducedER stress. We observed that protein tyrosine phosphatase (PTP inhibitors,sodium orthovanadate (SOV, and phenylarsine oxide(PAO significantly decreased expression of ER stress inducibleproteins, glucose-regulated protein 94 (GRP94, and CCAAT/ enhancer-binding-protein- related gene (GADD153, which was inducedby 2DG. In addition, we demonstrated that SOV and PAOnoticeably restored the expression and glycosylation of COX-2 aftertreatment with 2DG. These results suggest that protein phosphorylationof tyrosine residues plays an important role in theregulation of expression and glycosylation during 2DG-inducedER stress in rabbit articular chondrocytes. [BMB reports 2012;45(5: 317-322

  19. Primary structure and conformational analysis of peptide methionine-tyrosine, a peptide related to neuropeptide Y and peptide YY isolated from lamprey intestine

    DEFF Research Database (Denmark)

    Conlon, J M; Bjørnholm, B; Jørgensen, Flemming Steen

    1991-01-01

    A peptide belonging to the pancreatic-polypeptide-fold family of regulatory peptides has been isolated from the intestine of an Agnathan, the sea lamprey (Petromyzon marinus). The primary structure of the peptide (termed peptide methionine-tyrosine) was established as Met-Pro-Pro-Lys-Pro-Asp-Asn-......A peptide belonging to the pancreatic-polypeptide-fold family of regulatory peptides has been isolated from the intestine of an Agnathan, the sea lamprey (Petromyzon marinus). The primary structure of the peptide (termed peptide methionine-tyrosine) was established as Met......%) or with pig pancreatic polypeptide (42%). Molecular modelling and dynamic simulation, based upon sequence similarity with turkey pancreatic polypeptide, indicates that the conformations of the polyproline-helix-like region (residues 1-8) and the alpha-helical region (residues 15-30) in turkey pancreatic...... polypeptide are conserved in peptide methionine-tyrosine, and that non-bonded interactions between these domains have preserved the overall polypeptide fold in the molecule. The substitution of the otherwise totally conserved Gly9 residue by serine in lamprey peptide methionine-tyrosine, however, results...

  20. Structure and Molecular Dynamics Simulations of Protein Tyrosine Phosphatase Non-Receptor 12 Provide Insights into the Catalytic Mechanism of the Enzyme

    Directory of Open Access Journals (Sweden)

    Hui Dong

    2017-12-01

    Full Text Available Protein tyrosine phosphatase non-receptor 12 (PTPN12 is an important protein tyrosine phosphatase involved in regulating cell adhesion and migration as well as tumorigenesis. Here, we solved a crystal structure of the native PTPN12 catalytic domain with the catalytic cysteine (residue 231 in dual conformation (phosphorylated and unphosphorylated. Combined with molecular dynamics simulation data, we concluded that those two conformations represent different states of the protein which are realized during the dephosphorylation reaction. Together with docking and mutagenesis data, our results provide a molecular basis for understanding the catalytic mechanism of PTPN12 and its role in tumorigenesis.

  1. Deep residual networks of residual networks for image super-resolution

    Science.gov (United States)

    Wei, Xueqi; Yang, Fumeng; Wu, Congzhong

    2017-11-01

    Single image super-resolution (SISR), which aims at obtaining a high-resolution image from a single low-resolution image, is a classical problem in computer vision. In this paper, we address this problem based on a deep learning method with residual learning in an end-to-end manner. We propose a novel residual-network architecture, Residual networks of Residual networks (RoR), to promote the learning capability of residual networks for SISR. In residual network, the signal can be directly propagated from one unit to any other units in both forward and backward passes when using identity mapping as the skip connections. Based on it, we add level-wise connections upon original residual networks, to dig the optimization ability of residual networks. Our experiments demonstrate the effectiveness and versatility of RoR, it can get a faster convergence speed and gain higher resolution accuracy from considerably increased depth.

  2. Identification by Hydrogen/Deuterium Exchange of Structural Changes in Tyrosine Hydroxylase Associated with Regulation

    Science.gov (United States)

    Wang, Shanzhi; Sura, Giri R.; Dangott, Lawrence J.; Fitzpatrick, Paul F.

    2009-01-01

    The activity of tyrosine hydroxylase is regulated by reversible phosphorylation of serine residues in an N-terminal regulatory domain and catecholamine inhibition at the active site. Catecholamines such as dopamine bind very tightly to the resting enzyme; phosphorylation of Ser40 decreases the affinity for catecholamines by three orders of magnitude. The effects of dopamine binding and phosphorylation of Ser40 on the kinetics of deuterium incorporation into peptide bonds were examined by mass spectrometry. When dopamine is bound, three peptic peptides show significantly slower deuterium incorporation, 35-41 and 42-71 in the regulatory domain and 295-299 in the catalytic domain. In the phosphorylated enzyme, peptide 295-299 shows more rapid incorporation of deuterium, while 35-41 and 42-71 can not be detected. These results are consistent with tyrosine hydroxylase existing in two different conformations. In the closed conformation, the regulatory domain lies across the active site loop containing residues 295-298; this is stabilized when dopamine is bound in the active site. In the open conformation, the regulatory domain has moved out of the active site, allowing substrates access; this conformation is favored by phosphorylation of Ser40. PMID:19371093

  3. Adaptation of phenylalanine and tyrosine catabolic pathway to hibernation in bats.

    Science.gov (United States)

    Pan, Yi-Hsuan; Zhang, Yijian; Cui, Jie; Liu, Yang; McAllan, Bronwyn M; Liao, Chen-Chung; Zhang, Shuyi

    2013-01-01

    Some mammals hibernate in response to harsh environments. Although hibernating mammals may metabolize proteins, the nitrogen metabolic pathways commonly activated during hibernation are not fully characterized. In contrast to the hypothesis of amino acid preservation, we found evidence of amino acid metabolism as three of five key enzymes, including phenylalanine hydroxylase (PAH), homogentisate 1,2-dioxygenase (HGD), fumarylacetoacetase (FAH), involved in phenylalanine and tyrosine catabolism were co-upregulated during hibernation in two distantly related species of bats, Myotis ricketti and Rhinolophus ferrumequinum. In addition, the levels of phenylalanine in the livers of these bats were significantly decreased during hibernation. Because phenylalanine and tyrosine are both glucogenic and ketogenic, these results indicate the role of this catabolic pathway in energy supply. Since any deficiency in the catabolism of these two amino acids can cause accumulations of toxic metabolites, these results also suggest the detoxification role of these enzymes during hibernation. A higher selective constraint on PAH, HPD, and HGD in hibernators than in non-hibernators was observed, and hibernators had more conserved amino acid residues in each of these enzymes than non-hibernators. These conserved amino acid residues are mostly located in positions critical for the structure and activity of the enzymes. Taken together, results of this work provide novel insights in nitrogen metabolism and removal of harmful metabolites during bat hibernation.

  4. Phosphatase of regenerating liver-3 directly interacts with integrin β1 and regulates its phosphorylation at tyrosine 783

    Directory of Open Access Journals (Sweden)

    Tian Wei

    2012-10-01

    Full Text Available Abstract Background Phosphatase of regenerating liver-3 (PRL-3 or PTP4A3 has been implicated in controlling cancer cell proliferation, motility, metastasis, and angiogenesis. Deregulated expression of PRL-3 is highly correlated with cancer progression and predicts poor survival. Although PRL-3 was categorized as a tyrosine phosphatase, its cellular substrates remain largely unknown. Results We demonstrated that PRL-3 interacts with integrin β1 in cancer cells. Recombinant PRL-3 associates with the intracellular domain of integrin β1 in vitro. Silencing of integrin α1 enhances PRL-3-integrin β1 interaction. Furthermore, PRL-3 diminishes tyrosine phosphorylation of integrin β1 in vitro and in vivo. With site-specific anti-phosphotyrosine antibodies against residues in the intracellular domain of integrin β1, tyrosine-783, but not tyrosine-795, is shown to be dephosphorylated by PRL-3 in a catalytic activity-dependant manner. Phosphorylation of Y783 is potentiated by ablation of PRL-3 or by treatment with a chemical inhibitor of PRL-3. Conversely, depletion of integrin α1 decreases the phosphorylation of this site. Conclusions Our results revealed a direct interaction between PRL-3 and integrin β1 and characterized Y783 of integrin β1 as a bona fide substrate of PRL-3, which is negatively regulated by integrin α1.

  5. c-Abl mediated tyrosine phosphorylation of paxillin regulates LPS-induced endothelial dysfunction and lung injury

    Science.gov (United States)

    Usatyuk, Peter V.; Lele, Abhishek; Harijith, Anantha; Gregorio, Carol C.; Garcia, Joe G. N.; Salgia, Ravi; Natarajan, Viswanathan

    2015-01-01

    Paxillin is phosphorylated at multiple residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and acute lung injury (ALI) remains unclear. We used siRNA and site-specific nonphosphorylable mutants of paxillin to abrogate the function of paxillin to determine its role in lung endothelial permeability and ALI. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (HLMVECs) resulted in enhanced tyrosine phosphorylation of paxillin at Y31 and Y118 with no significant change in Y181 and significant barrier dysfunction. Knockdown of paxillin with siRNA attenuated LPS-induced endothelial barrier dysfunction and destabilization of VE-cadherin. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase, but not by Src and focal adhesion kinase. c-Abl siRNA significantly reduced LPS-induced endothelial barrier dysfunction. Transfection of HLMVECs with paxillin Y31F, Y118F, and Y31/118F double mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization. In vivo, the c-Abl inhibitor AG957 attenuated LPS-induced pulmonary permeability in mice. Together, these results suggest that c-Abl mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury. PMID:25795725

  6. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    LENUS (Irish Health Repository)

    Meehan, Maria

    2012-02-05

    Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA\\'s primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  7. Application of an M13 bacteriophage displaying tyrosine on the surface for detection of Fe(3+) and Fe(2+) ions.

    Science.gov (United States)

    Guo, Xiaohua; Niu, Chuncheng; Wu, Yunhua; Liang, Xiaosheng

    2015-12-01

    Ferric and ferrous ion plays critical roles in bioprocesses, their influences in many fields have not been fully explored due to the lack of methods for quantification of ferric and ferrous ions in biological system or complex matrix. In this study, an M13 bacteriophage (phage) was engineered for use as a sensor for ferric and ferrous ions via the display of a tyrosine residue on the P8 coat protein. The interaction between the specific phenol group of tyrosine and Fe(3+) / Fe(2+) was used as the sensor. Transmission electron microscopy showed aggregation of the tyrosine-displaying phages after incubation with Fe(3+) and Fe(2+). The aggregated phages infected the host bacterium inefficiently. This phenomenon could be utilized for detection of ferric and ferrous ions. For ferric ions, a calibration curve ranging from 200 nmol/L to 8 μmol/L with a detection limit of 58 nmol/L was acquired. For ferrous ions, a calibration curve ranging from 800 nmol/L to 8 μmol/L with a detection limit of 641.7 nmol/L was acquired. The assay was specific for Fe(3+) and Fe(2+) when tested against Ni(2+), Pb(2+), Zn(2+), Mn(2+), Co(2+), Ca(2+), Cu(2+), Cr(3+), Ba(2+), and K(+). The tyrosine displaying phage to Fe(3+) and Fe(2+) interaction would have plenty of room in application to biomaterials and bionanotechnology.

  8. Measurement of optical purity of p-BPA-Tyrosine dipeptide

    Energy Technology Data Exchange (ETDEWEB)

    Yoshino, K.; Sato, N.; Kitta, K.; Saitake, Y. [Shinshu Univ., Faculty of Science, Matsumoto, Nagano (Japan); Hiratsuka, J. [Kawasaki Medical School, Dept. of Radiation Oncology, Kurashiki, Okayama (Japan); Ichihashi, M. [Kobe Univ. (Japan). School of Medicine

    2000-10-01

    Melanin biosynthesis is very active in melanoma cells, and tyrosine is one of the substrates of the melanin biosynthesis. Tyrosine is oxidized to dopa by tyrosinase at the beginning of melanin biosynthesis process. Therefore, p-boronophenylalanine (BPA)-tyrosine dipeptide is expected to be a substrate of melanin biosynthesis process, and the peptide will be incorporated in melanoma cells, and then tumor boron concentration lasts in their cells for long time. Since p-BPA tyrosine are amino acids, they have D, L isomers. Therefore, we have tried to synthesize four isomers (L-L, L-D, D-L, D-D) of p-BPA-Tyrosine dipeptide, and have measured their optical purity with HPLC. (author)

  9. Characterization of individual ice residual particles by the single droplet freezing method: a case study in the Asian dust outflow region

    Science.gov (United States)

    Iwata, Ayumi; Matsuki, Atsushi

    2018-02-01

    pure or fresh calcite, Ca(NO3)2, and (NH4)2SO4 particles were more often found in the non-active fraction. In this study, we demonstrated the capability of the combined single droplet freezing method and thorough individual particle analysis to characterize the ice nucleation activity of atmospheric aerosols. We also found that dramatic changes in the particle mixing states during long-range transport had a complex effect on the ice nucleation activity of the host aerosol particles. A case study in the Asian dust outflow region highlighted the need to consider particle mixing states, which can dramatically influence ice nucleation activity.

  10. Characterization of individual ice residual particles by the single droplet freezing method: a case study in the Asian dust outflow region

    Directory of Open Access Journals (Sweden)

    A. Iwata

    2018-02-01

    dust particles. Also, relatively pure or fresh calcite, Ca(NO32, and (NH42SO4 particles were more often found in the non-active fraction. In this study, we demonstrated the capability of the combined single droplet freezing method and thorough individual particle analysis to characterize the ice nucleation activity of atmospheric aerosols. We also found that dramatic changes in the particle mixing states during long-range transport had a complex effect on the ice nucleation activity of the host aerosol particles. A case study in the Asian dust outflow region highlighted the need to consider particle mixing states, which can dramatically influence ice nucleation activity.

  11. Location of 3-hydroxyproline residues in collagen types I, II, III, and V/XI implies a role in fibril supramolecular assembly.

    Science.gov (United States)

    Weis, Mary Ann; Hudson, David M; Kim, Lammy; Scott, Melissa; Wu, Jiann-Jiu; Eyre, David R

    2010-01-22

    Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro(986) in A-clade chains alpha1(I), alpha1(II), and alpha2(V). Two partially modified sites (A2 and A3) were found at Pro(944) in alpha1(II) and alpha2(V) and Pro(707) in alpha2(I) and alpha2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro(470) in alpha2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian alpha1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.

  12. Protein Tyrosine Nitration: Selectivity, physicochemical and biological consequences, denitration and proteomics methods for the identification of tyrosine-nitrated proteins

    NARCIS (Netherlands)

    Abello, N.; Kerstjens, H.A.M.; Postma, D.S; Bischoff, Rainer

    2009-01-01

    Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO2). In the present article, we review the main nitration reactions and elucidate

  13. Protein Tyrosine Nitration : Selectivity, Physicochemical and Biological Consequences, Denitration, and Proteomics Methods for the Identification of Tyrosine-Nitrated Proteins

    NARCIS (Netherlands)

    Abello, Nicolas; Kerstjens, Huib A. M.; Postma, Dirkje S.; Bischoff, Rainer

    Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO(2)). In the present article, we review the main nitration reactions and

  14. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine......-phosphorylated and associated in vivo with the Grb2 protein. This association can be reproduced in stably and transiently transfected cells, as well as in vitro using recombinant Grb2 protein. Association requires the presence of an intact SH2 domain in Grb2, as well as tyrosine phosphorylation of R-PTP-alpha. This observation...... links a receptor tyrosine phosphatase with a key component of a central cellular signalling pathway and provides a basis for addressing R-PTP-alpha function....

  15. Coupled regulation by the juxtamembrane and sterile α motif (SAM) linker is a hallmark of Ephrin tyrosine kinase evolution.

    Science.gov (United States)

    Kwon, Annie; John, Mihir; Ruan, Zheng; Kannan, Natarajan

    2018-02-12

    Ephrin (Eph) receptor tyrosine kinases have evolutionarily diverged from other tyrosine kinases to respond to specific activation and regulatory signals that requires close coupling of kinase catalytic and regulatory functions. However, the evolutionary basis for such functional coupling is not fully understood. We employed an evolutionary systems approach involving statistical mining of large sequence and structural datasets to define the hallmarks of Eph kinase evolution and functional specialization. We find that some of the most distinguishing Eph- specific residues structurally tether the flanking juxtamembrane and sterile α motif (SAM) linker regions to the kinase domain, and substitutions of these residues in EphA3 result in faster kinase activation. We report for the first time that the SAM domain linker is functionally coupled to the juxtamembrane through co-conserved residues in the kinase domain, and that together these residues provide a structural framework for coupling catalytic and regulatory functions. The unique organization of Eph-specific tethering networks and the identification of other Eph-specific sequence features of unknown functions provide new hypotheses for future functional studies and new clues to disease mutations altering Eph kinase-specific functions. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Tyrosine aminotransferase contributes to benzylisoquinoline alkaloid biosynthesis in opium poppy.

    Science.gov (United States)

    Lee, Eun-Jeong; Facchini, Peter J

    2011-11-01

    Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5'-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and α-ketoglutarate, with apparent K(m) values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.

  17. Degradation of poly(glutamic acid-CO-tyrosine) in the solid state by gamma-radiation at 77 and 303 K

    International Nuclear Information System (INIS)

    Hill, D.J.T.; Ho, S.Y.; O'Donnell, J.H.; Pomery, P.J.

    1990-01-01

    The degradation of poly-L-glutamic acid, two copolymers of L-glutamic acid and L-tyrosine and poly-L-tyrosine by γ-radiation at 77 and 303 K has been investigated. The volatile radiolysis products and their yields have been measured at 303 K and the radical intermediates have been identified at 77 and 303 K, and the radical yields determined from ESR spectra. The scission and crosslinking yields have been determined from molecular weight measurements by gel permeation chromatography. The tyrosine residues in the copolymers lead to reduced yields of volatile products, radical intermediates and scission as a result of the protective effect of these aromatic units. (author)

  18. Bacillus subtilis single-stranded DNA-binding protein SsbA is phosphorylated at threonine 38 by the serine/threonine kinase YabT

    DEFF Research Database (Denmark)

    Derouiche, Abderahmane; Petranovic, Dina; Macek, Boris

    2016-01-01

    Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA-binding pro......Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA...... assays.Results: In addition to the known tyrosine phosphorylation of SsbA on tyrosine 82, we identified a new phosphorylation site: threonine 38. The in vitro assays demonstrated that SsbA is preferentially phosphorylated by the B. subtilis Hanks-type kinase YabT, and phosphorylation of threonine 38...... leads to enhanced cooperative binding to DNA.Conclusions: Our findings contribute to the emerging picture that bacterial proteins, exemplified here by SsbA, undergo phosphorylation at multiple residues. This results in a complex regulation of cellular functions, and suggests that the complexity...

  19. The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

    DEFF Research Database (Denmark)

    Voena, Claudia; Conte, Chiara; Ambrogio, Chiara

    2007-01-01

    , leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine......), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416...... in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration....

  20. The role of small adaptor proteins in the control of oncogenic signaling driven by tyrosine kinases in human cancer

    Science.gov (United States)

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-01-01

    Protein phosphorylation on tyrosine (Tyr) residues has evolved as an important mechanism to coordinate cell communication in multicellular organisms. The importance of this process has been revealed by the discovery of the prominent oncogenic properties of tyrosine kinases (TK) upon deregulation of their physiological activities, often due to protein overexpression and/or somatic mutation. Recent reports suggest that TK oncogenic signaling is also under the control of small adaptor proteins. These cytosolic proteins lack intrinsic catalytic activity and signal by linking two functional members of a catalytic pathway. While most adaptors display positive regulatory functions, a small group of this family exerts negative regulatory functions by targeting several components of the TK signaling cascade. Here, we review how these less studied adaptor proteins negatively control TK activities and how their loss of function induces abnormal TK signaling, promoting tumor formation. We also discuss the therapeutic consequences of this novel regulatory mechanism in human oncology. PMID:26788993

  1. The role of small adaptor proteins in the control of oncogenic signalingr driven by tyrosine kinases in human cancer.

    Science.gov (United States)

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-03-08

    Protein phosphorylation on tyrosine (Tyr) residues has evolved as an important mechanism to coordinate cell communication in multicellular organisms. The importance of this process has been revealed by the discovery of the prominent oncogenic properties of tyrosine kinases (TK) upon deregulation of their physiological activities, often due to protein overexpression and/or somatic mutation. Recent reports suggest that TK oncogenic signaling is also under the control of small adaptor proteins. These cytosolic proteins lack intrinsic catalytic activity and signal by linking two functional members of a catalytic pathway. While most adaptors display positive regulatory functions, a small group of this family exerts negative regulatory functions by targeting several components of the TK signaling cascade. Here, we review how these less studied adaptor proteins negatively control TK activities and how their loss of function induces abnormal TK signaling, promoting tumor formation. We also discuss the therapeutic consequences of this novel regulatory mechanism in human oncology.

  2. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    Directory of Open Access Journals (Sweden)

    Ayrapetov Marina K

    2005-11-01

    Full Text Available Abstract Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1 binds to ATP, and the other (M2 acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator.

  3. Differential evolutionary wiring of the tyrosine kinase Btk.

    Directory of Open Access Journals (Sweden)

    Hossain M Nawaz

    Full Text Available BACKGROUND: A central question within biology is how intracellular signaling pathways are maintained throughout evolution. Btk29A is considered to be the fly-homolog of the mammalian Bruton's tyrosine kinase (Btk, which is a non-receptor tyrosine-kinase of the Tec-family. In mammalian cells, there is a single transcript splice-form and the corresponding Btk-protein plays an important role for B-lymphocyte development with alterations within the human BTK gene causing the immunodeficiency disease X-linked agammaglobulinemia in man and a related disorder in mice. In contrast, the Drosophila Btk29A locus encodes two splice-variants, where the type 2-form is the more related to the mammalian Btk gene product displaying more than 80% homology. In Drosophila, Btk29A displays a dynamic pattern of expression through the embryonic to adult stages. Complete loss-of-function of both splice-forms is lethal, whereas selective absence of the type 2-form reduces the adult lifespan of the fly and causes developmental abnormalities in male genitalia. METHODOLOGY/PRINCIPAL FINDINGS: Out of 7004-7979 transcripts expressed in the four sample groups, 5587 (70-79% were found in all four tissues and strains. Here, we investigated the role of Btk29A type 2 on a transcriptomic level in larval CNS and adult heads. We used samples either selectively defective in Btk29A type 2 (Btk29A(ficP or revertant flies with restored Btk29A type 2-function (Btk29A(fic Exc1-16. The whole transcriptomic profile for the different sample groups revealed Gene Ontology patterns reflecting lifespan abnormalities in adult head neuronal tissue, but not in larvae. CONCLUSIONS: In the Btk29A type 2-deficient strains there was no significant overlap between transcriptomic alterations in adult heads and larvae neuronal tissue, respectively. Moreover, there was no significant overlap of the transcriptomic changes between flies and mammals, suggesting that the evolutionary conservation is confined

  4. Mechanistic studies of ionizing radiation and oxidative mutagenesis: Genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome

    Energy Technology Data Exchange (ETDEWEB)

    Wood, M.L.; Essigmann, J.M. (Massachusetts Institute of Technology, Cambridge (USA)); Dizdaroglu, M.; Gajewski, E. (National Institute of Standards and Technology, Gaithersburg, MD (USA))

    1990-07-31

    T4 RNA ligase was used to construct a deoxypentanucleotide containing a single 8-hydroxyguanine (7-hydro-8-oxoguanine; G{sup 8-OH}) residue, which is one of the putatively mutagenic DNA adducts produced by oxidants and ionizing radiation. The pentamer d(GCTAG{sup 8-OH})p was prepared by the ligation of a chemically synthesized acceptor molecule, d(GCTA), to an adducted donor, 8-hydroxy-2{prime}-deoxyguanosine 5{prime},3{prime}-bisphosphate. Following 3{prime}-dephosphorylation, the pentamer was characterized by UV spectroscopy, by high-pressure liquid chromatography, and by gas chromatography-mass spectrometry of the nucleosides released by enzymatic hydrolysis. Both d(GCTAG{sup 8-OH}) and an unmodified control were 5{prime}-phosphorylated by using ({gamma}-{sup 32}P)ATP and incorporated covalently by DNA ligase into a five-base gap at a unique NheI restriction site in the otherwise duplex genome of an M13mp19 derivative. The adduct was part of a nonsense codon in a unique restriction site in order to facilitate the identification and selection of mutants generated by the replication of the modified genome in Escherichia coli. Both control and adducted pentamers ligated into the genome at 50% of the maximum theoretical efficiency, and nearly all of the site-specifically adducted products possessed pentanucleotides that were covalently linked at both 5{prime} and 3{prime} termini. Transformation of E. coli strain DL7 with the uniquely modified single-stranded genome resulted in {approximately}0.5-1.0% of the progeny phase showing the G {yields} T transversion mutation at the original position of G{sup 8-OH}. The vector containing G{sup 8-OH} also transformed 50-90% as efficiently as the unmodified control, indicating that the adduct can be both weakly cytotoxic and mutagenic to the phase genome.

  5. rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

    Science.gov (United States)

    Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J

    1994-04-08

    We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

  6. Myocardial blood flow rate and capillary permeability for 99mTc-DTPA in patients with angiographically normal coronary arteries. Evaluation of the single-injection, residue detection method with intracoronary indicator bolus injection and the use of a mobile gamma camera

    DEFF Research Database (Denmark)

    Svendsen, Jesper Hastrup; Kelbaek, H; Efsen, F

    1994-01-01

    The aims of the present study were to quantitate myocardial perfusion and capillary permeability in the human heart by means of the single-injection, residue detection method using a mobile gamma camera. With this method, the intravascular mean transit time and the capillary extraction fraction (...

  7. Cytochrome c Is Tyrosine 97 Phosphorylated by Neuroprotective Insulin Treatment

    Czech Academy of Sciences Publication Activity Database

    Sanderson, T. H.; Mahapatra, G.; Pecina, Petr; Ji, Q.; Yu, K.; Sinkler, Ch.; Varughese, A.; Kumar, R.; Bukowski, M. J.; Tousignant, R. N.; Salomon, A. R.; Lee, I.; Hüttemann, M.

    2013-01-01

    Roč. 8, č. 11 (2013), e78627 E-ISSN 1932-6203 Institutional support: RVO:67985823 Keywords : cytochrome c * tyrosine phosphorylation * brain ischemia * insulin Subject RIV: ED - Physiology Impact factor: 3.534, year: 2013

  8. Tyrosine biosynthesis, metabolism, and catabolism in plants.

    Science.gov (United States)

    Schenck, Craig A; Maeda, Hiroshi A

    2018-05-01

    L-Tyrosine (Tyr) is an aromatic amino acid (AAA) required for protein synthesis in all organisms, but synthesized de novo only in plants and microorganisms. In plants, Tyr also serves as a precursor of numerous specialized metabolites that have diverse physiological roles as electron carriers, antioxidants, attractants, and defense compounds. Some of these Tyr-derived plant natural products are also used in human medicine and nutrition (e.g. morphine and vitamin E). While the Tyr biosynthesis and catabolic pathways have been extensively studied in microbes and animals, respectively, those of plants have received much less attention until recently. Accumulating evidence suggest that the Tyr biosynthetic pathways differ between microbes and plants and even within the plant kingdom, likely to support the production of lineage-specific plant specialized metabolites derived from Tyr. The interspecies variations of plant Tyr pathway enzymes can now be used to enhance the production of Tyr and Tyr-derived compounds in plants and other synthetic biology platforms. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Manzamenones Inhibit T-Cell Protein Tyrosine Phosphatase

    Directory of Open Access Journals (Sweden)

    Jun'ichi Kobayashi

    2006-02-01

    Full Text Available Abstract: Manzamenones A~C (1~3 and E~F (5~6, unique oxylipin metabolites isolated from a marine sponge Plakortis sp., have been found to exhibit inhibitory activity against Tcell protein tyrosine phosphatase (TCPTP. The inhibitory activity of 2 and 5 against TCPTP was 4 times more potent than that against protein tyrosine phosphatase-1B (PTP1B.

  10. Co-conserved features associated with cis regulation of ErbB tyrosine kinases.

    Directory of Open Access Journals (Sweden)

    Amar Mirza

    Full Text Available BACKGROUND: The epidermal growth factor receptor kinases, or ErbB kinases, belong to a large sub-group of receptor tyrosine kinases (RTKs, which share a conserved catalytic core. The catalytic core of ErbB kinases have functionally diverged from other RTKs in that they are activated by a unique allosteric mechanism that involves specific interactions between the kinase core and the flanking Juxtamembrane (JM and COOH-terminal tail (C-terminal tail. Although extensive studies on ErbB and related tyrosine kinases have provided important insights into the structural basis for ErbB kinase functional divergence, the sequence features that contribute to the unique regulation of ErbB kinases have not been systematically explored. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we use a Bayesian approach to identify the selective sequence constraints that most distinguish ErbB kinases from other receptor tyrosine kinases. We find that strong ErbB kinase-specific constraints are imposed on residues that tether the JM and C-terminal tail to key functional regions of the kinase core. A conserved RIxKExE motif in the JM-kinase linker region and a glutamine in the inter-lobe linker are identified as two of the most distinguishing features of the ErbB family. While the RIxKExE motif tethers the C-terminal tail to the N-lobe of the kinase domain, the glutamine tethers the C-terminal tail to hinge regions critical for inter-lobe movement. Comparison of the active and inactive crystal structures of ErbB kinases indicates that the identified residues are conformationally malleable and can potentially contribute to the cis regulation of the kinase core by the JM and C-terminal tail. ErbB3, and EGFR orthologs in sponges and parasitic worms, diverge from some of the canonical ErbB features, providing insights into sub-family and lineage-specific functional specialization. CONCLUSION/SIGNIFICANCE: Our analysis pinpoints key residues for mutational analysis, and

  11. Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity

    DEFF Research Database (Denmark)

    Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno

    2004-01-01

    or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone......, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX......[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570...

  12. Cryopreservation of common carp (Cyprinus carpio L.) sperm induces protein phosphorylation in tyrosine and threonine residues

    Czech Academy of Sciences Publication Activity Database

    Li, P.; Hulák, M.; Li, Z.; H.; Šulc, Miroslav; Pšenička, M.; Rodina, M.; Gela, D.; Linhart, O.

    2013-01-01

    Roč. 80, č. 2 (2013), s. 84-89 ISSN 0093-691X Institutional support: RVO:61388971 Keywords : Cryopreservation * Sperm * Phosphorylation Subject RIV: CE - Biochemistry Impact factor: 1.845, year: 2013

  13. Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis

    DEFF Research Database (Denmark)

    Pedersen, A.K.; Guo, X.; Møller, K.B.

    2004-01-01

    , in comparison with Phe(182)-PTPs, have significantly decreased k(cat) values, and to a lesser degree, decreased k(cat)/K-m values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His(182) is due to interactions with Asp(181) and with Gln(262). We...

  14. Salivary peptide tyrosine-tyrosine 3-36 modulates ingestive behavior without inducing taste aversion.

    Science.gov (United States)

    Hurtado, Maria D; Sergeyev, Valeriy G; Acosta, Andres; Spegele, Michael; La Sala, Michael; Waler, Nickolas J; Chiriboga-Hurtado, Juan; Currlin, Seth W; Herzog, Herbert; Dotson, Cedrick D; Gorbatyuk, Oleg S; Zolotukhin, Sergei

    2013-11-20

    Hormone peptide tyrosine-tyrosine (PYY) is secreted into circulation from the gut L-endocrine cells in response to food intake, thus inducing satiation during interaction with its preferred receptor, Y2R. Clinical applications of systemically administered PYY for the purpose of reducing body weight were compromised as a result of the common side effect of visceral sickness. We describe here a novel approach of elevating PYY in saliva in mice, which, although reliably inducing strong anorexic responses, does not cause aversive reactions. The augmentation of salivary PYY activated forebrain areas known to mediate feeding, hunger, and satiation while minimally affecting brainstem chemoreceptor zones triggering nausea. By comparing neuronal pathways activated by systemic versus salivary PYY, we identified a metabolic circuit associated with Y2R-positive cells in the oral cavity and extending through brainstem nuclei into hypothalamic satiety centers. The discovery of this alternative circuit that regulates ingestive behavior without inducing taste aversion may open the possibility of a therapeutic application of PYY for the treatment of obesity via direct oral application.

  15. Identification of Tyrosine Kinase Src Responsible for Antimicrobial Peptides Production in Bombyx mori.

    Science.gov (United States)

    Li, Xianyang; Hua, Xiaoting; Song, Liang; Xia, Qingyou; Wang, Fei

    2017-01-01

    Src is a non-receptor protein tyrosine kinase ubiquitously expressed in animals. It is involved in various cellular processes, including the innate immune response in mammals. However, less is known about the function of insect Src. Here we presented a homologue of Src in silkworm (Bombyx mori), named as BmSrc by phylogenetic analysis, homologous comparison and domain prediction. BmSrc contains the conserved phosphorylation residues and possesses tyrosine kinase activity. The expression pattern of BmSrc mRNA was specific in developmental stages and tissues. The highest expression of BmSrc was detected in moth stage, and the gonads showed the highest expression during larval stage. We then found over-expression of BmSrc in BmE cell resulted in an increase of p38 mitogen-activated protein kinase (p38 MAPK) and Akt phosphorylation but a decrease in extracellular signal-regulated kinase (ERK) phosphorylation. Finally, we demonstrated that BmSrc promoted the production of antimicrobial peptides (AMPs). These results implied that BmSrc is involved in immune response of silkworm possibly through activating p38 MAPK and Akt signaling pathway. Our study may provide reference for further investigation of the biological function of BmSrc in Bombyx mori. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Implications of tyrosine phosphoproteomics in cervical carcinogenesis

    Directory of Open Access Journals (Sweden)

    DeFord James

    2008-01-01

    Full Text Available Abstract Background Worldwide cervical cancer remains a leading cause of mortality from gynecologic malignancies. The link between cervical cancer and persistent infection with HPV has been established. At a molecular level little is known about the transition from the precancerous state to invasive cancer. To elucidate this process, cervical biopsies from human specimens were obtained from precancerous state to stage III disease. Methods Cervical biopsies were obtained from patients with a diagnosis of cervical cancer undergoing definitive surgery or staging operation. Biopsies were obtained from patients with precancerous lesions at the time of their excisional procedure. Control samples were obtained from patients undergoing hysterectomy for benign conditions such as fibroids. Samples were subjected to proteomic profiling using two dimensional gel electrophoresis with subsequent trypsin digestion followed by MALDI-TOF protein identification. Candidate proteins were then further studied using western blotting, immunoprecipitation and immunohistochemistry. Results Annexin A1 and DNA-PKcs were found to be differentially expressed. Phosphorylated annexin A1 was up regulated in diseased states in comparison to control and its level was strongly detected in the serum of cervical cancer patients compared to controls. DNA-PKcs was noted to be hyperphosphorylated and fragmented in cancer when compared to controls. By immunohistochemistry annexin A1 was noted in the vascular environment in cancer and certain precancerous samples. Conclusion This study suggests a probable role for protein tyrosine phosphorylation in cervical carcinogenesis. Annexin A1 and DNA-PK cs may have synergistic effects with HPV infection. Precancerous lesions that may progress to cervical cancer may be differentiated from lesions that will not base on similar immunohistochemical profile to invasive squamous cell carcinoma.

  17. Sequential Proton Loss Electron Transfer in Deactivation of Iron(IV) Binding Protein by Tyrosine Based Food Components

    DEFF Research Database (Denmark)

    Tang, Ning; Skibsted, Leif Horsfelt

    2017-01-01

    to understand the relationship between density functional theory calculated molecular descriptors and kinetic data, which was further modeled by partial least squares for quantitative structure-activity relationship analysis. In addition, a three tyrosine residue containing protein, lysozyme, was also found...... by protonation of ferrylmyoglobin and facilitated proton transfer at acidic conditions. Enthalpy-entropy compensation effects were observed for the activation parameters (ΔH† and ΔS†), indicating the common reaction mechanism. Moreover, principal component analysis combined with heat map were performed...... to be able to reduce ferrylmyoglobin with a second order rate constant of 66 ± 28 L/mol/s as determined by a competitive kinetic method....

  18. Multiple forms of the human tyrosine phosphatase RPTP alpha. Isozymes and differences in glycosylation

    DEFF Research Database (Denmark)

    Daum, G; Regenass, S; Sap, J

    1994-01-01

    -linked carbohydrates. Pulse-chase experiments demonstrated that the smaller RPTP alpha protein is a precursor of the larger one. A high affinity antibody was generated that recognizes the immature protein only; however, both proteins can be detected by Western blot analysis after a simple chemical hydrolysis......Among all the receptor-linked protein-tyrosine-phosphatase RPTP alpha clones described from mammalian tissues, one differed in that it encoded a 9-amino-acid insert 3 residues upstream from the transmembrane segment (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R. Ravera, M., Ricca, G....... Following Superose 12 chromatography, the 100- and 130-kDa species of RPTP alpha emerged as 200- and 340-kDa proteins, respectively. Both species exhibited similar enzymatic activities as determined with a peptide substrate in immunoprecipitates....

  19. Water molecule network and active site flexibility of apo protein tyrosine phosphatase 1B

    DEFF Research Database (Denmark)

    Pedersen, A.K.; Peters, Günther H.J.; Møller, K.B.

    2004-01-01

    the conformation and flexibility of active-site residues as well as the water-molecule network, is a key issue in understanding ligand binding and enzyme kinetics and in structure-based drug design. A 1.95 Angstrom apo PTP1B structure has been obtained, showing four highly coordinated water molecules in the active......Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including......-site pocket of the enzyme; hence, the active site is highly solvated in the apo state. Three of the water molecules are located at positions that approximately correspond to the positions of the phosphate O atoms of the natural substrate phosphotyrosine and form a similar network of hydrogen bonds. The active...

  20. In Vitro Characterization of the Bacillus subtilis Protein Tyrosine Phosphatase YwqE

    Science.gov (United States)

    Mijakovic, Ivan; Musumeci, Lucia; Tautz, Lutz; Petranovic, Dina; Edwards, Robert A.; Jensen, Peter Ruhdal; Mustelin, Tomas; Deutscher, Josef; Bottini, Nunzio

    2005-01-01

    Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains one such CpsB-like PTP, YwqE, in addition to two class II Cys-based PTPs, YwlE and YfkJ. The substrates for both YwlE and YfkJ are presently unknown, while YwqE was shown to dephosphorylate two phosphotyrosine-containing proteins implicated in UDP-glucuronate biosynthesis, YwqD and YwqF. In this study, we characterize YwqE, compare the activities of the three B. subtilis PTPs (YwqE, YwlE, and YfkJ), and demonstrate that the two B. subtilis class II PTPs do not dephosphorylate the physiological substrates of YwqE. PMID:15866923

  1. Leptogenesis and residual CP symmetry

    International Nuclear Information System (INIS)

    Chen, Peng; Ding, Gui-Jun; King, Stephen F.

    2016-01-01

    We discuss flavour dependent leptogenesis in the framework of lepton flavour models based on discrete flavour and CP symmetries applied to the type-I seesaw model. Working in the flavour basis, we analyse the case of two general residual CP symmetries in the neutrino sector, which corresponds to all possible semi-direct models based on a preserved Z 2 in the neutrino sector, together with a CP symmetry, which constrains the PMNS matrix up to a single free parameter which may be fixed by the reactor angle. We systematically study and classify this case for all possible residual CP symmetries, and show that the R-matrix is tightly constrained up to a single free parameter, with only certain forms being consistent with successful leptogenesis, leading to possible connections between leptogenesis and PMNS parameters. The formalism is completely general in the sense that the two residual CP symmetries could result from any high energy discrete flavour theory which respects any CP symmetry. As a simple example, we apply the formalism to a high energy S 4 flavour symmetry with a generalized CP symmetry, broken to two residual CP symmetries in the neutrino sector, recovering familiar results for PMNS predictions, together with new results for flavour dependent leptogenesis.

  2. Mechanisms underlying the inhibitory effects of arsenic compounds on protein tyrosine phosphatase (PTP)

    Energy Technology Data Exchange (ETDEWEB)

    Rehman, Kanwal [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Chen, Zhe [Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou (China); Wang, Wen Wen; Wang, Yan Wei [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Sakamoto, Akira [Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260‐8675 (Japan); Zhang, Yan Fang [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Naranmandura, Hua, E-mail: narenman@zju.edu.cn [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Suzuki, Noriyuki [Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260‐8675 (Japan)

    2012-09-15

    Arsenic binding to biomolecules is considered one of the major toxic mechanisms, which may also be related to the carcinogenic risks of arsenic in humans. At the same time, arsenic is also known to activate the phosphorylation-dependent signaling pathways including the epidermal growth factor receptor, the mitogen-activated protein kinase and insulin/insulin-like growth factor-1 pathways. These signaling pathways originate at the level of receptor tyrosine kinases whose phosphorylation status is regulated by opposing protein tyrosine phosphatase (PTP) activity. Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases and phosphatases, regulates important signaling pathways that are involved in the control of cell proliferation, adhesion and migration. In the present study, we have focused on the interaction of cellular PTPs with toxic trivalent arsenite (iAs{sup III}) and its intermediate metabolites such as monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) in vitro, and then determined the arsenic binding site in PTP by the use of recombinant PTPs (e.g., PTP1B and CD45). Interestingly, the activities of PTP1B (cytoplasm-form) or CD45 (receptor-linked form) were observed to be strongly inhibited by both methylated metabolites (i.e., MMA{sup III} and DMA{sup III}) but not by iAs{sup III}. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMA{sup III} directly bound to the active site cysteine residue of PTP1B (e.g., Cys215), resulting in inhibition of enzyme activity. These results suggest that arsenic exposure may disturb the cellular signaling pathways through PTP inactivation. Highlights: ► This study focused on the interaction of PTPs with trivalent arsenicals in vitro. ► We for the first time confirmed that DMA{sup III} strongly inhibited activity of PTP1B. ► DMA{sup III} directly

  3. Crystal Structure of Human Dual-Specificity Tyrosine-Regulated Kinase 3 Reveals New Structural Features and Insights into its Auto-phosphorylation.

    Science.gov (United States)

    Kim, Kuglae; Cha, Jeong Seok; Cho, Yong-Soon; Kim, Hoyoung; Chang, Nienping; Kim, Hye-Jung; Cho, Hyun-Soo

    2018-04-07

    Dual-specificity tyrosine-regulated kinases (DYRKs) auto-phosphorylate a critical tyrosine residue in their activation loop and phosphorylate their substrate on serine and threonine residues. The auto-phosphorylation occurs intramolecularly and is a one-off event. DYRK3 is selectively expressed at a high level in hematopoietic cells and attenuates erythroblast development, leading to anemia. In the present study, we determined the crystal structure of the mature form of human DYRK3 in complex with harmine, an ATP competitive inhibitor. The crystal structure revealed a phosphorylation site, residue S350, whose phosphorylation increases the stability of DYRK3 and enhances its kinase activity. In addition, our structural and biochemical assays suggest that the N-terminal auto-phosphorylation accessory domain stabilizes the DYRK3 protein, followed by auto-phosphorylation of the tyrosine of the activation loop, which is important for kinase activity. Finally, our docking analysis provides information for the design of novel and potent therapeutics to treat anemia. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Insulin and Metabolic Stress Stimulate Multisite Serine/Threonine Phosphorylation of Insulin Receptor Substrate 1 and Inhibit Tyrosine Phosphorylation*

    Science.gov (United States)

    Hançer, Nancy J.; Qiu, Wei; Cherella, Christine; Li, Yedan; Copps, Kyle D.; White, Morris F.

    2014-01-01

    IRS1 and IRS2 are key substrates of the insulin receptor tyrosine kinase. Mass spectrometry reveals more than 50 phosphorylated IRS1 serine and threonine residues (Ser(P)/Thr(P) residues) in IRS1 from insulin-stimulated cells or human tissues. We investigated a subset of IRS1 Ser(P)/Thr(P) residues using a newly developed panel of 25 phospho-specific monoclonal antibodies (αpS/TmAbIrs1). CHO cells overexpressing the human insulin receptor and rat IRS1 were stimulated with insulin in the absence or presence of inhibitors of the PI3K → Akt → mechanistic target of rapamycin (mTOR) → S6 kinase or MEK pathways. Nearly all IRS1 Ser(P)/Thr(P) residues were stimulated by insulin and significantly suppressed by PI3K inhibition; fewer were suppressed by Akt or mTOR inhibition, and none were suppressed by MEK inhibition. Insulin-stimulated Irs1 tyrosine phosphorylation (Tyr(P)Irs1) was enhanced by inhibition of the PI3K → Akt → mTOR pathway and correlated with decreased Ser(P)-302Irs1, Ser(P)-307Irs1, Ser(P)-318Irs1, Ser(P)-325Irs1, and Ser(P)-346Irs1. Metabolic stress modeled by anisomycin, thapsigargin, or tunicamycin increased many of the same Ser(P)/Thr(P) residues as insulin, some of which (Ser(P)-302Irs1, Ser(P)-307Irs1, and four others) correlated significantly with impaired insulin-stimulated Tyr(P)Irs1. Thus, IRS1 Ser(P)/Thr(P) is an integrated response to insulin stimulation and metabolic stress, which associates with reduced Tyr(P)Irs1 in CHOIR/IRS1 cells. PMID:24652289

  5. Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase.

    Science.gov (United States)

    Cieńska, Małgorzata; Labus, Karolina; Lewańczuk, Marcin; Koźlecki, Tomasz; Liesiene, Jolanta; Bryjak, Jolanta

    2016-01-01

    Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH2), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH2. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH2 complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity.

  6. Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase.

    Directory of Open Access Journals (Sweden)

    Małgorzata Cieńska

    Full Text Available Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA by immobilized tyrosinase in the presence of ascorbic acid (AH2, which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH2. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH2 complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native to 30% (immobilized enzyme. To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme and 70% (immobilized. A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity.

  7. Identification of a fungi-specific lineage of protein kinases closely related to tyrosine kinases.

    Science.gov (United States)

    Zhao, Zhongtao; Jin, Qiaojun; Xu, Jin-Rong; Liu, Huiquan

    2014-01-01

    Tyrosine kinases (TKs) specifically catalyze the phosphorylation of tyrosine residues in proteins and play essential roles in many cellular processes. Although TKs mainly exist in animals, recent studies revealed that some organisms outside the Opisthokont clade also contain TKs. The fungi, as the sister group to animals, are thought to lack TKs. To better understand the origin and evolution of TKs, it is important to investigate if fungi have TK or TK-related genes. We therefore systematically identified possible TKs across the fungal kingdom by using the profile hidden Markov Models searches and phylogenetic analyses. Our results confirmed that fungi lack the orthologs of animal TKs. We identified a fungi-specific lineage of protein kinases (FslK) that appears to be a sister group closely related to TKs. Sequence analysis revealed that members of the FslK clade contain all the conserved protein kinase sub-domains and thus are likely enzymatically active. However, they lack key amino acid residues that determine TK-specific activities, indicating that they are not true TKs. Phylogenetic analysis indicated that the last common ancestor of fungi may have possessed numerous members of FslK. The ancestral FslK genes were lost in Ascomycota and Ustilaginomycotina and Pucciniomycotina of Basidiomycota during evolution. Most of these ancestral genes, however, were retained and expanded in Agaricomycetes. The discovery of the fungi-specific lineage of protein kinases closely related to TKs helps shed light on the origin and evolution of TKs and also has potential implications for the importance of these kinases in mushroom fungi.

  8. Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240.

    Science.gov (United States)

    Fenton, Tim R; Nathanson, David; Ponte de Albuquerque, Claudio; Kuga, Daisuke; Iwanami, Akio; Dang, Julie; Yang, Huijun; Tanaka, Kazuhiro; Oba-Shinjo, Sueli Mieko; Uno, Miyuki; Inda, Maria del Mar; Wykosky, Jill; Bachoo, Robert M; James, C David; DePinho, Ronald A; Vandenberg, Scott R; Zhou, Huilin; Marie, Suely K N; Mischel, Paul S; Cavenee, Webster K; Furnari, Frank B

    2012-08-28

    Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

  9. Fundamentals on the biochemistry of peroxynitrite and protein tyrosine nitration

    Directory of Open Access Journals (Sweden)

    Silvina Bartesaghi

    2018-04-01

    Full Text Available In this review we provide an analysis of the biochemistry of peroxynitrite and tyrosine nitration. Peroxynitrite is the product of the diffusion-controlled reaction between superoxide (O2•- and nitric oxide (•NO. This process is in competition with the enzymatic dismutation of O2•- and the diffusion of •NO across cells and tissues and its reaction with molecular targets (e.g. guanylate cyclase. Understanding the kinetics and compartmentalization of the O2•- / •NO interplay is critical to rationalize the shift of •NO from a physiological mediator to a cytotoxic intermediate. Once formed, peroxynitrite (ONOO- and ONOOH; pKa = 6,8 behaves as a strong one and two-electron oxidant towards a series of biomolecules including transition metal centers and thiols. In addition, peroxynitrite anion can secondarily evolve to secondary radicals either via its fast reaction with CO2 or through proton-catalyzed homolysis. Thus, peroxynitrite can participate in direct (bimolecular and indirect (through secondary radical intermediates oxidation reactions; through these processes peroxynitrite can participate as cytotoxic effector molecule against invading pathogens and/or as an endogenous pathogenic mediator. Peroxynitrite can cause protein tyrosine nitration in vitro and in vivo. Indeed, tyrosine nitration is a hallmark of the reactions of •NO-derived oxidants in cells and tissues and serves as a biomarker of oxidative damage. Protein tyrosine nitration can mediate changes in protein structure and function that affect cell homeostasis. Tyrosine nitration in biological systems is a free radical process that can be promoted either by peroxynitrite-derived radicals or by other related •NO-dependent oxidative processes. Recently, mechanisms responsible of tyrosine nitration in hydrophobic biostructures such as membranes and lipoproteins have been assessed and involve the parallel occurrence and connection with lipid

  10. Identifying proteins that can form tyrosine-cysteine crosslinks.

    Science.gov (United States)

    Martinie, Ryan J; Godakumbura, Pahan I; Porter, Elizabeth G; Divakaran, Anand; Burkhart, Brandon J; Wertz, John T; Benson, David E

    2012-10-01

    Protein cofactors represent a unique class of redox active posttranslational protein modifications formed in or by metalloproteins. Once formed, protein cofactors provide a one-electron oxidant, which is tethered to the protein backbone. Twenty-five proteins are known to contain protein cofactors, but this number is likely limited by the use of crystallography as the identification technique. In order to address this limitation, a search of all reported protein structures for chemical environments conducive to forming a protein cofactor through tyrosine and cysteine side chain crosslinking yielded three hundred candidate proteins. Using hydrogen bonding and metal center proximity, the three hundred proteins were narrowed to four highly viable candidates. An orphan metalloprotein (BF4112) was examined to validate this methodology, which identifies proteins capable of crosslinking tyrosine and cysteine sidechains. A tyrosine-cysteine crosslink was formed in BF4112 using copper-dioxygen chemistry, as in galactose oxidase. Liquid chromatography-MALDI mass spectrometry and optical spectroscopy confirmed tyrosine-cysteine crosslink formation in BF4112. This finding demonstrates the efficacy of these predictive methods and the minimal constraints, provided by the BF4112 protein structure, in tyrosine-cysteine crosslink formation. This search method, when coupled with physiological evidence for crosslink formation and function as a cofactor, could identify additional protein-derived cofactors.

  11. RADIATION-INDUCED INHIBITION OF TUMOR-GROWTH AS MONITORED BY PET USING L-[1-C-11]TYROSINE AND FLUORINE-18-FLUORODEOXYGLUCOSE

    NARCIS (Netherlands)

    DAEMEN, BJG; ELSINGA, PH; PAANS, AMJ; WIERINGA, AR; KONINGS, AWT; VAALBURG, W

    The potential use of PET to monitor radiotherapeutic effects on tumors has been evaluated with L-[1-C-11]tyrosine and 18FDG. Single x-ray doses of 10, 30, or 50 Gy have been applied to rhabdomyosarcoma tumors growing in the flank of rats. Dose-dependent reductions of tracer uptake were registered by

  12. Structure determination of T-cell protein-tyrosine phosphatase

    DEFF Research Database (Denmark)

    Iversen, L.F.; Møller, K. B.; Pedersen, A.K.

    2002-01-01

    Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly...... homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co...

  13. Steric Hindrance as a Basis for Structure-Based Design of Selective Inhibitors of Protein-Tyrosine Phosphatases

    DEFF Research Database (Denmark)

    Iversen, L. F.; Andersen, H. S.; Møller, K. B.

    2001-01-01

    Utilizing structure-based design, we have previously demonstrated that it is possible to obtain selective inhibitors of protein-tyrosine phosphatase 1B (PTP1B). A basic nitrogen was introduced into a general PTP inhibitor to form a salt bridge to Asp48 in PTP1B and simultaneously cause repulsion...... residues in the same position in other PTPs cause steric hindrance and reduced substrate recognition capacity [Peters, G. H., et al. (2000) J. Biol. Chem. 275, 18201−18209]. The current study was undertaken to investigate the feasibility of structure-based design, utilizing these differences...... in accessibility to the active site among various PTPs. We show that a general, low-molecular weight PTP inhibitor can be developed into a highly selective inhibitor for PTP1B and TC-PTP by introducing a substituent, which is designed to address the region around residues 258 and 259. Detailed enzyme kinetic...

  14. Novel tyrosine phosphorylation sites in rat skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS

    DEFF Research Database (Denmark)

    Zhang, Xiangmin; Højlund, Kurt; Luo, Moulun

    2012-01-01

    Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (...

  15. Evaluation of an Adsorbent Based on Agricultural Waste (Corn Cobs for Removal of Tyrosine and Phenylalanine from Aqueous Solutions

    Directory of Open Access Journals (Sweden)

    Cibele C. O. Alves

    2013-01-01

    Full Text Available Adsorption of phenolic amino acids, such as phenylalanine and tyrosine, is quite relevant for the production of protein hydrolysates used as dietary formulations for patients suffering from congenital disorders of amino acid metabolism, such as phenylketonuria. In this study, an adsorbent prepared from corn cobs was evaluated for the removal of tyrosine (Tyr from both a single component solution and a binary aqueous solution with phenylalanine (Phe. The adsorption behavior of tyrosine was similar to that of phenylalanine in single component solutions, however, with a much lower adsorption capacity (14 mg g−1 for Tyr compared to 109 mg g−1 for Phe. Tyr adsorption kinetics was satisfactorily described by a pseudosecond-order model as it was for Phe. In adsorption equilibrium studies for binary mixtures, the presence of Tyr in Phe solutions favored Phe faster adsorption whereas the opposite behavior was observed for the presence of Phe in Tyr solutions. Such results indicate that, in binary systems, Phe will be adsorbed preferably to Tyr, and this is a welcome feature when employing the prepared adsorbent for the removal of Phe from protein hydrolysates to be used in dietary formulations for phenylketonuria treatment.

  16. Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage

    Science.gov (United States)

    Narula, Gagandeep; Tse-Dinh, Yuk-Ching

    2012-01-01

    Bacterial and archaeal topoisomerase I display selectivity for a cytosine base 4 nt upstream from the DNA cleavage site. Recently, the solved crystal structure of Escherichia coli topoisomerase I covalently linked to a single-stranded oligonucleotide revealed that R169 and R173 interact with the cytosine base at the −4 position via hydrogen bonds while the phenol ring of Y177 wedges between the bases at the −4 and the −5 position. Substituting R169 to alanine changed the selectivity of the enzyme for the base at the −4 position from a cytosine to an adenine. The R173A mutant displayed similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity. Mutation of Y177 to serine or alanine rendered the enzyme inactive. Although mutation of each of these residues led to different outcomes, R169, R173 and Y177 work together to interact with a cytosine base at the −4 position to facilitate DNA cleavage. These strictly conserved residues might act after initial substrate binding as a Molecular Ruler to form a protein–DNA complex with the scissile phosphate positioned at the active site for optimal DNA cleavage by the tyrosine hydroxyl nucleophile to facilitate DNA cleavage in the reaction pathway. PMID:22833607

  17. Expression of tyrosine kinase gene in mouse thymic stromal cells

    NARCIS (Netherlands)

    Rinke de Wit, T. F.; Izon, D. J.; Revilla, C.; Oosterwegel, M.; Bakker, A. Q.; van Ewijk, W.; Kruisbeek, A. M.

    1996-01-01

    Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both

  18. Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances

    NARCIS (Netherlands)

    B.C. Tilly (Bernard); N. van den Berghe (Nina); L.G. Tertoolen; M.J. Edixhoven (Marcel); H.R. de Jonge (Hugo)

    1993-01-01

    textabstractUsing the human Intestine 407 cell line as a model, we investigated a possible role for tyrosine kinase(s) in regulating the ion efflux pathways induced by hyposmotic stimulation (regulatory volume decrease, RVD). Pretreatment of 125I(-)-and 86Rb(+)-loaded

  19. Auto-thiophosphorylation activity of Src tyrosine kinase.

    Science.gov (United States)

    Cabail, M Zulema; Chen, Emily I; Koller, Antonius; Miller, W Todd

    2016-07-07

    Intermolecular autophosphorylation at Tyr416 is a conserved mechanism of activation among the members of the Src family of nonreceptor tyrosine kinases. Like several other tyrosine kinases, Src can catalyze the thiophosphorylation of peptide and protein substrates using ATPγS as a thiophosphodonor, although the efficiency of the reaction is low. Here, we have characterized the ability of Src to auto-thiophosphorylate. Auto-thiophosphorylation of Src at Tyr416 in the activation loop proceeds efficiently in the presence of Ni(2+), resulting in kinase activation. Other tyrosine kinases (Ack1, Hck, and IGF1 receptor) also auto-thiophosphorylate in the presence of Ni(2+). Tyr416-thiophosphorylated Src is resistant to dephosphorylation by PTP1B phosphatase. Src and other tyrosine kinases catalyze auto-thiophosphorylation in the presence of Ni(2+). Thiophosphorylation of Src occurs at Tyr416 in the activation loop, and results in enhanced kinase activity. Tyr416-thiophosphorylated Src could serve as a stable, persistently-activated mimic of Src.

  20. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  1. Role of Bruton's tyrosine kinase in B cells and malignancies

    NARCIS (Netherlands)

    Pal Singh, S. (Simar); F. Dammeijer (Floris); R.W. Hendriks (Rudi)

    2018-01-01

    textabstractBruton's tyrosine kinase (BTK) is a non-receptor kinase that plays a crucial role in oncogenic signaling that is critical for proliferation and survival of leukemic cells in many B cell malignancies. BTK was initially shown to be defective in the primary immunodeficiency X-linked

  2. Understanding proton affinity of tyrosine sidechain in hydrophobic ...

    Indian Academy of Sciences (India)

    Tyrosine (Tyr), like histidine (His), is known to play a crucial role in a wide range of chemical and biochemi- cal processes3 primarily through (a) deprotonation of the phenolic –OH group present in its sidechain and. #Dedicated to Prof. N Sathyamurthy on his 60th birthday. ∗. For correspondence. (b) formation of hydrogen ...

  3. Isolation of a tyrosine-activating enzyme from baker's yeast

    NARCIS (Netherlands)

    Ven, A.M. van de; Koningsberger, V.V.; Overbeek, J.Th.G.

    1958-01-01

    The extracts of ether-CO2-frozen baker's yeast contain enzymes that catalyze the ATP-linked amino acid activation by way of pyrophosphate elimination. From the extract a tyrosine-activating enzyme could be isolated, which, judging from ultracentrifugation and electrophoretic data, was about 70% pure

  4. D-tyrosine negatively regulates melanin synthesis by competitively inhibiting tyrosinase activity.

    Science.gov (United States)

    Park, Jisu; Jung, Hyejung; Kim, Kyuri; Lim, Kyung-Min; Kim, Ji-Young; Jho, Eek-Hoon; Oh, Eok-Soo

    2017-11-09

    Although L-tyrosine is well known for its melanogenic effect, the contribution of D-tyrosine to melanin synthesis was previously unexplored. Here, we reveal that, unlike L-tyrosine, D-tyrosine dose-dependently reduced the melanin contents of human MNT-1 melanoma cells and primary human melanocytes. In addition, 500 μM of D-tyrosine completely inhibited 10 μM L-tyrosine-induced melanogenesis, and both in vitro assays and L-DOPA staining MNT-1 cells showed that tyrosinase activity is reduced by D-tyrosine treatment. Thus, D-tyrosine appears to inhibit L-tyrosine-mediated melanogenesis by competitively inhibiting tyrosinase activity. Furthermore, we found that D-tyrosine inhibited melanogenesis induced by α-MSH treatment or UV irradiation, which are the most common environmental factors responsible for melanin synthesis. Finally, we confirmed that D-tyrosine reduced melanin synthesis in the epidermal basal layer of a 3D human skin model. Taken together, these data suggest that D-tyrosine negatively regulates melanin synthesis by inhibiting tyrosinase activity in melanocyte-derived cells. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Exploring the sensitivity of ZnO nanotubes to tyrosine nitration: A DFT approach

    Energy Technology Data Exchange (ETDEWEB)

    Maddahi, Pari Sadat [Department of Physics, Ferdowsi University of Mashhad, Mashhad (Iran, Islamic Republic of); Nanoresearch Center, Ferdowsi University of Mashhad, Mashhad (Iran, Islamic Republic of); Shahtahmassebi, Nasser, E-mail: Nasser@um.ac.ir [Department of Physics, Ferdowsi University of Mashhad, Mashhad (Iran, Islamic Republic of); Nanoresearch Center, Ferdowsi University of Mashhad, Mashhad (Iran, Islamic Republic of); Rezaee Roknabadi, Mahmood [Department of Physics, Ferdowsi University of Mashhad, Mashhad (Iran, Islamic Republic of); Moosavi, Fatemeh [Department of Chemistry, Ferdowsi University of Mashhad, Mashhad (Iran, Islamic Republic of)

    2016-05-27

    Due to association of protein tyrosine nitration (PTN) with development of some serious human disorders and diseases, in this paper, the possible applications of ZnO-based nanobiosensors in nitrated tyrosine (nTyr) detection were explored within the density functional framework. With this motivation, the interaction of nTyr with ZnO single walled nanotubes via all possible active sites of nTyr was investigated. The results show the tendency of nTyr to interact through its nitro site (forming nitro-site configuration) with ZnO SWNTs as it has the highest binding energy; while, the charge–solvent configuration involving the interaction of nTyr's phenolic ring has the second place in terms of binding energy magnitude. Regardless of which active site contributes in interaction, the binding energies exhibit an ascending trend with decrease of SWNTs' curvature. Electronic properties analysis indicates that nTyr interaction via its nitro group results in formation of some flat bands inside the band gap region leading to significant reduction of overall band gap energy. Similar behavior is also observed in charge–solvent configuration but the band gap energy is larger. These red shifts are mainly attributed to contribution of 2p orbitals of species present in nTyr. Also, the hybridization of 3d orbital of Zn atom with 2p orbitals of nitro group atomic species is found responsible for bonding formation in bioconjugated system possessing the highest binding energy. Comparison of the electronic band structure of ZnO SWNT–Tyr with that of ZnO SWNT–nTyr indicates the sensitivity of ZnO SWNTs toward tyrosine nitration hence, a considerable change in its optical spectra is expectable. This introduces ZnO SWNTs as a promising candidate for PTN detection. - Highlights: • Physical properties of ZnO SWNT conjugated with nTyr is studied by DFT method. • nTyr prefers to interact with ZnO SWNTs via the nitro site. • An ascending trend is observed in binding

  6. Lack of protein-tyrosine sulfation disrupts photoreceptor outer segment morphogenesis, retinal function and retinal anatomy.

    Science.gov (United States)

    Sherry, David M; Murray, Anne R; Kanan, Yogita; Arbogast, Kelsey L; Hamilton, Robert A; Fliesler, Steven J; Burns, Marie E; Moore, Kevin L; Al-Ubaidi, Muayyad R

    2010-11-01

    To investigate the role(s) of protein-tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1(-/-) /Tpst2 (-/-) ) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein-tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  7. Tyrosine hydroxylase positive nerves and mast cells in the porcine gallbladder

    Directory of Open Access Journals (Sweden)

    I. Stefanov

    2017-03-01

    Full Text Available The aim of this study was to detect the localisation of tyrosine hydroxylase (TH positive nerve fibres (THN and distribution of tyrosine hydroxylase positive mast cells (THMC in the wall of porcine gallbladder. THN were observed as single fibres, nerve fibres forming perivascular plexuses and nerve fibres grouped within the nerve fascicles. In the gallbladder`s fundus, body and neck, the TH+ fibres formed mucosal, muscular and serosal nonganglionated nerve plexuses. Toluidine blue (TB staining was used to confirm that the TH positive cells were mast cells. The number of THMC in the propria of gallbladder`s fundus, body and neck was significantly higher than in the muscular and serosal layers in both genders. The number of mast cells in the musculature was higher than in the serosa. The density and location of the THMC were similar to the TB positive (with gamma meta-chromasia mast cells in both males and females, and statistically significant difference was not established. In conclusion, original data concerning the existence and localisation of catecholaminergic nerves and THMC distribution in the porcine gallbladder’s wall are presented. The results could con-tribute to the body of knowledge of functional communication between autonomic nerves and mast cells in the gallbladder.

  8. Residual gas analysis

    International Nuclear Information System (INIS)

    Berecz, I.

    1982-01-01

    Determination of the residual gas composition in vacuum systems by a special mass spectrometric method was presented. The quadrupole mass spectrometer (QMS) and its application in thin film technology was discussed. Results, partial pressure versus time curves as well as the line spectra of the residual gases in case of the vaporization of a Ti-Pd-Au alloy were demonstrated together with the possible construction schemes of QMS residual gas analysers. (Sz.J.)

  9. Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules.

    Science.gov (United States)

    Jorge-Finnigan, Ana; Kleppe, Rune; Jung-Kc, Kunwar; Ying, Ming; Marie, Michael; Rios-Mondragon, Ivan; Salvatore, Michael F; Saraste, Jaakko; Martinez, Aurora

    2017-08-25

    Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Separate domains of the insulin receptor contain sites of autophosphorylation and tyrosine kinase activity

    International Nuclear Information System (INIS)

    Goren, H.J.; White, M.F.; Khan, C.R.

    1987-01-01

    The authors have studied the structure and function of the solubilized insulin receptor before and after partial proteolytic digestion to define domains in the β-subunit that undergo autophosphorylation and contain the tyrosine kinase activity. Wheat germ agglutinin purified insulin receptor from Fao cells was digested briefly at 22 0 C with low concentrations of trypsin, staphylococcal V8 protease, or elastase. Autophosphorylation of the β-subunit was carried out before and after digestion, and the [ 32 P]phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and analyzed by tryptic peptide mapping by use of reverse-phase high-performance liquid chromatography. The 85-kDa fragment was not immunoprecipitated by an antibody directed against the C-terminal domain of the β-subunit (αPep-1), indicating that this region of the receptor was lost. The 85-kDa fragment contained about half of the [ 32 P]phosphate originally found in the β-subunit, and tryptic peptide mapping showed that two major tryptic phosphopeptides (previously called pY2 and pY3) were removed. Three other tryptic phosphopeptides (pY1, pY1a, and pY4) were found in the 85- and 70-kDa fragments. To determined the structural requirements for kinase activity, the insulin receptor was subjected to tryptic digestion for 30 s-30 min, such that the receptor was composed exclusively of 85- and 70-kDa fragments of the β-subunit. The 85-kDa fragment exhibited autophosphorylation at pY1, pY1a, and pY4. Both the 85- and 70-kDa fragments phosphorylated tyrosine residues in a synthetic decapeptide that has the sequence of the C-terminal domain of the β-subunit of human insulin rare in the receptor

  11. Protein tyrosine phosphatase-1B contributes to LPS-induced leptin resistance in male rats

    Science.gov (United States)

    Borges, Beatriz de Carvalho; Rorato, Rodrigo C.; Uchoa, Ernane Torres; Marangon, Paula B.; Elias, Carol F.; Antunes-Rodrigues, Jose

    2014-01-01

    Leptin resistance is induced by the feedback inhibitors tyrosine phosphatase-1B (PTP1B) and decreased Src homology 2 domain-containing tyrosine phosphatase-2 (SHP-2) signaling. To investigate the participation of PTP1B and SHP-2 in LPS-induced leptin resistance, we injected repeated (6-LPS) intraperitoneal LPS doses (100 μg/kg ip) for comparison with a single (1-LPS) treatment and evaluated the expression of SHP-2, PTP1B, p-ERK1/2, and p-STAT3 in the hypothalamus of male Wistar rats. The single LPS treatment increased the expression of p-STAT3 and PTP1B but not SHP-2. The repeated LPS treatment reduced SHP-2, increased PTP1B, and did not change p-STAT3. We observed that the PTP1B expression induced by the endotoxin was highly colocalized with leptin receptor cells in the hypothalamus of LepRb-IRES-Cre-tdTomato reporter mice. The single, but not the repeated, LPS treatment decreased the food intake and body weight. Leptin had no stimulatory effect on the hypophagia, body weight loss, or pSTAT3 expression in 6-LPS rats, indicating leptin unresponsiveness. Notably, the PTP1B inhibitor (3.0 nmol/rat in 5 μl icv) restored the LPS-induced hypophagia in 6-LPS rats and restored the ability of leptin to reduce food intake and body weight as well as to phosphorylate STAT3 in the arcuate, paraventricular, and ventromedial nuclei of the hypothalamus. The present data suggest that an increased PTP1B expression in the hypothalamus underlies the development of leptin resistance during repeated exposure to LPS. Our findings contribute to understanding the mechanisms involved in leptin resistance during low-grade inflammation as seen in obesity. PMID:25352433

  12. L-tyrosine hydrochloride crystals under high pressures via Raman Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Santos, C.A.A.S.; Facanha Filho, P.F.; Santos, A.O. dos; Ribeiro, L.H.L.; Victor, F.M.S.; Abreu, D.C.; Carvalho, J.O.; Soares, R.A.; Sousa, J.C.F.; Lima, R.C.; Cavaignac, A.O. [Universidade Federal do Maranhao (UFMA), MA (Brazil)

    2016-07-01

    Full text: Amino acid single crystals have been attracted researchers in recent years due to their potential applications as second harmonic generator. The goal of this work is to produce semi organic single crystals of L-tyrosine hydrochloride (LTHCl) and verify the behavior of their vibrational normal modes under high pressures and the stability of material in these conditions extremes. The LTHCl single crystals were produced for solubilization of amino acid L-tyrosine in hydrochloric acid by slow evaporation technique of the solvent in room temperature. The technique of X-ray diffraction (XRD) and the refinement of structure by the Rietveld method were used to confirm the crystal structure. The LTHCl crystal belongs to the monoclinic crystal system having two molecules per unit cell. The refinement by the Rietveld method showed good results with Rwp = 8.49% and Rp = 6.29% with S = 1.13. Raman scattering measurements as a function of pressure was performed in a piece of crystal from the ambient pressure to 7.2 GPa and Nujol was used as pressure medium. It was observed the appearance of a weak band around 163 cm-1 between pressures of 0.5 and 1.0 GPa, which characterize an phase transition undergone by the crystal. Moreover, this band gains intensity as pressure increases while gradual decreasing relative intensity of the very strong band at 123 cm-1 for all range of pressure also was observed. In fact, almost all bands of the spectra have undergone strong decreasing up to 7.2 GPa. However, on release of pressure the crystal has reached the original phase again. Therefore, the results showed this material cannot be suitable for the application (NLO) in this range of pressure. (author)

  13. Protein tyrosine phosphatase-1B contributes to LPS-induced leptin resistance in male rats.

    Science.gov (United States)

    Borges, Beatriz de Carvalho; Rorato, Rodrigo C; Uchoa, Ernane Torres; Marangon, Paula B; Elias, Carol F; Antunes-Rodrigues, Jose; Elias, Lucila L K

    2015-01-01

    Leptin resistance is induced by the feedback inhibitors tyrosine phosphatase-1B (PTP1B) and decreased Src homology 2 domain-containing tyrosine phosphatase-2 (SHP-2) signaling. To investigate the participation of PTP1B and SHP-2 in LPS-induced leptin resistance, we injected repeated (6-LPS) intraperitoneal LPS doses (100 μg/kg ip) for comparison with a single (1-LPS) treatment and evaluated the expression of SHP-2, PTP1B, p-ERK1/2, and p-STAT3 in the hypothalamus of male Wistar rats. The single LPS treatment increased the expression of p-STAT3 and PTP1B but not SHP-2. The repeated LPS treatment reduced SHP-2, increased PTP1B, and did not change p-STAT3. We observed that the PTP1B expression induced by the endotoxin was highly colocalized with leptin receptor cells in the hypothalamus of LepRb-IRES-Cre-tdTomato reporter mice. The single, but not the repeated, LPS treatment decreased the food intake and body weight. Leptin had no stimulatory effect on the hypophagia, body weight loss, or pSTAT3 expression in 6-LPS rats, indicating leptin unresponsiveness. Notably, the PTP1B inhibitor (3.0 nmol/rat in 5 μl icv) restored the LPS-induced hypophagia in 6-LPS rats and restored the ability of leptin to reduce food intake and body weight as well as to phosphorylate STAT3 in the arcuate, paraventricular, and ventromedial nuclei of the hypothalamus. The present data suggest that an increased PTP1B expression in the hypothalamus underlies the development of leptin resistance during repeated exposure to LPS. Our findings contribute to understanding the mechanisms involved in leptin resistance during low-grade inflammation as seen in obesity. Copyright © 2015 the American Physiological Society.

  14. Sequence-specific backbone 1H, 13C and 15N assignments of the catalytic domain of the Escherichia coli protein tyrosine kinase, Wzc

    Science.gov (United States)

    Temel, Deniz B.; Dutta, Kaushik; Ghose, Ranajeet

    2012-01-01

    Protein tyrosine kinases in bacteria are structurally and functionally distinct from their eukaryotic counterparts. The largest family of bacterial tyrosine kinases, the BY-kinase family, is highly conserved in Gram-negative and Gram-positive species, and plays a central role in biofilm and capsule formation. In E. coli the BY-kinase, Wzc, is a critical component of the machinery responsible for the synthesis and export of the exo-polysaccharide colanic acid, a key constituent of biofilms. Here we present the main-chain 1HN, 15N, 13C′ and 13Cα, side-chain 13Cβ resonance assignments for a construct that encodes the entire 274-residue cytosolic catalytic domain of Wzc. PMID:23225198

  15. Agricultural pesticide residues

    International Nuclear Information System (INIS)

    Fuehr, F.

    1984-01-01

    The utilization of tracer techniques in the study of agricultural pesticide residues is reviewed under the following headings: lysimeter experiments, micro-ecosystems, translocation in soil, degradation of pesticides in soil, biological availability of soil-applied substances, bound residues in the soil, use of macro- and microautography, double and triple labelling, use of tracer labelling in animal experiments. (U.K.)

  16. Phosphoproteomics identifies driver tyrosine kinases in sarcoma cell lines and tumors.

    Science.gov (United States)

    Bai, Yun; Li, Jiannong; Fang, Bin; Edwards, Arthur; Zhang, Guolin; Bui, Marilyn; Eschrich, Steven; Altiok, Soner; Koomen, John; Haura, Eric B

    2012-05-15

    Driver tyrosine kinase mutations are rare in sarcomas, and patterns of tyrosine phosphorylation are poorly understood. To better understand the signaling pathways active in sarcoma, we examined global tyrosine phosphorylation in sarcoma cell lines and human tumor samples. Anti-phosphotyrosine antibodies were used to purify tyrosine phosphorylated peptides, which were then identified by liquid chromatography and tandem mass spectrometry. The findings were validated with RNA interference, rescue, and small-molecule tyrosine kinase inhibitors. We identified 1,936 unique tyrosine phosphorylated peptides, corresponding to 844 unique phosphotyrosine proteins. In sarcoma cells alone, peptides corresponding to 39 tyrosine kinases were found. Four of 10 cell lines showed dependence on tyrosine kinases for growth and/or survival, including platelet-derived growth factor receptor (PDGFR)α, MET, insulin receptor/insulin-like growth factor receptor signaling, and SRC family kinase signaling. Rhabdomyosarcoma samples showed overexpression of PDGFRα in 13% of examined cases, and sarcomas showed abundant tyrosine phosphorylation and expression of a number of tyrosine phosphorylated tyrosine kinases, including DDR2, EphB4, TYR2, AXL, SRC, LYN, and FAK. Together, our findings suggest that integrating global phosphoproteomics with functional analyses with kinase inhibitors can identify drivers of sarcoma growth and survival. ©2012 AACR.

  17. D-tyrosine affects aggregation behavior of Pantoea agglomerans.

    Science.gov (United States)

    Yang, Jing; Yu, Jiajia; Jiang, Jing; Liang, Chen; Feng, Yongjun

    2017-02-01

    D-amino acids have been proved to disassemble biofilms by disassociating the matrix. Pantoea agglomerans is characterized by the formation of another kind of multicellular structure called symplasmata, which also remains the ability to form biofilms. In this study, a rice diazotrophic endophyte P. agglomerans YS19 was selected as a model strain to explore the effects of D-amino acids on these two kinds of cell aggregate structures. It was discovered that D-tyrosine disassociates biofilm, yet promotes symplasmata formation. D-tyrosine showed no influence on bacterial growth yet promoted the bacterial motility and inhibited the expression of cellular MalE and OmpF proteins, which enriched our knowledge of the biological effect of D-amino acids and expanded the research ideas of symplasmata formation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Receptor tyrosine kinases: the emerging tip of systems control.

    Science.gov (United States)

    Seger, R; Rodeck, U; Yarden, Y

    2008-01-01

    Receptor tyrosine kinases (RTKs) are transmembrane allosteric enzymes: binding of ligand growth factors to their ectodomains stimulates a cytoplasm-facing tyrosine kinase activity, which initiates a plethora of cellular processes. The enormous complexity of RTK signalling, along with rich involvement in pathologies (e.g. cancer and diabetes), motivated the establishment of the international, multi-disciplinary RTK consortium (http://www.rtkconsort.org/) in 2005. In collaboration with the British Society for Proteome Research and the European Bioinformatics Institute, the Consortium held on July 23rd and 24th a Workshop on Proteomics and Phosphoproteomics of RTK Signalling Networks (Hinxton Hall Conference Centre, Cambridge, UK). As highlighted below, systems control (a layered web of regulatory loops summarised in Fig.1) emerged throughout the workshop as a common theme of many presentations.

  19. Novel Bruton's tyrosine kinase inhibitors currently in development

    Directory of Open Access Journals (Sweden)

    D'Cruz OJ

    2013-03-01

    Full Text Available Osmond J D'Cruz,1 Fatih M Uckun1,21Children's Center for Cancer and Blood Diseases, Children's Hospital Los Angeles, Los Angeles, CA, USA; 2Department of Pediatrics, University of Southern California, Los Angeles, CA, USAAbstract: Bruton's tyrosine kinase (Btk is intimately involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is overexpressed and constitutively active in several B-lineage lymphoid malignancies. Btk has emerged as a new antiapoptotic molecular target for treatment of B-lineage leukemias and lymphomas. Preclinical and early clinical results indicate that Btk inhibitors may be useful in the treatment of leukemias and lymphomas.Keywords: tyrosine kinase, personalized therapy, kinase inhibitors, Btk, leukemia, lymphoma

  20. Stress signaling by Tec tyrosine kinase in the ischemic myocardium.

    Science.gov (United States)

    Zhang, Michael J; Franklin, Sarah; Li, Yifeng; Wang, Sujing; Ru, Xiaochen; Mitchell-Jordan, Scherise A; Mano, Hiroyuki; Stefani, Enrico; Ping, Peipei; Vondriska, Thomas M

    2010-09-01

    Nonreceptor tyrosine kinases have an increasingly appreciated role in cardiac injury and protection. To investigate novel tasks for members of the Tec family of nonreceptor tyrosine kinases in cardiac phenotype, we examined the behavior of the Tec isoform in myocardial ischemic injury. Ischemia-reperfusion, but not cardiac protective agents, induced altered intracellular localization of Tec, highlighting distinct actions of this protein compared with other isoforms, such as Bmx, in the same model. Tec is abundantly expressed in cardiac myocytes and assumes a diffuse intracellular localization under basal conditions but is recruited to striated structures upon various stimuli, including ATP. To characterize Tec signaling targets in vivo, we performed an exhaustive proteomic analysis of Tec-binding partners. These experiments expand the role of the Tec family in the heart, identifying the Tec isoform as an ischemic injury-induced isoform, and map the subproteome of its interactors in isolated cells.

  1. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    and Bmp-2a loci. The corresponding mRNA (3.0 kilobases) is expressed in most murine tissues and most abundantly expressed in brain and kidney. Antibodies against a synthetic peptide of R-PTP-alpha identified a 130-kDa protein in cells transfected with the R-PTP-alpha cDNA.......We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  2. Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

    International Nuclear Information System (INIS)

    Ecarot-Charrier, B.; Bouchard, F.; Delloye, C.

    1989-01-01

    Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with [35S] sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I

  3. Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization

    International Nuclear Information System (INIS)

    Berod, A.; Biguet, N.F.; Dumas, S.; Bloch, B.; Mallet, J.

    1987-01-01

    cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as control probe. Using the tyrosine hydroxylase probe, the authors ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of 35 S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed

  4. Meroterpenoids with Protein Tyrosine Phosphatase 1B Inhibitory Activity from a Hyrtios sp. Marine Sponge.

    Science.gov (United States)

    Wang, Jie; Mu, Feng-Rong; Jiao, Wei-Hua; Huang, Jian; Hong, Li-Li; Yang, Fan; Xu, Ying; Wang, Shu-Ping; Sun, Fan; Lin, Hou-Wen

    2017-09-22

    Three new meroterpenoids, hyrtiolacton A (1), nakijinol F (2), and nakijinol G (3), along with three known ones, nakijinol B (4), nakijinol E (5), and dactyloquinone A (6), were isolated and characterized from a Hyrtios sp. marine sponge collected from the South China Sea. The new structures were determined based on extensive analysis of HRESIMS and NMR data, and their absolute configurations were assigned by a combination of single-crystal X-ray diffraction and electronic circular dichroism analyses. Hyrtiolacton A (1) represents an unprecedented meroterpenoid featuring an unusual 2-pyrone attached to the sesquiterpene core, which is the first example of a pyrone-containing 4,9-friedodrimane-type sesquiterpene. These compounds were evaluated for their protein tyrosine phosphatase (PTP1B) inhibitory and cytotoxic activities. Nakijinol G (3) showed PTP1B inhibitory activity with an IC 50 value of 4.8 μM but no cytotoxicity against four human cancer cell lines.

  5. Proteomic analysis of tyrosine phosphorylation during human liver transplantation

    Directory of Open Access Journals (Sweden)

    Boutros Tarek

    2007-01-01

    Full Text Available Abstract Background Ischemia-reperfusion (I/R causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. Results Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. Conclusion Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

  6. Global alteration of the drug-binding pocket of human P-glycoprotein (ABCB1) by substitution of fifteen conserved residues reveals a negative correlation between substrate size and transport efficiency.

    Science.gov (United States)

    Vahedi, Shahrooz; Chufan, Eduardo E; Ambudkar, Suresh V

    2017-11-01

    P-glycoprotein (P-gp), an ATP-dependent efflux pump, is linked to the development of multidrug resistance in cancer cells. However, the drug-binding sites and translocation pathways of this transporter are not yet well-characterized. We recently demonstrated the important role of tyrosine residues in regulating P-gp ATP hydrolysis via hydrogen bond formations with high affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, in this study we investigated the global effect of enrichment of tyrosine residues in the drug-binding pocket on the drug binding and transport function of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine (F72, F303, F314, F336, F732, F759, F770, F938, F942, F983, F994), two leucine (L339, L975), one isoleucine (I306), and one methionine (M949). Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size (transport large (>1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased. This was further supported by the physico-chemical characterization of seventeen tested substrates, which revealed a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant. Published by Elsevier Inc.

  7. Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Peaucellier, G.; Andersen, A.C.; Kinsey, W.H. (Univ. of Miami School of Medicine, FL (USA))

    1990-04-01

    We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.

  8. Handling of Solid Residues

    International Nuclear Information System (INIS)

    Medina Bermudez, Clara Ines

    1999-01-01

    The topic of solid residues is specifically of great interest and concern for the authorities, institutions and community that identify in them a true threat against the human health and the atmosphere in the related with the aesthetic deterioration of the urban centers and of the natural landscape; in the proliferation of vectorial transmitters of illnesses and the effect on the biodiversity. Inside the wide spectrum of topics that they keep relationship with the environmental protection, the inadequate handling of solid residues and residues dangerous squatter an important line in the definition of political and practical environmentally sustainable. The industrial development and the population's growth have originated a continuous increase in the production of solid residues; of equal it forms, their composition day after day is more heterogeneous. The base for the good handling includes the appropriate intervention of the different stages of an integral administration of residues, which include the separation in the source, the gathering, the handling, the use, treatment, final disposition and the institutional organization of the administration. The topic of the dangerous residues generates more expectation. These residues understand from those of pathogen type that are generated in the establishments of health that of hospital attention, until those of combustible, inflammable type, explosive, radio-active, volatile, corrosive, reagent or toxic, associated to numerous industrial processes, common in our countries in development

  9. Synthesis and characterization of L-tyrosine hydrochloride crystals submitted to high and low temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Santos, C.A.A.S.; Facanha Filho, P.F.; Ribeiro, L.H.L.; Victor, F.M.S.; Abreu, D.C.; Santos, A.O. dos; Carvalho, J.O.; Soares, R.A.; Sousa, J.C.F.; Lima, R.C.; Cavaignac, A.O. [Universidade Federal do Maranhao (UFMA), MA (Brazil)

    2016-07-01

    Full text: New materials are emerging and generate advances in nonlinear optics that studies the phenomena related to changes in optical properties when occurs interaction of light with the matter. Semi organic crystals present such properties. The goal is this work is to produce semi organic single crystal of L-tyrosine hydrochloride (LTHCl) and verify their thermal stability when subjected to high and low temperatures. The single crystals of LTHCl were produced for solubilization of amino acid L-tyrosine in hydrochloric acid using slow solvent evaporation technique at a constant temperature of 25 deg C. The X-ray diffraction (XRD) and refining by the Rietveld method were used to confirm the structure of the material. The thermal stability was investigated using DSC, TGA-DTA. The LTHCl crystal belongs to the monoclinic system, with two molecules per unit cell. The refinement by the Rietveld method showed good results with Rwp= 8.49% and Rp= 6.29% with S=1.13. Thermal analysis shown an endothermic event at about 160°C, which can be associated with phase transition occurred in LTHCl crystal. It was also observed that the crystal melting point occurs at a temperature of 230°C. No water of crystallization was found in the crystal structure, which was confirmed by Raman spectroscopy and thermal analysis. From the Raman spectroscopy experiments in function of temperature, no significant changes was observe in the behavior of vibrational normal modes between temperatures of -253 and 170 deg C. Finally, a monoclinic crystal system LTHCl is stable up to 160°C at high temperatures and -253°C at low temperatures. Therefore, our investigation has proved that LTHCl crystals can be used in this range of temperature without the lost of their nonlinear optical properties. (author)

  10. Single-walled carbon nanotubes covalently functionalized with polytyrosine: A new material for the development of NADH-based biosensors.

    Science.gov (United States)

    Eguílaz, Marcos; Gutierrez, Fabiana; González-Domínguez, Jose Miguel; Martínez, María T; Rivas, Gustavo

    2016-12-15

    We report for the first time the use of single-walled carbon nanotubes (SWCNT) covalently functionalized with polytyrosine (Polytyr) (SWCNT-Polytyr) as a new electrode material for the development of nicotinamide adenine dinucleotide (NADH)-based biosensors. The oxidation of glassy carbon electrodes (GCE) modified with SWCNT-Polytyr at potentials high enough to oxidize the tyrosine residues have allowed the electrooxidation of NADH at low potentials due to the catalytic activity of the quinones generated from the primary oxidation of tyrosine without any additional redox mediator. The amperometric detection of NADH at 0.200V showed a sensitivity of (217±3)µAmM(-1)cm(-2) and a detection limit of 7.9nM. The excellent electrocatalytic activity of SWCNT-Polytyr towards NADH oxidation has also made possible the development of a sensitive ethanol biosensor through the immobilization of alcohol dehydrogenase (ADH) via Nafion entrapment, with excellent analytical characteristics (sensitivity of (5.8±0.1)µAmM(-1)cm(-2), detection limit of 0.67µM) and very successful application for the quantification of ethanol in different commercial beverages. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Effects of hemorrhagic hypotension on tyrosine concentrations in rat spinal cord and plasma

    Science.gov (United States)

    Conlay, L. A.; Maher, T. J.; Roberts, C. H.; Wurtman, R. J.

    1988-01-01

    Tyrosine is the precursor for catecholamine neurotransmitters. When catecholamine-containing neurons are physiologically active (as sympathoadrenal cells are in hypotension), tyrosine administration increases catecholamine synthesis and release. Since hypotension can alter plasma amino acid composition, the effects of an acute hypotensive insult on tyrosine concentrations in plasma and spinal cord were examined. Rats were cannulated and bled until the systolic blood pressure was 50 mmHg, or were kept normotensive for 1 h. Tyrosine and other large neutral amino acids (LNAA) known to compete with tyrosine for brain uptake were assayed in plasma and spinal cord. The rate at which intra-arterial (H-3)tyrosine disappeared from the plasma was also estimated in hemorrhaged and control rats. In plasma of hemorrhaged animals, both the tyrosine concentration and the tyrosine/LNAA ratio was elevated; moreover, the disappearance of (H-3)tyrosine was slowed. Tyrosine concentrations also increased in spinal cords of hemorrhaged-hypotensive rats when compared to normotensive controls. Changes in plasma amino acid patterns may thus influence spinal cord concentrations of amino acid precursors for neurotransmitters during the stress of hemorrhagic shock.

  12. Residual stress field of ballised holes

    International Nuclear Information System (INIS)

    Lai, Man On; He, Zhimin

    2012-01-01

    Ballising, involving pushing a slightly over-sized ball made of hard material through a hole, is a kind of cold working process. Applying ballising process to fastener holes produces compressive residual stress on the edge of the holes, and therefore increases the fatigue life of the components or structures. Quantification of the residual stress field is critical to define and precede the ballising process. In this article, the ballised holes are modeled as cold-expanded holes. Elastic-perfectly plastic theory is employed to analyze the holes with cold expansion process. For theoretical simplification, an axially symmetrical thin plate with a cold expanded hole is assumed. The elasticplastic boundaries and residual stress distribution surrounding the cold expanded hole are derived. With the analysis, the residual stress field can be obtained together with actual cold expansion process in which only the diameters of hole before and after cold expansion need to be measured. As it is a non-destructive method, it provides a convenient way to estimate the elastic-plastic boundaries and residual stresses of cold worked holes. The approach is later extended to the case involving two cold-worked holes. A ballised hole is looked upon as a cold expanded hole and therefore is investigated by the approach. Specimens ballised with different interference levels are investigated. The effects of interference levels and specimen size on residual stresses are studied. The overall residual stresses of plates with two ballised holes are obtained by superposing the residual stresses induced on a single ballised hole. The effects of distance between the centers of the two holes with different interference levels on the residual stress field are revealed

  13. TENORM: Wastewater Treatment Residuals

    Science.gov (United States)

    Water and wastes which have been discharged into municipal sewers are treated at wastewater treatment plants. These may contain trace amounts of both man-made and naturally occurring radionuclides which can accumulate in the treatment plant and residuals.

  14. Residuation in orthomodular lattices

    Directory of Open Access Journals (Sweden)

    Chajda Ivan

    2017-04-01

    Full Text Available We show that every idempotent weakly divisible residuated lattice satisfying the double negation law can be transformed into an orthomodular lattice. The converse holds if adjointness is replaced by conditional adjointness. Moreover, we show that every positive right residuated lattice satisfying the double negation law and two further simple identities can be converted into an orthomodular lattice. In this case, also the converse statement is true and the corresponence is nearly one-to-one.

  15. Toxoplasma growth in vitro is dependent on exogenous tyrosine and is independent of AAH2 even in tyrosine-limiting conditions.

    Science.gov (United States)

    Marino, Nicole D; Boothroyd, John C

    2017-05-01

    Toxoplasma gondii is an obligate intracellular parasite capable of infecting virtually all nucleated cell types in almost all warm-blooded animals. Interestingly, Toxoplasma has a relatively full repertoire of amino acid biosynthetic machinery, perhaps reflecting its broad host range and, consequently, its need to adapt to a wide array of amino acid resources. Although Toxoplasma has been shown to be auxotrophic for tryptophan and arginine, it has not previously been determined if Toxoplasma is also auxotrophic for tyrosine. Toxoplasma tachyzoites and bradyzoites were recently found to express an amino acid hydroxylase (AAH2) that is capable of synthesizing tyrosine and dihydroxyphenylalanine (DOPA) from phenylalanine; however, the role of AAH2 in tachyzoite and bradyzoite infection has not yet been identified. To determine if Toxoplasma requires exogenous tyrosine for growth, we performed growth assays on tachyzoites and bradyzoites in nutrient-rich media titrated with varying amounts of tyrosine. We found that Toxoplasma tachyzoites form significantly smaller plaques in tyrosine-limiting media in a dose-dependent manner and that this phenotype is not affected by deletion of TgAAH2. To determine if bradyzoites require exogenous tyrosine for growth, we induced differentiation from tachyzoites in vitro in tyrosine-limiting media and found that replication and vacuole number are all decreased in tyrosine-deficient media. Importantly, culture of confluent human fibroblasts in tyrosine-deficient media does not affect their viability, indicating that, at least in vitro, the need for tyrosine is at the level of Toxoplasma, not the host cell supporting its growth. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Characterization of Hospital Residuals

    International Nuclear Information System (INIS)

    Blanco Meza, A.; Bonilla Jimenez, S.

    1997-01-01

    The main objective of this investigation is the characterization of the solid residuals. A description of the handling of the liquid and gassy waste generated in hospitals is also given, identifying the source where they originate. To achieve the proposed objective the work was divided in three stages: The first one was the planning and the coordination with each hospital center, in this way, to determine the schedule of gathering of the waste can be possible. In the second stage a fieldwork was made; it consisted in gathering the quantitative and qualitative information of the general state of the handling of residuals. In the third and last stage, the information previously obtained was organized to express the results as the production rate per day by bed, generation of solid residuals for sampled services, type of solid residuals and density of the same ones. With the obtained results, approaches are settled down to either determine design parameters for final disposition whether for incineration, trituration, sanitary filler or recycling of some materials, and storage politics of the solid residuals that allow to determine the gathering frequency. The study concludes that it is necessary to improve the conditions of the residuals handling in some aspects, to provide the cleaning personnel of the equipment for gathering disposition and of security, minimum to carry out this work efficiently, and to maintain a control of all the dangerous waste, like sharp or polluted materials. In this way, an appreciable reduction is guaranteed in the impact on the atmosphere. (Author) [es

  17. Mapping Residual Structure in Intrinsically Disordered Proteins at Residue Resolution Using Millisecond Hydrogen/Deuterium Exchange and Residue Averaging

    Science.gov (United States)

    Keppel, Theodore R.; Weis, David D.

    2015-04-01

    Measurement of residual structure in intrinsically disordered proteins can provide insights into the mechanisms by which such proteins undergo coupled binding and folding. The present work describes an approach to measure residual structure in disordered proteins using millisecond hydrogen/deuterium (H/D) exchange in a conventional bottom-up peptide-based workflow. We used the exchange mid-point, relative to a totally deuterated control, to quantify the rate of H/D exchange in each peptide. A weighted residue-by-residue average of these midpoints was used to map the extent of residual structure at near single-residue resolution. We validated this approach both by simulating a disordered protein and experimentally using the p300 binding domain of ACTR, a model disordered protein already well-characterized by other approaches. Secondary structure elements mapped in the present work are in good agreement with prior nuclear magnetic resonance measurements. The new approach was somewhat limited by a loss of spatial resolution and subject to artifacts because of heterogeneities in intrinsic exchange. Approaches to correct these limitations are discussed.

  18. Hydrogen exchange rate of tyrosine hydroxyl groups in proteins as studied by the deuterium isotope effect on C(zeta) chemical shifts.

    Science.gov (United States)

    Takeda, Mitsuhiro; Jee, Jungoo; Ono, Akira Mei; Terauchi, Tsutomu; Kainosho, Masatsune

    2009-12-30

    We describe a new NMR method for monitoring the individual hydrogen exchange rates of the hydroxyl groups of tyrosine (Tyr) residues in proteins. The method utilizes (2S,3R)-[beta(2),epsilon(1,2)-(2)H(3);0,alpha,beta,zeta-(13)C(4);(15)N]-Tyr, zeta-SAIL Tyr, to detect and assign the (13)C(zeta) signals of Tyr rings efficiently, either by indirect (1)H-detection through 7-8 Hz (1)H(delta)-(13)C(zeta) spin couplings or by direct (13)C(zeta) observation. A comparison of the (13)C(zeta) chemical shifts of three Tyr residues of an 18.2 kDa protein, EPPIb, dissolved in H(2)O and D(2)O, revealed that all three (13)C(zeta) signals in D(2)O appeared at approximately 0.13 ppm ( approximately 20 Hz at 150.9 MHz) higher than those in H(2)O. In a H(2)O/D(2)O (1:1) mixture, however, one of the three signals for (13)C(zeta) appeared as a single peak at the averaged chemical shifts, and the other two appeared as double peaks at exactly the same chemical shifts in H(2)O and D(2)O, in 50 mM phosphate buffer (pH 6.6) at 40 degrees C. These three peaks were assigned to Tyr-36, Tyr-120, and Tyr-30, from the lower to higher chemical shifts, respectively. The results indicate that the hydroxyl proton of Tyr-120 exchanges faster than a few milliseconds, whereas those of Tyr-30 and Tyr-36 exchange more slowly. The exchange rate of the Tyr-30 hydroxyl proton, k(ex), under these conditions was determined by (13)C NMR exchange spectroscopy (EXSY) to be 9.2 +/- 1.1 s(-1). The Tyr-36 hydroxyl proton, however, exchanges too slowly to be determined by EXSY. These profound differences among the hydroxyl proton exchange rates are closely related to their relative solvent accessibility and the hydrogen bonds associated with the Tyr hydroxyl groups in proteins.

  19. Insights into the smooth-to-rough transitioning in Mycobacterium bolletii unravels a functional Tyr residue conserved in all mycobacterial MmpL family members.

    Science.gov (United States)

    Bernut, Audrey; Viljoen, Albertus; Dupont, Christian; Sapriel, Guillaume; Blaise, Mickaël; Bouchier, Christiane; Brosch, Roland; de Chastellier, Chantal; Herrmann, Jean-Louis; Kremer, Laurent

    2016-03-01

    In mycobacteria, MmpL proteins represent key components that participate in the biosynthesis of the complex cell envelope. Whole genome analysis of a spontaneous rough morphotype variant of Mycobacterium abscessus subsp. bolletii identified a conserved tyrosine that is crucial for the function of MmpL family proteins. Isogenic smooth (S) and rough (R) variants differed by a single mutation linked to a Y842H substitution in MmpL4a. This mutation caused a deficiency in glycopeptidolipid production/transport in the R variant and a gain in the capacity to produce cords in vitro. In zebrafish, increased virulence of the M. bolletii R variant over the parental S strain was found, involving massive production of serpentine cords, abscess formation and rapid larval death. Importantly, this finding allowed us to demonstrate an essential role of Tyr842 in several different MmpL proteins, including Mycobacterium tuberculosis MmpL3. Structural homology models of MmpL4a and MmpL3 identified two additional critical residues located in the transmembrane regions TM10 and TM4 that are facing each other. We propose that these central residues are part of the proton-motive force that supplies the energy for substrate transport. Hence, we provide important insights into mechanistic/structural aspects of MmpL proteins as lipid transporters and virulence determinants in mycobacteria. © 2015 John Wiley & Sons Ltd.

  20. Asperterpenoid A, a new sesterterpenoid as an inhibitor of Mycobacterium tuberculosis protein tyrosine phosphatase B from the culture of Aspergillus sp. 16-5c.

    Science.gov (United States)

    Huang, Xishan; Huang, Hongbo; Li, Hanxiang; Sun, Xuefeng; Huang, Huarong; Lu, Yongjun; Lin, Yongcheng; Long, Yuhua; She, Zhigang

    2013-02-15

    Asperterpenoid A (1), a novel sesterterpenoid with a new carbon skeleton, has been isolated from a mangrove endophytic fungus Aspergillus sp. 16-5c. Its structure was characterized by extensive spectroscopic methods, and the absolute configuration was determined by single crystal X-ray diffraction analysis. Asperterpenoid A (1) exhibited strong inhibitory activity against Mycobacterium tuberculosis protein tyrosine phosphatase B (mPTPB) with an IC(50) value of 2.2 μM.

  1. Novel Mixed-Type Inhibitors of Protein Tyrosine Phosphatase 1B. Kinetic and Computational Studies

    Directory of Open Access Journals (Sweden)

    Marie Jazmín Sarabia-Sánchez

    2017-12-01

    Full Text Available The Atlas of Diabetes reports 415 million diabetics in the world, a number that has surpassed in half the expected time the twenty year projection. Type 2 diabetes is the most frequent form of the disease; it is characterized by a defect in the secretion of insulin and a resistance in its target organs. In the search for new antidiabetic drugs, one of the principal strategies consists in promoting the action of insulin. In this sense, attention has been centered in the protein tyrosine phosphatase 1B (PTP1B, a protein whose overexpression or increase of its activity has been related in many studies with insulin resistance. In the present work, a chemical library of 250 compounds was evaluated to determine their inhibition capability on the protein PTP1B. Ten molecules inhibited over the 50% of the activity of the PTP1B, the three most potent molecules were selected for its characterization, reporting Ki values of 5.2, 4.2 and 41.3 µM, for compounds 1, 2, and 3, respectively. Docking and molecular dynamics studies revealed that the three inhibitors made interactions with residues at the secondary binding site to phosphate, exclusive for PTP1B. The data reported here support these compounds as hits for the design more potent and selective inhibitors against PTP1B in the search of new antidiabetic treatment.

  2. Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding.

    Science.gov (United States)

    Kleiman, Laura B; Maiwald, Thomas; Conzelmann, Holger; Lauffenburger, Douglas A; Sorger, Peter K

    2011-09-02

    Epidermal growth factor receptors (ErbB1-4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100-1000 times in the course of a typical immediate-early response to ligand. Rapid phospho-turnover is also observed for EGF-activated ErbB2 and ErbB3, unrelated RTKs, and multiple intracellular adaptor proteins and signaling kinases. Thus, the complexes formed on the cytoplasmic tail of active receptors and the downstream signaling kinases they control are highly dynamic and antagonized by potent phosphatases. We develop a kinetic scheme for binding of anti-ErbB1 drugs to receptors and show that rapid phospho-turnover significantly impacts their mechanisms of action. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos

    Directory of Open Access Journals (Sweden)

    Stabel Silvia

    2002-04-01

    Full Text Available Abstract Background The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro. Results We generated recombinant 124-v-Mos using the baculovirus expression system in Spodoptera frugiperda cells and demonstrated constitutive kinase activity by the ability of 124-v-Mos to auto-phosphorylate and to phosphorylate vimentin, a known substrate of c-Mos. Using this approach we analyzed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B (PTP1B, alpha-casein and beta-casein. We controlled mos-specific phosphorylation of PTP1B and casein in comparative assays using a synthetic kinase-inactive 124-v-Mos mutant and further, tryptic digests of mos-phosphorylated beta-casein identified a phosphopeptide specifically targeted by wild-type 124-v-Mos. Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues. Conclusion The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

  4. Detection of Sequence-Specific Tyrosine Nitration of Manganese SOD and SERCA in Cardiovascular Disease and Aging

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Shanqin; Ying, Jia; Jiang, Bingbing; Guo, Wei; Adachi, Takeshi; Sharov, Victor; Lazar, Harold; Menzoian, James; Knyushko, Tanya V.; Bigelow, Diana J.; Schoneich, Christian; Cohen, Richard

    2006-06-01

    Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies towards site-specific nY-modified proteins and to use histochemical and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies towards peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with angiotensin II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite that chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site, but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates, but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.

  5. Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases.

    Science.gov (United States)

    Niehrs, C; Huttner, W B; Carvallo, D; Degryse, E

    1990-06-05

    Hirudin, a tyrosine-sulfated protein secreted by the leech Hirudo medicinalis, is one of the most potent anticoagulants known. The hirudin cDNA has previously been cloned and has been expressed in yeast, but the resulting recombinant protein was found to be produced in the unsulfated form, which is known to have an at least 10 times lower affinity for thrombin than the naturally occurring tyrosine-sulfated hirudin. Here we describe the in vitro tyrosine sulfation of recombinant hirudin by leech and bovine tyrosylprotein sulfotransferase (TPST). With both enzymes, in vitro sulfation of recombinant hirudin occurred at the physiological site (Tyr-63) and rendered the protein biochemically and biologically indistinguishable from natural hirudin. However, leech TPST had an over 20-fold lower apparent Km value for recombinant hirudin than bovine TPST. Further differences in the catalytic properties of leech and bovine TPSTs were observed when synthetic peptides were tested as substrates. Moreover, a synthetic peptide corresponding to the 9 carboxyl-terminal residues of hirudin (which include Tyr-63) was sulfated by leech TPST with a similar apparent Km value as full length hirudin, indicating that structural determinants residing in the immediate vicinity of Tyr-63 are sufficient for sulfation to occur.

  6. Sphingosine 1-Phosphate Induces Platelet/Endothelial Cell Adhesion Molecule-1 Tyrosine Phosphorylation in Bovine Aortic Endothelial Cells through a PP2-Inhibitable Mechanism

    Directory of Open Access Journals (Sweden)

    Yu-Ting Huang

    2007-12-01

    Full Text Available Sphingosine-1-phosphate (S1P is a low-molecular-weight phospholipid derivative released by activated platelets. S1P transduces signals through a family of G protein-coupled receptors to modulate various physiological behaviors of endothelial cells. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31 is a 130-kDa protein expressed on the surfaces of leukocytes, platelets, and endothelial cells. Upon PECAM-1 activation, its cytoplasmic tyrosine residues become phosphorylated and bind with SH2 domain-containing proteins, thus leading to the downstream functions mediated by PECAM-1. In the present study, we found that S1P induced PECAM-1 tyrosine phosphorylation and SHP-2 association in bovine aortic endothelial cells (BAECs by immunoprecipitation and western blotting. The pretreatment of BAECs with a series of chemical inhibitors to determine the signaling pathway showed that the PECAM-1 phosphorylation was inhibited by PP2, indicating the participation of Src family kinases. These results demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in BAECs through mediation of Src family kinases, and this may regulate the physiological behaviors of endothelial cells.

  7. Tyrosine-rich crystals associated with oncocytic salivary gland neoplasms.

    Science.gov (United States)

    Gilcrease, M Z; Nelson, F S; Guzman-Paz, M

    1998-07-01

    Crystalloids have been identified ultrastructurally within the epithelial cells of Warthin's tumors, but there have been no studies characterizing crystals or crystalloids in Warthin's tumors by light microscopy. The finding of abundant needle-shaped crystals in a fine-needle aspirate of a cystadenoma of the parotid prompted us to examine the prevalence of crystals and crystalloids in oncocytic salivary gland neoplasms. Ninety-seven oncocytic neoplasms (93 Warthin's tumors, 3 cystadenomas, and 1 oncocytoma) excised at our institution between 1950 and 1996 were examined, to identify crystals. Neoplasms with crystals were further characterized by means of a variety of histochemical stains and electron microscopy. Ninety-nine pleomorphic adenomas were similarly reviewed. Seven cases with crystals were identified. Five of these were Warthin's tumors, 1 was a cystadenoma, and 1 was an oncocytoma. The crystals were noted within tumor cysts but were not limited to the neoplasms. The crystals were predominantly either needle-shaped or tabular, but some cases contained mixtures of both as well as intermediate forms. They stained pink with hematoxylin-eosin, although the tabular forms also exhibited a focal yellow hue. The crystals were not discernible under polarized light. They stained a red-brown color with Millon's reagent, which indicated the presence of tyrosine. Trichrome, periodic acid-Schiff stain with diastase, alcian blue (pH 2.5), and Congo red stains were negative. Electron microscopy revealed sharply defined, elongate, electron-dense structures with periodicity, both extracellular and within epithelial cells. No crystals or crystalloids were identified in any of 99 pleomorphic adenomas reviewed. The findings indicate that tyrosine-rich crystals associated with several oncocytic salivary gland neoplasms are morphologically, histochemically, and ultrastructurally distinct from previously described tyrosine-rich crystalloids and collagenous crystalloids of

  8. Genomic organization of Bruton`s tyrosine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Rohrer, J.; Conley, M.E. [Univ. of Tennessee, Memphis, TN (United States)

    1994-09-01

    Bruton`s tyrosine kinase (Btk), is a nonreceptor tyrosine kinase that has been identified as the defective gene in X-linked agammaglobulinemia (XLA). XLA patients have profound hypogammaglobulinemia and markedly reduced numbers of B cells while their T cell and phagocyte numbers remain normal. To determine the genomic organization of Btk, intron/exon borders were identified by sequencing cosmid DNA using cDNA primers. Nineteen exons spanning 37 kb of genomic DNA were identified. All the intron/exon splice junctions followed the GT/AG rule. The translational ATG start codon was in exon 2 which was 6 kb downstream of exon 1. Exon 19, 519 bp in length and 3.8 kb distal to exon 18, was the largest exon and included the 450 bp of the 3{prime} untranslated region. Exons 6 through 18 formed the largest cluster of exons with no intron being longer than 1550 bp. There was no apparent correlation between the exon boundaries of Btk and the functional domains of the protein or the exon boundaries of src, the nonreceptor protein tyrosine kinase prototype. The region 500 bp upstream of the presumed transcriptional start site was sequenced and found to have a G+C content of 52%. No TATA-type promoter elements in the -20 bp to -30 bp region were identified. However, at position -48 bp, a TGTGAA motif was found that bears some similarity to the TATA box. This sequence was preceded by a perfect inverted CCAAT box at position -90 bp. Three retinoic acid binding sites were also identified at positions -50 bp, -83 bp and -197 bp. Defining the genomic structure of Btk will permit us to identify regulatory elements in this gene and to identify mutations in genomic DNA of patients with XLA.

  9. Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations

    Energy Technology Data Exchange (ETDEWEB)

    Mehere, P.; Robinson, H.; Han, Q.; Lemkul, J. A.; Vavricka, C. J.; Bevan, D. R.; Li, J.

    2010-11-01

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  10. Tyrosine Aminotransferase: Biochemical and Structural Properties and Molecular Dynamics Simulations

    Energy Technology Data Exchange (ETDEWEB)

    P Mehere; Q Han; J Lemkul; C Vavricka; H Robinson; D Bevan; J Li

    2011-12-31

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  11. Management of NORM Residues

    International Nuclear Information System (INIS)

    2013-06-01

    The IAEA attaches great importance to the dissemination of information that can assist Member States in the development, implementation, maintenance and continuous improvement of systems, programmes and activities that support the nuclear fuel cycle and nuclear applications, and that address the legacy of past practices and accidents. However, radioactive residues are found not only in nuclear fuel cycle activities, but also in a range of other industrial activities, including: - Mining and milling of metalliferous and non-metallic ores; - Production of non-nuclear fuels, including coal, oil and gas; - Extraction and purification of water (e.g. in the generation of geothermal energy, as drinking and industrial process water; in paper and pulp manufacturing processes); - Production of industrial minerals, including phosphate, clay and building materials; - Use of radionuclides, such as thorium, for properties other than their radioactivity. Naturally occurring radioactive material (NORM) may lead to exposures at some stage of these processes and in the use or reuse of products, residues or wastes. Several IAEA publications address NORM issues with a special focus on some of the more relevant industrial operations. This publication attempts to provide guidance on managing residues arising from different NORM type industries, and on pertinent residue management strategies and technologies, to help Member States gain perspectives on the management of NORM residues

  12. Annatto seed residue (Bixa orellana L.: nutritional quality

    Directory of Open Access Journals (Sweden)

    Melissa Alessandra Valério

    2015-06-01

    Full Text Available Considering that annatto seeds are rich in protein, the present work aimed to evaluate the biological quality of this nutrient in the meal residue originating from annatto seed processing. We determined the general composition, mineral levels, amino acid composition and chemical scores, antinutritional factors, and protein quality using biological assays. The following values were obtained: 11.50% protein, 6.74% moisture, 5.22% ash, 2.22% lipids, 42.19% total carbohydrates and 28.45% fiber. The residue proved to be a food rich in fiber and also a protein source. Antinutritional factors were not detected. The most abundant amino acids were lysine, phenylalanine + tyrosine, leucine and isoleucine. Valine was the most limiting amino acid (chemical score 0.22. The protein quality of the seed residue and the isolated protein showed no significant differences. The biological value was lower than that of the control protein but higher than that found in other vegetables. Among the biochemical analyses, only creatinine level was decreased in the two test groups compared to the control group. Enzyme tests did not indicate liver toxicity. The results showed favorable aspects for the use of annatto seed residue in the human diet, meriting further research.

  13. Phenylalanine as substrate for tyrosine hydroxylase in bovine adrenal chromaffin cells.

    OpenAIRE

    Fukami, M H; Haavik, J; Flatmark, T

    1990-01-01

    Incubation of bovine chromaffin cells with L-[14C]phenylalanine resulted in label accumulation in catecholamines at about 30% of the rate seen with L-tyrosine as precursor. Studies with purified tyrosine hydroxylase (EC 1.14.16.2) showed that the enzyme catalysed the hydroxylation of L-phenylalanine first to L-p-tyrosine and then to 3,4-dihydroxyphenylalanine (DOPA). No evidence for a significant involvement of an L-m-tyrosine intermediate in DOPA formation was found.

  14. PTP1B Inhibition Causes Rac1 Activation by Enhancing Receptor Tyrosine Kinase Signaling

    Directory of Open Access Journals (Sweden)

    Ayako Tsuchiya

    2014-04-01

    Full Text Available Background/Aims: The present study investigated the signaling pathway underlying Rac1 activation induced by the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl-cyclopropyl]-octanoic acid (DCP-LA. Methods: Activity of protein tyrosine phosphatase 1B (PTP1B was assayed under cell-free conditions. Western blot was carried out to quantify phosphorylation of insulin receptor substrate-1 (IRS-1 and Akt in PC-12 cells. Rac1 activity was monitored in the föerster resonance energy transfer (FRET analysis using living and fixed PC-12 cells. Results: DCP-LA markedly suppressed PTP1B activity in a concentration (100 pM-100 µM-dependent manner. In the DCP-LA binding assay, fluorescein-conjugated DCP-LA produced a single fluorescent signal band at 60 kDa, corresponding to the molecule of PTP1B, and the signal was attenuated or abolished by co-treatment or pretreatment with non-conjugated DCP-LA. DCP-LA significantly enhanced nerve growth factor (NGF-stimulated phosphorylation of IRS-1 at Tyr1222 and Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells. In the FRET analysis, DCP-LA significantly enhanced NGF-stimulated Rac1 activation, which is abrogated by the phosphatidylinositol 3 kinase (PI3K inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase-1 (PDK1 inhibitor BX912, or the Akt inhibitor MK2206. Conclusion: The results of the present study show that DCP-LA-induced PTP1B inhibition, possibly through its direct binding, causes Rac1 activation by enhancing a pathway along a receptor tyrosine kinase (RTK/IRS-1/PI3K/Akt/Rac1 axis.

  15. A novel protein tyrosine kinase Tec identified in lamprey, Lampetra japonica.

    Science.gov (United States)

    Li, Ranran; Su, Peng; Liu, Chang; Zhang, Qiong; Zhu, Ting; Pang, Yue; Liu, Xin; Li, Qingwei

    2015-08-01

    Protein tyrosine kinase Tec, a kind of non-receptor tyrosine kinase, is primarily found to be expressed in T cells, B cells, hematopoietic cells, and liver cells as a cytoplasmic protein. Tec has been proved to be a critical modulator of T cell receptor signaling pathway. In the present study, a homolog of Tec was identified in the lamprey, Lampetra japonica. The full-length Tec cDNA of L. japonica (Lja-Tec) contains a 1923 bp open reading frame that encodes a 641-amino acid protein. The multi-alignment of the deduced amino acid sequence of Lja-Tec with typical vertebrate Tecs showed that it possesses all conserved domains of the Tec family proteins, indicating that an ortholog of Tec exists in the extant jawless vertebrate. In the phylogenetic tree that was reconstructed with 24 homologs of jawless and jawed vertebrates, the Tecs from lampreys and hagfish were clustered as a single clade. The genetic distance between the outgroup and agnathan Tecs' group is closer than that between outgroup and gnathostome Tecs' group, indicating that its origin was far earlier than any of the jawed vertebrates. The mRNA levels of Lja-Tec in lymphocyte-like cells and gills were detected by real-time quantitative polymerase chain reaction. Results showed that it was significantly upregulated under stimulation with mixed pathogens. This result was further confirmed by western blot analysis. All these results indicated that Lja-Tec plays an important role in immune response. Our data will provide a reference for the further study of lamprey Tec and its immunological function in jawless vertebrates. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

  16. Tyrosine Kinase Inhibitor Treatment for Newly Diagnosed Chronic Myeloid Leukemia.

    Science.gov (United States)

    Radich, Jerald P; Mauro, Michael J

    2017-08-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder that accounts for approximately 10% of new cases of leukemia. The introduction of tyrosine kinase inhibitors has led to a reduction in mortalities. Thus, the estimated prevalence of CML is increasing. The National Comprehensive Cancer Network and the European Leukemia Net guidelines incorporate frequent molecular monitoring of the fusion BCR-ABL transcript to ensure that patients reach and keep treatment milestones. Most patients with CML are diagnosed in the chronic phase, and approximately 10% to 30% of these patients will at some time in their course meet definition criteria of resistance to imatinib. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Second-generation inhibitors of Bruton tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Jingjing Wu

    2016-09-01

    Full Text Available Abstract Bruton tyrosine kinase (BTK is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib, ONO/GS-4059, and BGB-3111.

  18. Second-generation inhibitors of Bruton tyrosine kinase.

    Science.gov (United States)

    Wu, Jingjing; Liu, Christina; Tsui, Stella T; Liu, Delong

    2016-09-02

    Bruton tyrosine kinase (BTK) is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib), ONO/GS-4059, and BGB-3111.

  19. Residual-stress measurements

    Energy Technology Data Exchange (ETDEWEB)

    Ezeilo, A.N.; Webster, G.A. [Imperial College, London (United Kingdom); Webster, P.J. [Salford Univ. (United Kingdom)

    1997-04-01

    Because neutrons can penetrate distances of up to 50 mm in most engineering materials, this makes them unique for establishing residual-stress distributions non-destructively. D1A is particularly suited for through-surface measurements as it does not suffer from instrumental surface aberrations commonly found on multidetector instruments, while D20 is best for fast internal-strain scanning. Two examples for residual-stress measurements in a shot-peened material, and in a weld are presented to demonstrate the attractive features of both instruments. (author).

  20. Catabolism of L-tyrosine in Trichosporon cutaneum.

    Science.gov (United States)

    Sparnins, V L; Burbee, D G; Dagley, S

    1979-01-01

    Protocatechuic acid was a catabolite in the degradation of L-tyrosine by Trichosporon cutaneum. Intact cells oxidized to completion various compounds proposed as intermediates in this conversion, but they did not readily oxidize catabolites of the homogentisate and homoprotocatechuate metabolic pathways, which are known to function in other organisms. Cell extracts converted tyrosine first to 4-hydroxycinnamic acid and then to 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid. The proposed hydration product of 4-hydroxycinnamic acid, namely, beta-(4-hydroxyphenyl)-hydracrylic acid, was synthesized chemically, and its enzymatic degradation to 4-hydroxybenzaldehyde was shown to be dependent upon additions of adenosine triphosphate and coenzyme A. The hydroxylase that attacked 4-hydroxybenzoate showed a specific requirement for reduced nicotinamide adenine dinucleotide phosphate. Protocatechuate, the product of this reaction, was oxidized by cell extracts supplemented with reduced nicotinamide adenine dinucleotide or, less effectively, with reduced nicotinamide adenine dinucleotide phosphate, but these extracts contained no ring fission dioxygenase for protocatechuate. Evidence is presented that the principal hydroxylation product of protocatechuate was hydroxyquinol, the benzene nucleus of which was cleaved oxidatively to give maleylacetic acid. PMID:571434

  1. Dielectric Relaxation Behavior of Tyrosine-Derived Polycarbonate.

    Science.gov (United States)

    Suarez, N.; Laredo, E.; Bello, A.; Kohn, J.

    1996-03-01

    Tyrosine-derived polycarbonates represent a new family of polymers that were specifically designed for medical applications. In this work we have used Thermally Stimulated Depolarization Currents (TSDC) to study for the first time the dielectric relaxation behavior of a series of degradable tyrosine-derived polycarbonates. The test polymers differed only in the length of the pendent chain which was increased from two to eight carbons by the use of ethyl, butyl, hexyl and octyl esters as C-terminus protecting groups. The high temperature zone of the spectra shows the glass transition relaxation located at decreasing temperatures as the length of the pendent chain is increased. The low temperature spectrum exhibits a complex dielectric relaxation composed of 4 peaks. The relative intensities of these four peaks are sensitive to packing and entanglement effects caused by the variation in the length of the pendent chain. For data analysis, the Direct Signal Analysis (DSA) procedure was used. This procedure allows the precise determination of the relaxation parameters without having to use peak cleaning techniques. To analyze the whole spectra the Vogel-Fulcher temperature dependence of the relaxation time was used for the glass transition relaxation, and the Arrhenius dependence for the remaining relaxations.

  2. Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis

    Directory of Open Access Journals (Sweden)

    Feng Liu

    2016-06-01

    Full Text Available Renal fibrosis can be induced in different renal diseases, but ultimately progresses to end stage renal disease. Although the pathophysiologic process of renal fibrosis have not been fully elucidated, it is characterized by glomerulosclerosis and/or tubular interstitial fibrosis, and is believed to be caused by the proliferation of renal inherent cells, including glomerular epithelial cells, mesangial cells, and endothelial cells, along with defective kidney repair, renal interstitial fibroblasts activation, and extracellular matrix deposition. Receptor tyrosine kinases (RTKs regulate a variety of cell physiological processes, including metabolism, growth, differentiation, and survival. Many studies from in vitro and animal models have provided evidence that RTKs play important roles in the pathogenic process of renal fibrosis. It is also showed that tyrosine kinases inhibitors (TKIs have anti-fibrotic effects in basic research and clinical trials. In this review, we summarize the evidence for involvement of specific RTKs in renal fibrosis process and the employment of TKIs as a therapeutic approach for renal fibrosis.

  3. Tyrosine Sulfation as a Protein Post-Translational Modification

    Directory of Open Access Journals (Sweden)

    Yuh-Shyong Yang

    2015-01-01

    Full Text Available Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.

  4. Dietary Tyrosine Protects Striatal Dopamine Receptors from the Adverse Effects of REM Sleep Deprivation.

    Science.gov (United States)

    Hamdi, A; Brock, J W; Payne, S; Ross, K D; Bond, S P; Prasad, C

    1998-01-01

    L-Tyrosine is a non-essential amino acid that is produced as an intermediary metabolite in the conversion of phenylalanine to 3,4-dihyroxyphenylalanine (DOPA), and is a precursor of the neurotransmitter dopamine. In previous studies, tyrosine pretreatment was shown to protect against the neurochemical and behavioral deficits of acute stress caused by tail shock or cold exposure in rodents. The present study addressed the hypothesis that tyrosine administration may be an effective counter-measure to dopamine-mediated behaviors induced by rapid eye-movement sleep deprivation (RSD). In order to test the hypothesis, Sprague-Dawley rats were divided into 9 treatment groups: RSD-treated rats on normal-protein diet (20% casein: 1% tyrosine, 1% valine); tank control (TC) rats on a normal diet; cage control (CC) rats on normal diet; RSD-treated rats on 4% tyrosine diet; TC rats on 4% tyrosine diet; CC rats on 4% tyrosine diet; RSD-treated rats on 4% valine diet; TC rats on 4% valine diet; CC rats on 4% valine diet. In the RSD group receiving tyrosine, there was no apparent change in Bmax for binding of the dopamine D2 receptor ligand [(3)H]YM-09151-2 in the striata as compared to the respective TC and CC groups; whereas RSD-treated rats maintained on the normal diet and valine supplementation demonstrated expected increases in Bmax for ligand binding. The TC group on the tyrosine diet showed attenuated catalepsy compared to the corresponding CC group, while the RSD group consuming tyrosine showed a catalepsy that was significantly increased, and similar to that of cage control animais on a control diet. These data suggest that the tyrosine-supplemented diet significantly attenuated RSD-induced changes in striatal dopamine D2 receptors, and the effect appeared sufficient to influence RSD-induced behaviors.

  5. Composition of carbonization residues

    Energy Technology Data Exchange (ETDEWEB)

    Hupfer; Leonhardt

    1943-11-27

    This report compared the composition of samples from Wesseling and Leuna. In each case the sample was a residue from carbonization of the residues from hydrogenation of the brown coal processed at the plant. The composition was given in terms of volatile components, fixed carbon, ash, water, carbon, hydrogen, oxygen, nitrogen, volatile sulfur, and total sulfur. The result of carbonization was given in terms of (ash and) coke, tar, water, gas and losses, and bitumen. The composition of the ash was given in terms of silicon dioxide, ferric oxide, aluminum oxide, calcium oxide, magnesium oxide, potassium and sodium oxides, sulfur trioxide, phosphorus pentoxide, chlorine, and titanium oxide. The most important difference between the properties of the two samples was that the residue from Wesseling only contained 4% oil, whereas that from Leuna had about 26% oil. Taking into account the total amount of residue processed yearly, the report noted that better carbonization at Leuna could save 20,000 metric tons/year of oil. Some other comparisons of data included about 33% volatiles at Leuna vs. about 22% at Wesseling, about 5 1/2% sulfur at Leuna vs. about 6 1/2% at Leuna, but about 57% ash for both. Composition of the ash differed quite a bit between the two. 1 table.

  6. Designing with residual materials

    NARCIS (Netherlands)

    Walhout, W.; Wever, R.; Blom, E.; Addink-Dölle, L.; Tempelman, E.

    2013-01-01

    Many entrepreneurial businesses have attempted to create value based on the residual material streams of third parties. Based on ‘waste’ materials they designed products, around which they built their company. Such activities have the potential to yield sustainable products. Many of such companies

  7. ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK)

    Science.gov (United States)

    Morris, S W; Naeve, C; Mathew, P; James, P L; Kirstein, M N; Cui, X; Witte, D P

    1997-05-08

    Anaplastic Lymphoma Kinase (ALK) was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases that acquires transforming capability when truncated and fused to nucleophosmin (NPM) in the t(2;5) chromosomal rearrangement associated with non-Hodgkin's lymphoma, but further insights into its normal structure and function are lacking. Here, we characterize a full-length normal human ALK cDNA and its product, and determine the pattern of expression of its murine homologue in embryonic and adult tissues as a first step toward the functional assessment of the receptor. Analysis of the 6226 bp ALK cDNA identified an open reading frame encoding a 1620-amino acid (aa) protein of predicted mass approximately 177 kDa that is most closely related to leukocyte tyrosine kinase (LTK), the two exhibiting 57% aa identity and 71% similarity over their region of overlap. Biochemical analysis demonstrated that the approximately 177 kDa ALK polypeptide core undergoes co-translational N-linked glycosylation, emerging in its mature form as a 200 kDa single chain receptor. Surface labeling studies indicated that the 200 kDa glycoprotein is exposed at the cell membrane, consistent with the prediction that ALK serves as the receptor for an unidentified ligand(s). In situ hybridization studies revealed Alk expression beginning on embryonic day 11 and persisting into the neonatal and adult periods of development. Alk transcripts were confined to the nervous system and included several thalamic and hypothalamic nuclei; the trigeminal, facial, and acoustic cranial ganglia; the anterior horns of the spinal cord in the region of the developing motor neurons; the sympathetic chain; and the ganglion cells of the gut. Thus, ALK is a novel orphan receptor tyrosine kinase that appears to play an important role in the normal development and function of the nervous system.

  8. State-by-State Investigation of Destructive Interference in Resonance Raman Spectra of Neutral Tyrosine and the Tyrosinate Anion with the Simplified Sum-over-States Approach

    OpenAIRE

    Cabalo, Jerry B.; Saikin, Semion K.; Emmons, Erik D.; Rappoport, Dmitrij; Aspuru-Guzik, Alan

    2014-01-01

    UV resonance Raman scattering is uniquely sensitive to the molecular electronic structure as well as intermolecular interactions. To better understand the relationship between electronic structure and resonance Raman cross section, we carried out combined experimental and theoretical studies of neutral tyrosine and the tyrosinate anion. We studied the Raman cross sections of four vibrational modes as a function of excitation wavelength, and we analyzed them in terms of the contributions of th...

  9. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    Directory of Open Access Journals (Sweden)

    Belén Mezquita

    2014-02-01

    Full Text Available One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT and a truncated isoform of VEGFR-1 (i21-VEGFR-1, which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer.

  10. Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling

    DEFF Research Database (Denmark)

    Lund, I K; Hansen, J A; Andersen, H S

    2005-01-01

    Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators of this sign...

  11. Peptide Microarray Analysis of the Cross-talk Between O-GlcNAcylation and Tyrosine Phosphorylation

    NARCIS (Netherlands)

    Shi, Jie; Tomašič, Tihomir; Sharif, Suhela; Brouwer, Arwin J; Anderluh, Marko; Ruijtenbeek, Rob; Pieters, Roland J

    2017-01-01

    O-GlcNAcylation of proteins regulates important cellular processes. A few reports noted that O-GlcNAcylation exhibits cross-talk with tyrosine phosphorylation. With an activity-based microarray analysis of 256 tyrosine kinase peptide substrates, we found that phosphorylation of 6 peptides by Jak2

  12. The Relationship among Tyrosine Decarboxylase and Agmatine Deiminase Pathways in Enterococcus faecalis

    Directory of Open Access Journals (Sweden)

    Marta Perez

    2017-11-01

    Full Text Available Enterococci are considered mainly responsible for the undesirable accumulation of the biogenic amines tyramine and putrescine in cheeses. The biosynthesis of tyramine and putrescine has been described as a species trait in Enterococcus faecalis. Tyramine is formed by the decarboxylation of the amino acid tyrosine, by the tyrosine decarboxylase (TDC route encoded in the tdc cluster. Putrescine is formed from agmatine by the agmatine deiminase (AGDI pathway encoded in the agdi cluster. These biosynthesis routes have been independently studied, tyrosine and agmatine transcriptionally regulate the tdc and agdi clusters. The objective of the present work is to study the possible co-regulation among TDC and AGDI pathways in E. faecalis. In the presence of agmatine, a positive correlation between putrescine biosynthesis and the tyrosine concentration was found. Transcriptome studies showed that tyrosine induces the transcription of putrescine biosynthesis genes and up-regulates pathways involved in cell growth. The tyrosine modulation over AGDI route was not observed in the mutant Δtdc strain. Fluorescence analyses using gfp as reporter protein revealed PaguB (the promoter of agdi catabolic genes was induced by tyrosine in the wild-type but not in the mutant strain, confirming that tdc cluster was involved in the tyrosine induction of putrescine biosynthesis. This study also suggests that AguR (the transcriptional regulator of agdi was implicated in interaction among the two clusters.

  13. Bosutinib induced pleural effusions: Case report and review of tyrosine kinase inhibitors induced pulmonary toxicity

    Directory of Open Access Journals (Sweden)

    Natalia I. Moguillansky, MD

    2017-01-01

    Full Text Available Tyrosine kinase inhibitors are known to cause pulmonary complications. We report a case of bosutinib related bilateral pleural effusions in a patient with chronic myeloid leukemia. Characteristics of the pleural fluid are presented. We also discuss other tyrosine kinase inhibitors induced pulmonary toxicities, including pulmonary hypertension and interstitial lung disease.

  14. From immune response to cancer : a spot on the low molecular weight protein tyrosine phosphatase

    NARCIS (Netherlands)

    Souza, A. C. S.; Azoubel, S.; Queiroz, K. C. S.; Peppelenbosch, M. P.; Ferreira, C. V.

    Reversible tyrosine phosphorylation is a key posttranslational regulatory modification of proteins in all eukaryotic cells in normal and pathological processes. Recently a pivotal janus-faced biological role of the low molecular weight protein tyrosine phosphatase (LMWPTP) has become clear. On the

  15. Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma.

    Science.gov (United States)

    Marley, K; Maier, C S; Helfand, S C

    2012-09-01

    Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. © 2012 Blackwell Publishing Ltd.

  16. Loss of Function Studies in Mice and Genetic Association Link Receptor Protein Tyrosine Phosphatase a to Schizophrenia

    DEFF Research Database (Denmark)

    Takahashi, Nagahide; Nielsen, Karin Sandager; Aleksic, Branko

    2011-01-01

    Solid evidence links schizophrenia (SZ) susceptibility to neurodevelopmental processes involving tyrosine phosphorylation-mediated signaling. Mouse studies implicate the Ptpra gene, encoding protein tyrosine phosphatase RPTPa, in the control of radial neuronal migration, cortical cytoarchitecture...

  17. Residual stresses in material processing

    Science.gov (United States)

    Kozaczek, K. J.; Watkins, T. R.; Hubbard, C. R.; Wang, Xun-Li; Spooner, S.

    Material manufacturing processes often introduce residual stresses into the product. The residual stresses affect the properties of the material and often are detrimental. Therefore, the distribution and magnitude of residual stresses in the final product are usually an important factor in manufacturing process optimization or component life prediction. The present paper briefly discusses the causes of residual stresses. It then addresses the direct, nondestructive methods of residual stress measurement by X ray and neutron diffraction. Examples are presented to demonstrate the importance of residual stress measurement in machining and joining operations.

  18. Fate and Transport of Colloidal Energetic Residues

    Science.gov (United States)

    2015-07-01

    field) images of (b) a single and (c) an aggregated particle in the experimental solution . .............................................. 19  Figure...84  vii ACRONYM LIST ARW artificial rainwater °C degree Celsius cm centimeter d day DNX hexahydro-1,3-dinitroso-5- nitro ...munition constituents. This size fraction of residues had not been studied in any detail, yet it represents the most mobile and most rapidly

  19. Residue specific hydration of primary cell wall potato pectin identified by solid-state 13C single-pulse MAS and CP/MAS NMR spectroscopy

    DEFF Research Database (Denmark)

    Larsen, Flemming Hofmann; Chrestensen, Inge Byg; Damager, Iben

    2011-01-01

    Hydration of rhamnogalacturonan-I (RG-I) derived from potato cell wall was analyzed by 13C single-pulse (SP) magic-angle-spinning (MAS) and 13C cross-polarization (CP) MAS nuclear magnetic resonance (NMR) and supported by 2H SP/MAS NMR experiments. The study shows that the arabinan side chains...... hydrate more readily than the galactan side chains and suggests that the overall hydration properties can be controlled by modifying the ratio of these side chains. Enzymatic modification of native (NA) RG-I provided samples with reduced content of arabinan (sample DA), galactan (sample DG), or both side...... chains (sample DB). Results of these samples suggested that hydration properties were determined by the length and character of the side chains. NA and DA exhibited similar hydration characteristics, whereas DG and DB were difficult to hydrate because of the less hydrophilic properties of the rhamnose...

  20. Translation of tyrosine hydroxylase from poly(A)-mRNA in pheochromocytoma cells is enhanced by dexamethasone.

    OpenAIRE

    Baetge, E E; Kaplan, B B; Reis, D J; Joh, T H

    1981-01-01

    Polysomal poly(A)-mRNA was purified from a clonal cell line of rat pheochromocytoma (PC 12) and translated in a reticulocyte cell-free protein-synthesizing system. Tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahyropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] was isolated from other protein by immunoprecipitation and NaDodSO4/polyacrylamide gel electrophoresis. The molecular weight and relative proportion of tyrosine hydroxylase to other proteins synthesi...

  1. Composition of carbonization residues

    Energy Technology Data Exchange (ETDEWEB)

    Hupfer; Leonhardt

    1943-11-30

    This report gave a record of the composition of several samples of residues from carbonization of various hydrogenation residue from processing some type of coal or tar in the Bergius process. These included Silesian bituminous coal processed at 600 atm. with iron catalyst, in one case to produce gasoline and middle oil and in another case to produce heavy oil excess, Scholven coal processed at 250 atm. with tin oxalate and chlorine catalyst, Bruex tar processed in a 10-liter oven using iron catalyst, and a pitch mixture from Welheim processed in a 10-liter over using iron catalyst. The values gathered were compared with a few corresponding values estimated for Boehlen tar and Gelsenberg coal based on several assumptions outlined in the report. The data recorded included percentage of ash in the dry residue and percentage of carbon, hydrogen, oxygen, nitrogen, chlorine, total sulfur, and volatile sulfur. The percentage of ash varied from 21.43% in the case of Bruex tar to 53.15% in the case of one of the Silesian coals. Percentage of carbon varied from 44.0% in the case of Scholven coal to 78.03% in the case of Bruex tar. Percentage of total sulfur varied from 2.28% for Bruex tar to a recorded 5.65% for one of the Silesian coals and an estimated 6% for Boehlen tar. 1 table.

  2. PTEN is a protein tyrosine phosphatase for IRS1.

    Science.gov (United States)

    Shi, Yuji; Wang, Junru; Chandarlapaty, Sarat; Cross, Justin; Thompson, Craig; Rosen, Neal; Jiang, Xuejun

    2014-06-01

    The biological function of the PTEN tumor suppressor is mainly attributed to its lipid phosphatase activity. This study demonstrates that mammalian PTEN is a protein tyrosine phosphatase that selectively dephosphorylates insulin receptor substrate-1 (IRS1), a mediator of insulin and IGF signals. IGF signaling was defective in cells lacking NEDD4, a PTEN ubiquitin ligase, whereas AKT activation triggered by EGF or serum was unimpaired. Defective IGF signaling caused by NEDD4 deletion, including phosphorylation of IRS1 and AKT, was rescued by PTEN ablation. We demonstrate the nature of PTEN as an IRS1 phosphatase by direct biochemical analysis and cellular reconstitution, showing that NEDD4 supports insulin-mediated glucose metabolism and is required for the proliferation of IGF1 receptor-dependent but not EGF receptor-dependent tumor cells. Thus, PTEN is a protein phosphatase for IRS1, and its antagonism by NEDD4 promotes signaling by IGF and insulin.

  3. Effect of radiation on tyrosine and tetracycline in poultry meat

    International Nuclear Information System (INIS)

    Vachin, I.; Pavlov, A.; Lashev, L.

    1994-01-01

    The effect of gamma rays on tissue level changes of unbound tyrosine and tetracycline was examined in comparative trials with broiler chicken meat. White meat and dark meat samples were taken from the killed chicken and were frozen at -18 o C. The three experimental groups were gamma irradiated with doses of 1.0, 1.5 and 2 kGy. The contents of the unbound antibiotics were microbiologically tested on 24th hour and 45th day after irradiation using Bacillus subtilis ATCC 6633, respectively Bac. mycoides HB 2 as test organisms. It was found that on 25th hour after irradiation both antibiotics were not significantly changed compared to the controls. After 45 days of storage a tendency towards decreasing the antibiotic concentrations in both the white and dark meat had been detected. (author)

  4. Tyrosine Kinase Receptor Landscape in Lung Cancer: Therapeutical Implications

    Directory of Open Access Journals (Sweden)

    A. Quintanal-Villalonga

    2016-01-01

    Full Text Available Lung cancer is a heterogeneous disease responsible for the most cases of cancer-related deaths. The majority of patients are clinically diagnosed at advanced stages, with a poor survival rate. For this reason, the identification of oncodrivers and novel biomarkers is decisive for the future clinical management of this pathology. The rise of high throughput technologies popularly referred to as “omics” has accelerated the discovery of new biomarkers and drivers for this pathology. Within them, tyrosine kinase receptors (TKRs have proven to be of importance as diagnostic, prognostic, and predictive tools and, due to their molecular nature, as therapeutic targets. Along this review, the role of TKRs in the different lung cancer histologies, research on improvement of anti-TKR therapy, and the current approaches to manage anti-TKR resistance will be discussed.

  5. The Receptor Tyrosine Kinase AXL in Cancer Progression

    Directory of Open Access Journals (Sweden)

    Erinn B. Rankin

    2016-11-01

    Full Text Available The AXL receptor tyrosine kinase (AXL has emerged as a promising therapeutic target for cancer therapy. Recent studies have revealed a central role of AXL signaling in tumor proliferation, survival, stem cell phenotype, metastasis, and resistance to cancer therapy. Moreover, AXL is expressed within cellular components of the tumor microenvironment where AXL signaling contributes to the immunosuppressive and protumorigenic phenotypes. A variety of AXL inhibitors have been developed and are efficacious in preclinical studies. These agents offer new opportunities for therapeutic intervention in the prevention and treatment of advanced disease. Here we review the literature that has illuminated the cellular and molecular mechanisms by which AXL signaling promotes tumor progression and we will discuss the therapeutic potential of AXL inhibition for cancer therapy.

  6. Design and Synthesis of Novel Macrocyclic Mer Tyrosine Kinase Inhibitors.

    Science.gov (United States)

    Wang, Xiaodong; Liu, Jing; Zhang, Weihe; Stashko, Michael A; Nichols, James; Miley, Michael J; Norris-Drouin, Jacqueline; Chen, Zhilong; Machius, Mischa; DeRyckere, Deborah; Wood, Edgar; Graham, Douglas K; Earp, H Shelton; Kireev, Dmitri; Frye, Stephen V

    2016-12-08

    Mer tyrosine kinase (MerTK) is aberrantly elevated in various tumor cells and has a normal anti-inflammatory role in the innate immune system. Inhibition of MerTK may provide dual effects against these MerTK-expressing tumors through reducing cancer cell survival and redirecting the innate immune response. Recently, we have designed novel and potent macrocyclic pyrrolopyrimidines as MerTK inhibitors using a structure-based approach. The most active macrocycles had an EC 50 below 40 nM in a cell-based MerTK phosphor-protein ELISA assay. The X-ray structure of macrocyclic analogue 3 complexed with MerTK was also resolved and demonstrated macrocycles binding in the ATP binding pocket of the MerTK protein as anticipated. In addition, the lead compound 16 (UNC3133) had a 1.6 h half-life and 16% oral bioavailability in a mouse PK study.

  7. Tyrosine kinase inhibitors induced immune thrombocytopenia in chronic myeloid leukemia?

    Directory of Open Access Journals (Sweden)

    Avital F. Barak

    2011-12-01

    Full Text Available The outcome and quality of life of chronic myeloid leukemia (CML patients has remarkably changed with the treatment of tyrosine kinase inhibitors (TKIs. Currently, hematopoietic stem cell transplantation (HSCT is considered mainly as a third line salvage therapy in cases of TKIs resistance or intolerance. Here we describe a patient with chronic phase CML who developed both resistance and late occurrence of s severe thrombocytopenia on first and second generation TKIs and eventually underwent HSCT. Although the mechanism of the myelosuppression is not fully understood, we showed for the first time the development of dose dependent platelet antibodies in the presence of TKIs, suggesting the possibility of TKIs induced thrombocytopenia. Our case emphasizes that late development of severe myelosuppression during imatinib treatment is probably an important indication for consideration of early HSCT.

  8. Conserved Molecular Mechanism of TyrA Dehydrogenase Substrate Specificity Underlying Alternative Tyrosine Biosynthetic Pathways in Plants and Microbes

    Directory of Open Access Journals (Sweden)

    Craig A. Schenck

    2017-11-01

    Full Text Available L-Tyrosine (Tyr is an aromatic amino acid synthesized de novo in plants and microbes. In animals, Tyr must be obtained through their diet or synthesized from L-phenylalanine. In addition to protein synthesis, Tyr serves as the precursor of neurotransmitters (e.g., dopamine and epinephrine in animals and of numerous plant natural products, which serve essential functions in both plants and humans (e.g., vitamin E and morphine. Tyr is synthesized via two alternative routes mediated by a TyrA family enzyme, prephenate, or arogenate dehydrogenase (PDH/TyrAp or ADH/TyrAa, typically found in microbes and plants, respectively. Although ADH activity is also found in some bacteria, the origin of arogenate-specific TyrAa enzymes is unknown. We recently identified an acidic Asp222 residue that confers ADH activity in plant TyrAs. In this study, structure-guided phylogenetic analyses identified bacterial homologs, closely-related to plant TyrAs, that also have an acidic 222 residue and ADH activity. A more distant archaeon TyrA that preferred PDH activity had a non-acidic Gln, whose substitution to Glu introduced ADH activity. These results indicate that the conserved molecular mechanism operated during the evolution of arogenate-specific TyrAa in both plants and microbes.

  9. Growth hormone-dependent phosphorylation of tyrosine 333 and/or 338 of the growth hormone receptor

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L

    1995-01-01

    Many signaling pathways initiated by ligands that activate receptor tyrosine kinases have been shown to involve the binding of SH2 domain-containing proteins to specific phosphorylated tyrosines in the receptor. Although the receptor for growth hormone (GH) does not contain intrinsic tyrosine...

  10. Identification and characterization of hydrophobic gate residues in TRP channels.

    Science.gov (United States)

    Zheng, Wang; Hu, Ruikun; Cai, Ruiqi; Hofmann, Laura; Hu, Qiaolin; Fatehi, Mohammad; Long, Wentong; Kong, Tim; Tang, Jingfeng; Light, Peter; Flockerzi, Veit; Cao, Ying; Chen, Xing-Zhen

    2018-02-01

    Transient receptor potential (TRP) channels, subdivided into 6 subfamilies in mammals, have essential roles in sensory physiology. They respond to remarkably diverse stimuli, comprising thermal, chemical, and mechanical modalities, through opening or closing of channel gates. In this study, we systematically substituted the hydrophobic residues within the distal fragment of pore-lining helix S6 with hydrophilic residues and, based on Xenopus oocyte and mammalian cell electrophysiology and a hydrophobic gate theory, identified hydrophobic gates in TRPV6/V5/V4/C4/M8. We found that channel activity drastically increased when TRPV6 Ala616 or Met617 or TRPV5 Ala576 or Met577 , but not any of their adjacent residues, was substituted with hydrophilic residues. Channel activity strongly correlated with the hydrophilicity of the residues at those sites, suggesting that consecutive hydrophobic residues TRPV6 Ala616-Met617 and TRPV5 Ala576-Met577 form a double-residue gate in each channel. By the same strategy, we identified a hydrophobic single-residue gate in TRPV4 Iso715 , TRPC4 Iso617 , and TRPM8 Val976 . In support of the hydrophobic gate theory, hydrophilic substitution at the gate site, which removes the hydrophobic gate seal, substantially increased the activity of TRP channels in low-activity states but had little effect on the function of activated channels. The double-residue gate channels were more sensitive to small changes in the gate's hydrophobicity or size than single-residue gate channels. The unconventional double-reside gating mechanism in TRP channels may have been evolved to respond especially to physiologic stimuli that trigger relatively small gate conformational changes.-Zheng, W., Hu, R., Cai, R., Hofmann, L., Hu, Q., Fatehi, M., Long, W., Kong, T., Tang, J., Light, P., Flockerzi, V., Cao, Y., Chen, X.-Z. Identification and characterization of hydrophobic gate residues in TRP channels.

  11. Spectroscopic analyses on interaction of o-Vanillin- D-Phenylalanine, o-Vanillin- L-Tyrosine and o-Vanillin- L-Levodopa Schiff Bases with bovine serum albumin (BSA)

    Science.gov (United States)

    Gao, Jingqun; Guo, Yuwei; Wang, Jun; Wang, Zhiqiu; Jin, Xudong; Cheng, Chunping; Li, Ying; Li, Kai

    2011-04-01

    In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin- D-Phenylalanine (o-VDP), o-Vanillin- L-Tyrosine (o-VLT) and o-Vanillin- L-Levodopa (o-VLL)) with alanine constituent were synthesized by direct reflux method in ethanol solution, and then were used to study the interaction to bovine serum albumin (BSA) molecules by fluorescence spectroscopy. Based on the fluorescence quenching calculation, the bimolecular quenching constant ( Kq), apparent quenching constant ( Ksv), effective binding constant ( KA) and corresponding dissociation constant ( KD) as well as binding site number ( n) were obtained. In addition, the binding distance ( r) was also calculated according to Foster's non-radioactive energy transfer theory. The results show that these three o-VSB can efficiently bind to BSA molecules, but the binding array order is o-VDP-BSA > o-VLT-BSA > o-VLL-BSA. Synchronous fluorescence spectroscopy indicates that the o-VDP is more accessibility to tryptophan (Trp) residues of BSA molecules than to tyrosine (Tyr) residues. Nevertheless, the o-VLT and o-VLL are more accessibility to Tyr residues than to Trp residues.

  12. The conserved tyrosine residue 940 plays a key structural role in membrane interaction of Bordetella adenylate cyclase toxin

    Czech Academy of Sciences Publication Activity Database

    Mašín, Jiří; Roderová, Jana; Osičková, Adriana; Novák, Petr; Bumba, Ladislav; Fišer, Radovan; Šebo, Peter; Osička, Radim

    2017-01-01

    Roč. 7, AUG 24 (2017), s. 1-14, č. článku 9330. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GA16-05919S; GA ČR GA15-09157S; GA ČR(CZ) GA15-11851S; GA ČR(CZ) GA13-14547S; GA MŠk(CZ) LM2015064; GA MŠk(CZ) LD15089; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : PERTUSSIS CYAA * BENZODIAZEPINE-RECEPTOR * ESCHERICHIA-COLI Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.259, year: 2016

  13. Protein tyrosine phosphatases: Ligand interaction analysis and optimisation of virtual screening.

    Science.gov (United States)

    Ghattas, Mohammad A; Atatreh, Noor; Bichenkova, Elena V; Bryce, Richard A

    2014-07-01

    Docking-based virtual screening is an established component of structure-based drug discovery. Nevertheless, scoring and ranking of computationally docked ligand libraries still suffer from many false positives. Identifying optimal docking parameters for a target protein prior to virtual screening can improve experimental hit rates. Here, we examine protocols for virtual screening against the important but challenging class of drug target, protein tyrosine phosphatases. In this study, common interaction features were identified from analysis of protein-ligand binding geometries of more than 50 complexed phosphatase crystal structures. It was found that two interactions were consistently formed across all phosphatase inhibitors: (1) a polar contact with the conserved arginine residue, and (2) at least one interaction with the P-loop backbone amide. In order to investigate the significance of these features on phosphatase-ligand binding, a series of seeded virtual screening experiments were conducted on three phosphatase enzymes, PTP1B, Cdc25b and IF2. It was observed that when the conserved arginine and P-loop amide interactions were used as pharmacophoric constraints during docking, enrichment of the virtual screen significantly increased in the three studied phosphatases, by up to a factor of two in some cases. Additionally, the use of such pharmacophoric constraints considerably improved the ability of docking to predict the inhibitor's bound pose, decreasing RMSD to the crystallographic geometry by 43% on average. Constrained docking improved enrichment of screens against both open and closed conformations of PTP1B. Incorporation of an ordered water molecule in PTP1B screening was also found to generally improve enrichment. The knowledge-based computational strategies explored here can potentially inform structure-based design of new phosphatase inhibitors using docking-based virtual screening. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Tyrosine Aminotransferase Contributes to Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy1[W

    Science.gov (United States)

    Lee, Eun-Jeong; Facchini, Peter J.

    2011-01-01

    Tyrosine aminotransferase (TyrAT) catalyzes the transamination of l-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and l-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5′-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for l-Tyr and α-ketoglutarate, with apparent Km values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of l-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde. PMID:21949209

  15. Molecular modeling and structural characterization of a high glycine-tyrosine hair keratin associated protein.

    Science.gov (United States)

    Singh, Rakesh S; Palmer, Jeremy C; Pudney, Paul D A; Paul, Prem K C; Johannessen, Christian; Debenedetti, Pablo G; Raut, Janhavi; Lee, Ken; Noro, Massimo; Tiemessen, David

    2017-03-22

    High glycine-tyrosine (HGT) proteins are an important constituent of the keratin associated proteins (KAPs) present in human hair. The glassy state physics of hair fibres are thought to be largely regulated by KAPs, which exist in an amorphous state and are readily affected by environmental conditions. However, there are no studies characterizing the individual KAPs. In this paper, we present the first step to fill this gap by computational modeling and experimental studies on a HGT protein, KAP8.1. In particular, we have modeled the three-dimensional structure of this 63-residue protein using homology information from an anti-freeze protein in snow flea. The model for KAP8.1 is characterized by four strands of poly-proline II (or PPII) type helical secondary structures, held together by two cysteine disulphide bridges. Computer simulations confirm the stability of the modelled structure and show that the protein largely samples the PPII and β-sheet conformations during the molecular dynamics simulations. Spectroscopic studies including Raman, IR and vibrational circular dichroism have also been performed on synthesized KAP8.1. The experimental studies suggest that KAP8.1 is characterised by β-sheet and PPII structures, largely consistent with the simulation studies. The model built in this work is a good starting point for further simulations to study in greater depth the glassy state physics of hair, including its water sorption isotherms, glass transition, and the effect of HGT proteins on KAP matrix plasticization. These results are a significant step towards our goal of understanding how the properties of hair can be affected and manipulated under different environmental conditions of temperature, humidity, ageing and small molecule additives.

  16. Epigenetic Reprogramming Sensitizes CML Stem Cells to Combined EZH2 and Tyrosine Kinase Inhibition.

    Science.gov (United States)

    Scott, Mary T; Korfi, Koorosh; Saffrey, Peter; Hopcroft, Lisa E M; Kinstrie, Ross; Pellicano, Francesca; Guenther, Carla; Gallipoli, Paolo; Cruz, Michelle; Dunn, Karen; Jorgensen, Heather G; Cassels, Jennifer E; Hamilton, Ashley; Crossan, Andrew; Sinclair, Amy; Holyoake, Tessa L; Vetrie, David

    2016-11-01

    A major obstacle to curing chronic myeloid leukemia (CML) is residual disease maintained by tyrosine kinase inhibitor (TKI)-persistent leukemic stem cells (LSC). These are BCR-ABL1 kinase independent, refractory to apoptosis, and serve as a reservoir to drive relapse or TKI resistance. We demonstrate that Polycomb Repressive Complex 2 is misregulated in chronic phase CML LSCs. This is associated with extensive reprogramming of H3K27me3 targets in LSCs, thus sensitizing them to apoptosis upon treatment with an EZH2-specific inhibitor (EZH2i). EZH2i does not impair normal hematopoietic stem cell survival. Strikingly, treatment of primary CML cells with either EZH2i or TKI alone caused significant upregulation of H3K27me3 targets, and combined treatment further potentiated these effects and resulted in significant loss of LSCs compared to TKI alone, in vitro, and in long-term bone marrow murine xenografts. Our findings point to a promising epigenetic-based therapeutic strategy to more effectively target LSCs in patients with CML receiving TKIs. In CML, TKI-persistent LSCs remain an obstacle to cure, and approaches to eradicate them remain a significant unmet clinical need. We demonstrate that EZH2 and H3K27me3 reprogramming is important for LSC survival, but renders LSCs sensitive to the combined effects of EZH2i and TKI. This represents a novel approach to more effectively target LSCs in patients receiving TKI treatment. Cancer Discov; 6(11); 1248-57. ©2016 AACR.See related article by Xie et al., p. 1237This article is highlighted in the In This Issue feature, p. 1197. ©2016 American Association for Cancer Research.

  17. Quadratic residues and non-residues selected topics

    CERN Document Server

    Wright, Steve

    2016-01-01

    This book offers an account of the classical theory of quadratic residues and non-residues with the goal of using that theory as a lens through which to view the development of some of the fundamental methods employed in modern elementary, algebraic, and analytic number theory. The first three chapters present some basic facts and the history of quadratic residues and non-residues and discuss various proofs of the Law of Quadratic Reciprosity in depth, with an emphasis on the six proofs that Gauss published. The remaining seven chapters explore some interesting applications of the Law of Quadratic Reciprocity, prove some results concerning the distribution and arithmetic structure of quadratic residues and non-residues, provide a detailed proof of Dirichlet’s Class-Number Formula, and discuss the question of whether quadratic residues are randomly distributed. The text is a valuable resource for graduate and advanced undergraduate students as well as for mathematicians interested in number theory.

  18. Determination of o-tyrosine as a marker for the detection of irradiated shrimps

    International Nuclear Information System (INIS)

    Hunková, J.; Simat, T.J.; Steinhart, H.

    2000-01-01

    o-tyrosine is proposed as a marker for the identification of irradiated protein-rich food. An HPLC method for qualitative and quantitative determination of non-protein bound o-tyrosine in shrimps (Crangon crangon) has been developed. For this purpose the o-tyrosine was extracted from non-irradiated as well as irradiated samples with perchloric acid, then separated isocratically (ammoniumformiat buffer, pH 4) on an RP-C18 column and detected by FLD (275/305 nm). The quantification of o-tyrosine was based on the use of alfa-methyl-p-tyrosine as internal standard. In non-irradiated shrimps a background level of 28.9 microg/kg was found. The content of o-tyrosine in 1 kGy irradiated shrimps was found to be 119.9 mikrog/kg, which was well 4-fold over the background level. The dependency between radiation dose and the amount of o-tyrosine was observed in the range of 0-5 kGy

  19. Production of tyrosine through phenylalanine hydroxylation bypasses the intrinsic feedback inhibition in Escherichia coli.

    Science.gov (United States)

    Huang, Jin; Lin, Yuheng; Yuan, Qipeng; Yan, Yajun

    2015-04-01

    Tyrosine is a proteinogenic aromatic amino acid that is often used as a supplement of food and animal feed, as well as a (bio-)synthetic precursor to various pharmaceutically or industrially important molecules. Extensive metabolic engineering efforts have been made towards the efficient and cost-effective microbial production of tyrosine. Conventional strategies usually focus on eliminating intrinsic feedback inhibition and redirecting carbon flux into the shikimate pathway. In this study, we found that continuous conversion of phenylalanine into tyrosine by the action of tetrahydromonapterin (MH4)-utilizing phenylalanine 4-hydroxylase (P4H) can bypass the feedback inhibition in Escherichia coli, leading to tyrosine accumulation in the cultures. First, expression of the P4H from Xanthomonas campestris in combination with an MH4 recycling system in wild-type E. coli allowed the strain to accumulate tyrosine at 262 mg/L. On this basis, enhanced expression of the key enzymes associated with the shikimate pathway and the MH4 biosynthetic pathway resulted in the elevation of tyrosine production up to 401 mg/L in shake flasks. This work demonstrated a novel approach to tyrosine production and verified the possibility to alleviate feedback inhibition by creating a phenylalanine sink.

  20. Role of RET protein-tyrosine kinase inhibitors in the treatment RET-driven thyroid and lung cancers.

    Science.gov (United States)

    Roskoski, Robert; Sadeghi-Nejad, Abdollah

    2018-02-01

    RET is a transmembrane receptor protein-tyrosine kinase that is required for the development of the nervous system and several other tissues. The mechanism of activation of RET by its glial-cell derived neurotrophic factor (GDNF) ligands differs from that of all other receptor protein-tyrosine kinases owing to the requirement for additional GDNF family receptor-α (GFRα) co-receptors (GFRα1/2/3/4). RET point mutations have been reported in multiple endocrine neoplasia (MEN2A, MEN2B) and medullary thyroid carcinoma. In contrast, RET fusion proteins have been reported in papillary thyroid and non-small cell lung adenocarcinomas. More than a dozen fusion partners of RET have been described in papillary thyroid carcinomas, most frequently CCDC6-RET and NCOA4-RET. RET-fusion proteins, commonly KIF5B-RET, have also been found in non-small cell lung cancer (NSCLC). Several drugs targeting RET have been approved by the FDA for the treatment of cancer: (i) cabozantinib and vandetanib for medullary thyroid carcinomas and (ii) lenvatinib and sorafenib for differentiated thyroid cancers. In addition, alectinib and sunitinib are approved for the treatment of other neoplasms. Each of these drugs is a multikinase inhibitor that has activity against RET. Previous X-ray studies indicated that vandetanib binds within the ATP-binding pocket and forms a hydrogen bond with A807 within the RET hinge and it makes hydrophobic contact with L881 of the catalytic spine which occurs in the floor of the adenine-binding pocket. Our molecular modeling studies indicate that the other antagonists bind in a similar fashion. All of these antagonists bind to the active conformation of RET and are therefore classified as type I inhibitors. The drugs also make variable contacts with other residues of the regulatory and catalytic spines. None of these drugs was designed to bind preferentially to RET and it is hypothesized that RET-specific antagonists might produce even better clinical outcomes

  1. Sharing Residual Liability

    DEFF Research Database (Denmark)

    Carbonara, Emanuela; Guerra, Alice; Parisi, Francesco

    2016-01-01

    Economic models of tort law evaluate the efficiency of liability rules in terms of care and activity levels. A liability regime is optimal when it creates incentives to maximize the value of risky activities net of accident and precaution costs. The allocation of primary and residual liability...... the virtues and limits of loss-sharing rules in generating optimal (second-best) incentives and allocations of risk. We find that loss sharing may be optimal in the presence of countervailing policy objectives, homogeneous risk avoiders, and subadditive risk, which potentially offers a valuable tool...

  2. Tyrosine Phosphorylation Pattern in Sperm Proteins Isolated from Normospermic and Teratospermic Men

    Science.gov (United States)

    Jabbari, Sepideh; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mahdi; Ebrahim Habibi, Azadeh; Amirjanati, Naser; Lakpour, Niknam; Asgharpour, Lima; Ardekani, Ali M.

    2009-01-01

    Introduction In mammalian system, spermatozoa are not able to fertilize the oocyte immediately upon ejaculation, thus they undergo a series of biochemical and molecular changes which is termed capacitation. During sperm capacitation, signal transduction pathways are activated which lead to protein tyrosine phosphorylation. Tyrosine phosphorylated proteins have an important role in sperm capacitation such as hyperactive motility, interaction with zona pellucida and acrosome reaction. Evaluation of tyrosine phosphorylation pattern is important for further understanding of molecular mechanisms of fertilization and the etiology of sperm dysfunctions and abnormalities such as teratospermia. The goal of this study is to characterize tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic infertile men attending Avicenna Infertility Clinic in Tehran. Materials and Methods Semen samples were collected and the spermatozoa were isolated using Percoll gradient centrifugation. Then the spermatozoa were incubated up to 6h at 37°C with 5% CO2 in 3% Bovine Serum Albumin-supplemented Ham's F-10 for capacitation to take place. The total proteins from spermatozoa were extracted and were subjected to SDS-PAGE before and after capacitation. To evaluate protein tyrosine phosphorylation pattern, western blotting with specific antibody against phosphorylated tyrosines was performed. Results The results upon western blotting showed: 1) at least six protein bands were detected before capacitation in the spermatozoa from normospermic samples. However, comparable levels of tyrosine phosphorylation was not observed in the spermatozoa from teratospermic samples. 2) The intensity of protein tyrosine phosphorylation appears to have been increased during capacitation in the normospermic relative to the teratospermic group. Conclusion For the first time, these findings demonstrate and suggest that the differences in the types of proteins and diminished

  3. Rates and energetics of tyrosine ring flips in yeast iso-2-cytochrome c

    International Nuclear Information System (INIS)

    Nall, B.T.; Zuniga, E.H.

    1990-01-01

    Isotope-edited nuclear magnetic resonance spectroscopy is used to monitor ring flip motion of the five tyrosine side chains in the oxidized and reduced forms of yeast iso-2-cytochrome c. With specifically labeled protein purified from yeast grown on media containing [3,5- 13 C]tyrosine, isotope-edited one-dimensional proton spectra have been collected over a 5-55 degree C temperature range. The spectra allow selective observation of the 10 3,5 tyrosine ring proton resonances and, using a two-site exchange model, allow estimation of the temperature dependence of ring flip rates from motion-induced changes in proton line shapes. For the reduced protein, tyrosines II and IV are in fast exchange throughout the temperature range investigated, or lack resolvable differences in static chemical shifts for the 3,5 ring protons. Tyrosines I, III, and V are in sloe exchange at low temperatures and in fast exchange at high temperatures. Spectral simulations give flip rates for individual tyrosines in a range of one flip per second at low temperatures to thousands of flips per second at high temperatures. Eyring plots show that two of the tyrosines (I and III) have essentially the same activation parameters. Tentative sequence-specific assignments for the tyrosines in reduced iso-2 are suggested by comparison to horse cytochrome c. For oxidized iso-2, five resonances are observed at high temperatures, suggesting flip rates for all five tyrosines sufficient to average static chemical shift differences. At lower temperatures, there is evidence of intermediate and slow flipping for some of the rings

  4. Sequence-specific backbone ¹H, ¹³C and ¹⁵N assignments of the catalytic domain of the Escherichia coli protein tyrosine kinase, Wzc.

    Science.gov (United States)

    Temel, Deniz B; Dutta, Kaushik; Ghose, Ranajeet

    2014-04-01

    Protein tyrosine kinases in bacteria are structurally and functionally distinct from their eukaryotic counterparts. The largest family of bacterial tyrosine kinases, the BY-kinase family, is highly conserved in Gram-negative and Gram-positive species, and plays a central role in biofilm and capsule formation. In Escherichia coli the BY-kinase, Wzc, is a critical component of the machinery responsible for the synthesis and export of the exo-polysaccharide colanic acid, a key constituent of biofilms. Here we present the main-chain (1)H(N), (15)N, (13)C' and (13)Cα, side-chain (13)Cβ resonance assignments for a construct that encodes the entire 274-residue cytosolic catalytic domain of Wzc.

  5. Congenital central hypoventilation syndrome: Mutation analysis of the receptor tyrosine kinase RET

    Energy Technology Data Exchange (ETDEWEB)

    Bolk, S.; Angrist, M.; Schwartz, S.; Chakravarti, A. [Case Western Reserve Univ., Cleveland, OH (United States)]|[University Hospitals, Cleveland, OH (United States)] [and others

    1996-06-28

    Congenital central hypoventilation syndrome (CCHS) usually occurs as an isolated phenotype. However, 16% of the index cases are also affected with Hirschsprung disease (HSCR). Complex segregation analysis suggests that CCHS is familial and has the same inheritance pattern with or without HSCR. We postulate that alteration of normal function of the receptor tyrosine kinase, RET, may contribute to CCHS based on RET`s expression pattern and the identification of RET mutations in HSCR patients. To further explore the nature of the inheritance of CCHS, we have undertaken two main routes of investigation: cytogenetic analysis and mutation detection. Cytogenetic analysis of metaphase chromosomes showed normal karyotypes in 13 of the 14 evaluated index cases; one index case carried a familial pericentric inversion on chromosome 2. Mutation analysis showed no sequence changes unique to index cases, as compared to control individuals, and as studied by single strand conformational polymorphism (SSCP) analysis of the coding region of RET. We conclude that point mutations in the RET coding region cannot account for a substantial fraction of CCHS in this patient population, and that other candidate genes involved in neural crest cell differentiation and development must be considered. 54 refs.

  6. Peripheral Lipopolysaccharide Challenge Induces Long-Term Changes in Tyrosine Hydroxylase Regulation in the Adrenal Medulla.

    Science.gov (United States)

    Ong, Lin Kooi; Page, Scott; Briggs, Gabrielle D; Guan, Liying; Dun, Matthew D; Verrills, Nicole M; Dunkley, Peter R; Dickson, Phillip W

    2017-08-01

    Immune activation can alter the activity of adrenal chromaffin cells. The effect of immune activation by lipopolysaccharide (LPS) on the regulation of tyrosine hydroxylase (TH) in the adrenal medulla in vivo was determined between 1 day and 6 months after LPS injection. The plasma levels of eleven cytokines were reduced 1 day after LPS injection, whereas the level for interleukin-10 was increased. The levels of all cytokines remained at control levels until 6 months when the levels of interleukin-6 and -4 were increased. One day after LPS injection, there was a decrease in TH-specific activity that may be due to decreased phosphorylation of serine 31 and 40. This decreased phosphorylation of serine 31 and 40 may be due to an increased activation of the protein phosphatase PP2A. One week after LPS injection, there was increased TH protein and increased phosphorylation of serine 40 that this was not accompanied by an increase in TH-specific activity. All TH parameters measured returned to basal levels between 1 month and 3 months. Six months after injection there was an increase in TH protein. This was associated with increased levels of the extracellular regulated kinase isoforms 1 and 2. This work shows that a single inflammatory event has the capacity to generate both short-term and long-term changes in TH regulation in the adrenal medulla of the adult animal. J. Cell. Biochem. 118: 2096-2107, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. Sulfone-stabilized carbanions for the reversible covalent capture of a posttranslationally-generated cysteine oxoform found in protein tyrosine phosphatase 1B (PTP1B).

    Science.gov (United States)

    Parsons, Zachary D; Ruddraraju, Kasi Viswanatharaju; Santo, Nicholas; Gates, Kent S

    2016-06-15

    Redox regulation of protein tyrosine phosphatase 1B (PTP1B) involves oxidative conversion of the active site cysteine thiolate into an electrophilic sulfenyl amide residue. Reduction of the sulfenyl amide by biological thiols regenerates the native cysteine residue. Here we explored fundamental chemical reactions that may enable covalent capture of the sulfenyl amide residue in oxidized PTP1B. Various sulfone-containing carbon acids were found to react readily with a model peptide sulfenyl amide via attack of the sulfonyl carbanion on the electrophilic sulfur center in the sulfenyl amide. Both the products and the rates of these reactions were characterized. The results suggest that capture of a peptide sulfenyl amide residue by sulfone-stabilized carbanions can slow, but not completely prevent, thiol-mediated generation of the corresponding cysteine-containing peptide. Sulfone-containing carbon acids may be useful components in the construction of agents that knock down PTP1B activity in cells via transient covalent capture of the sulfenyl amide oxoform generated during insulin signaling processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Elicitor-induced tyrosine decarboxylase in berberine-synthesizing suspension cultures of Thalictrum rugosum.

    Science.gov (United States)

    Gügler, K; Funk, C; Brodelius, P

    1988-01-04

    Tyrosine decarboxylase (EC 4.1.1.25) was induced in suspension cultures of Thalictrum rugosum by treatment with a yeast glucan elicitor. Maximum induction was observed at a carbohydrate concentration of 0.4 mg/g fresh weight of cells and maximum enzyme activity was reached 20 h after addition of elicitor. The enzyme was inducible in late exponential and early stationary growth phases. A good correlation between induced tyrosine decarboxylase activity and berberine biosynthesis has been established. It is suggested that tyrosine decarboxylase may be a key enzyme between primary and secondary metabolisms in the biosynthesis of norlaudanosoline-derived alkaloids.

  9. Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase.

    OpenAIRE

    Albert, K A; Helmer-Matyjek, E; Nairn, A C; Müller, T H; Haycock, J W; Greene, L A; Goldstein, M; Greengard, P

    1984-01-01

    Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12 cells. These actions of protein kinase C required the presence of calcium and phospholipid. This phosphorylation of tyrosine hydroxylase reduced the Km for the cofactor 6-methyltetrahydropterine from 0.45 mM to 0.11 mM, increased the Ki for dopamine from 4.2 microM to 47.5 microM, and produced no change in the Km for tyrosine. Lit...

  10. The use of the tyrosine phosphatase antagonist orthovanadate in the study of a cell proliferation inhibitor

    Science.gov (United States)

    Enebo, D. J.; Hanek, G.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Incubation of murine fibroblasts with orthovanadate, a global tyrosine phosphatase inhibitor, was shown to confer a "pseudo-transformed" phenotype with regard to cell morphology and growth characteristics. This alteration was manifested by both an increasing refractile appearance of the cells, consistent with many transformed cell lines, as well as an increase in maximum cell density was attained. Despite the abrogation of cellular tyrosine phosphatase activity, orthovanadate-treated cells remained sensitive to the biological activity of a naturally occurring sialoglycopeptide (SGP) cell surface proliferation inhibitor. The results indicated that tyrosine phosphatase activity, inhibited by orthovanadate, was not involved in the signal transduction pathway of the SGP.

  11. Processes for the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention generally relates to the field of biotechnology as it applies to the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity. More particularly, the present invention pertains to polypeptides having tyrosine ammonia lyase activity and high...... substrate specificity towards tyrosine, which makes them particularly suitable in the production of p-coumaric acid and other hydroxycinnamic acids. The present invention thus provides processes for the production of p-coumaric acid and other hydroxycinnamic acids employing these polypeptides as well...

  12. [Dasatinib. A novel tyrosine kinase inhibitor for the treatment of chronic myeloid leukaemia

    DEFF Research Database (Denmark)

    Dufva, I.H.; Stentoft, J.; Hasselbalch, H.C.

    2008-01-01

    Chronic myeloid leukaemia is characterized by an abnormal tyrosin kinase in the cytoplasm of the clonal cells. The enzyme is derived from a fusion gene on the Philadelphia-chromosome, evolved by a translocation between chromosomes 9 and 22. Understanding the biology of the tyrosin kinase led to t...... to targeted therapy, inhibiting the ATP-binding site by a small molecule--imatinib (Glivec). A novel 2nd generation tyrosin kinase inhibitor--dasatinib (Sprycel)--is now available in cases of insufficient response or intolerance to imatinib Udgivelsesdato: 2008/1/28...

  13. Prognostic impact of postoperative, pre-irradiation 18F-fluoroethyl-L-tyrosine uptake in glioblastoma patients treated with radiochemotherapy

    International Nuclear Information System (INIS)

    Piroth, Marc D.; Holy, Richard; Pinkawa, Michael; Stoffels, Gabriele; Kaiser, Hans J.; Galldiks, Norbert; Herzog, Hans; Coenen, Heinz H.; Eble, Michael J.; Langen, Karl J.

    2011-01-01

    Background and purpose: Resection is considered as essential for the efficacy of modern adjuvant treatment of glioblastoma multiforme (GBM). Previous studies have indicated that amino acid PET is more specific than contrast enhancement on MRI for detecting residual tumor tissue after surgery. In a prospective study we investigated the prognostic impact of postoperative tumor volume and tumor/brain ratios (TBR) in PET using O-(2-[ 18 F]fluoroethyl)-L-tyrosine (FET) in comparison with MRI. Materials and methods: Forty-four patients with GBM were investigated by FET PET and MRI after surgery. Tumor volume in FET PET with a tumor/brain ratio (TBR) > 1.6 and a TBR > 2, mean and maximum TBR and gadolinium contrast-enhancement on MRI (Gd-volume) were determined. Thereafter patients received a fractionated radiotherapy with concomitant temozolomide (RCX). The median follow-up was 15.4 (3-35) months. The prognostic value of postoperative residual tumor volume in FET PET, TBR mean, TBR max and Gd-volume was evaluated using Kaplan-Maier estimates for disease-free survival (DFS) and overall survival (OS). Results: Postoperative tumor volume in FET PET had a significant independent influence on OS and DFS (OS 20.0 vs. 6.9 months; DFS 9.6 vs. 5.1 months, p 18 F-FET PET. The TBR mean and TBR max of FET uptake had a significant influence on DFS (p < 0.05). Gd-volume in MRI had significant effect on OS and DFS in the univariate analysis. No independent significant influence in OS or DFS could be observed for Gd-volume in MRI. Conclusions: Our data indicate that the tumor volume in FET PET after surgery of GBM has a strong prognostic impact for these patients. FET PET appears to be helpful to determine the residual tumor volume after surgery of GBM and may serve as a valuable tool for optimal planning of radiation treatment.

  14. Bioenergy from sisal residues

    Energy Technology Data Exchange (ETDEWEB)

    Jungersen, G. [Dansk Teknologisk Inst. (Denmark); Kivaisi, A.; Rubindamayugi, M. [Univ. of Dar es Salaam (Tanzania, United Republic of)

    1998-05-01

    The main objectives of this report are: To analyse the bioenergy potential of the Tanzanian agro-industries, with special emphasis on the Sisal industry, the largest producer of agro-industrial residues in Tanzania; and to upgrade the human capacity and research potential of the Applied Microbiology Unit at the University of Dar es Salaam, in order to ensure a scientific and technological support for future operation and implementation of biogas facilities and anaerobic water treatment systems. The experimental work on sisal residues contains the following issues: Optimal reactor set-up and performance; Pre-treatment methods for treatment of fibre fraction in order to increase the methane yield; Evaluation of the requirement for nutrient addition; Evaluation of the potential for bioethanol production from sisal bulbs. The processing of sisal leaves into dry fibres (decortication) has traditionally been done by the wet processing method, which consumes considerable quantities of water and produces large quantities of waste water. The Tanzania Sisal Authority (TSA) is now developing a dry decortication method, which consumes less water and produces a waste product with 12-15% TS, which is feasible for treatment in CSTR systems (Continously Stirred Tank Reactors). (EG)

  15. Hydrogen bonding of tyrosine B10 to heme-bound oxygen in Ascaris hemoglobin. Direct evidence from UV resonance Raman spectroscopy.

    Science.gov (United States)

    Huang, S; Huang, J; Kloek, A P; Goldberg, D E; Friedman, J M

    1996-01-12

    The hemoglobin from Ascaris suum, a parasitic nematode, has a spontaneous dissociation rate for the dioxygen ligand that is 3 orders of magnitude less than for mammalian myoglobins or hemoglobins. In this hemoglobin, the distal histidine is replaced with a glutamine which is capable of forming a stabilizing hydrogen bond to the bound dioxygen. A single hydrogen bond from a glutamine is, under typical circumstances, not sufficient to account for the low off rate for oxygen. Several studies point to a second hydrogen bond to the heme-bound dioxygen originating from tyrosine B10 as the source of this unusual reactivity. In this study ultraviolet (UV) resonance Raman spectroscopy is used to directly observe the formation of this hydrogen bond upon oxygen binding. The study reveals that both oxygen and carbon monoxide induce similar conformational changes in the globin upon binding to the heme; however, in the case of oxygen, a strong hydrogen bond involving a tyrosine is also observed. Similar studies on the QE7L mutant of this Hb suggest that the glutamine plays a role in stabilizing a rigid tertiary structure associated with the distal heme pocket. This conformation maintains the tyrosine in an orientation conducive to hydrogen bond formation with a heme-bound dioxygen ligand.

  16. The effect of aspartate-lysine-isoleucine and aspartate-arginine-tyrosine mutations on the expression and activity of vasopressin V2 receptor gene.

    Science.gov (United States)

    Najafzadeh, Hossein; Safaeian, Leila; Mirmohammad Sadeghi, Hamid; Rabbani, Mohammad; Jafarian, Abbas

    2010-01-01

    Vasopressin type 2 receptor (V2R) plays an important role in the water reabsorption in the kidney collecting ducts. V2R is a G protein coupled receptor (GPCR) and the triplet of amino acids aspartate-arginine-histidine (DRH) in this receptor might significantly influence its activity similar to other GPCR. However, the role of this motif has not been fully confirmed. Therefore, the present study attempted to shed some more light on the role of DRH motif in G protein coupling and V2R function with the use of site-directed mutagenesis. Nested PCR using specific primers was used to produce DNA fragments containing aspartate-lysine-isoleucine and aspartate-arginine-tyrosine mutations with replacements of the arginine to lysine and histidine to tyrosine, respectively. After digestion, these inserts were ligated into the pcDNA3 vector and transformation into E. coli HB101 was performed using heat shock method. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using diethylaminoethyl-dextran method, the adenylyl cyclase activity assay was performed for functional study. The cell surface expression was analyzed by indirect ELISA method. The functional assay indicated that none of these mutations significantly altered cAMP production and cell surface expression of V2R in these cells. Since some substitutions in arginine residue have shown to lead to the inactive V2 receptor, further studies are required to define the role of this residue more precisely. However, it seems that the role of the histidine residue is not critical in the V2 receptor function.

  17. Geochemical Testing And Model Development - Residual Tank Waste Test Plan

    International Nuclear Information System (INIS)

    Cantrell, K.J.; Connelly, M.P.

    2010-01-01

    This Test Plan describes the testing and chemical analyses release rate studies on tank residual samples collected following the retrieval of waste from the tank. This work will provide the data required to develop a contaminant release model for the tank residuals from both sludge and salt cake single-shell tanks. The data are intended for use in the long-term performance assessment and conceptual model development.

  18. GEOCHEMICAL TESTING AND MODEL DEVELOPMENT - RESIDUAL TANK WASTE TEST PLAN

    Energy Technology Data Exchange (ETDEWEB)

    CANTRELL KJ; CONNELLY MP

    2010-03-09

    This Test Plan describes the testing and chemical analyses release rate studies on tank residual samples collected following the retrieval of waste from the tank. This work will provide the data required to develop a contaminant release model for the tank residuals from both sludge and salt cake single-shell tanks. The data are intended for use in the long-term performance assessment and conceptual model development.

  19. Process for measuring residual stresses

    International Nuclear Information System (INIS)

    Elfinger, F.X.; Peiter, A.; Theiner, W.A.; Stuecker, E.

    1982-01-01

    No single process can at present solve all problems. The complete destructive processes only have a limited field of application, as the component cannot be reused. However, they are essential for the basic determination of stress distributions in the field of research and development. Destructive and non-destructive processes are mainly used if investigations have to be carried out on original components. With increasing component size, the part of destructive tests becomes smaller. The main applications are: quality assurance, testing of manufactured parts and characteristics of components. Among the non-destructive test procedures, X-raying has been developed most. It gives residual stresses on the surface and on surface layers near the edges. Further development is desirable - in assessment - in measuring techniques. Ultrasonic and magnetic crack detection processes are at present mainly used in research and development, and also in quality assurance. Because of the variable depth of penetration and the possibility of automation they are gaining in importance. (orig./RW) [de

  20. Cytoplasmic aggregates of dynactin in iPSC-derived tyrosine hydroxylase-positive neurons from a patient with Perry syndrome.

    Science.gov (United States)

    Mishima, Takayasu; Ishikawa, Taizo; Imamura, Keiko; Kondo, Takayuki; Koshiba, Yasushi; Takahashi, Ryosuke; Takahashi, Jun; Watanabe, Akihiro; Fujii, Naoki; Tsuboi, Yoshio; Inoue, Haruhisa

    2016-09-01

    Perry syndrome is a rare autosomal dominant disorder clinically characterized by parkinsonism with depression/apathy, weight loss, and central hypoventilation. Eight mutations in DCTN1 gene have been reported. A novel disease model is required because the detailed pathogenesis remains unclear. To develop a novel model, we generated induced pluripotent stem cells (iPSCs) from a Perry syndrome patient with F52L mutation in DCTN1, and describe clinical and neuroimaging investigations. We differentiated iPSCs into tyrosine hydroxylase (TH)-positive neurons. Immunocytochemistry analyses of control and mutant were performed. The patient displayed levodopa responsive parkinsonism. Dopamine transporter single photon emission tomography showed markedly decreased uptake in the striatum, and metaiodobenzylguanidine cardiac scintigraphy also showed decreased uptake. Perry syndrome TH-positive neurons showed dynactin aggregates in cytoplasm. TH-positive neurons from Perry syndrome iPSCs recapitulated an aspect of the disease phenotype of Perry syndrome. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Homophilic interactions mediated by receptor tyrosine phosphatases mu and kappa. A critical role for the novel extracellular MAM domain

    DEFF Research Database (Denmark)

    Zondag, G C; Koningstein, G M; Jiang, Y P

    1995-01-01

    and is found in diverse transmembrane proteins, is not known. We previously reported that both RPTP mu and RPTP kappa can mediate homophilic cell interactions when expressed in insect cells. Here we show that despite their striking structural similarity, RPTP mu and RPTP kappa fail to interact......The receptor-like protein tyrosine phosphatases (RPTP) mu and RPTP kappa have a modular ectodomain consisting of four fibronectin type III-like repeats, a single Ig-like domain, and a newly identified N-terminal MAM domain. The function of the latter module, which comprises about 160 amino acids...... in a heterophilic manner. To examine the role of the MAM domain in homophilic binding, we expressed a mutant RPTP mu lacking the MAM domain in insect Sf9 cells. Truncated RPTP mu is properly expressed at the cell surface but fails to promote cell-cell adhesion. Homophilic cell adhesion is fully restored...

  2. Screening of selected starter cultures for the presence of DNA sequences coding for tyrosine decarboxylase

    Directory of Open Access Journals (Sweden)

    Radka Burdychová

    2006-01-01

    Full Text Available Here, seven different starter cultures used in the production of fermented sausages were screened for the presence or absence of specific DNA sequences coding for tyrosine decarboxylase. PCR with the a set of specific primers TDC2/TDC5 (COTON et al., 2004 was used. The PCR analysis of DNA from two starter cultures confirmed the presence of DNA sequences for tyrosine decarboxylase. A detailed analysis of the starter cultures showed that DNA sequences for tyrosine decarboxylase are contained in genomic DNA of Lactobacillus curvatus and Lactobacillus sakei. These results show suitability of the described PCR method for the screening of starter cultures for the presence of the gene for tyrosine decarboxylase that is responsible for the production of the biogenic amine tyramine.

  3. Lattice modes of the chirally pure and racemic phases of tyrosine crystals

    Energy Technology Data Exchange (ETDEWEB)

    Belyanchikov, M. A. [Moscow Institute of Physics and Technology (Russian Federation); Gorelik, V. S., E-mail: gorelik@sci.lebedev.ru [Russian Academy of Sciences, Lebedev Physical Institute (Russian Federation); Gorshunov, B. P. [Moscow Institute of Physics and Technology (Russian Federation); Pyatyshev, A. Yu., E-mail: jb-valensia@mail.ru [Bauman Moscow State Technical University (Russian Federation)

    2017-01-15

    High-Q librational modes have been found to be present in the infrared absorption and Raman spectra of chirally pure L-tyrosine. Such modes can serve as terahertz radiation detectors and generators in chirally pure biostructures.

  4. Administration of supplemental L-tyrosine with phenelzine: a clinical literature review

    Science.gov (United States)

    Hinz, Marty; Stein, Alvin; Cole, Ted; Ryan, Patricia

    2014-01-01

    The subject of this literature review is the alleged relationship between L-tyrosine, phenelzine, and hypertensive crisis. Phenelzine (Nardil®) prescribing information notes: “The potentiation of sympathomimetic substances and related compounds by MAO inhibitors may result in hypertensive crises (see WARNINGS). Therefore, patients being treated with NARDIL should not take […] L-tyrosine […]”. Interest in the scientific foundation of this claim was generated during routine patient care. A comprehensive literature search of Google Scholar and PubMed revealed no reported cases of hypertensive crisis associated with concomitant administration of L-tyrosine and phenelzine. Review of current US Food and Drug Administration nutritional guidelines relating to ongoing phenelzine studies reveals no mention and requires no consideration of L-tyrosine ingestion in combination with phenelzine. This paper is intended to provide an objective review of the science to then allow the reader to formulate the final opinion. PMID:25092999

  5. Reciprocal regulation of C-Maf tyrosine phosphorylation by Tec and Ptpn22.

    Science.gov (United States)

    Liu, Chih-Chun; Lai, Chen-Yen; Yen, Wei-Feng; Lin, Yu-Hsien; Chang, Hui-Hsin; Tai, Tzong-Shyuan; Lu, Yu-Jung; Tsao, Hsiao-Wei; Ho, I-Cheng; Miaw, Shi-Chuen

    2015-01-01

    C-Maf plays an important role in regulating cytokine production in TH cells. Its transactivation of IL-4 is optimized by phosphorylation at Tyr21, Tyr92, and Tyr131. However, the molecular mechanism regulating its tyrosine phosphorylation remains unknown. In this study, we demonstrate that Tec kinase family member Tec, but not Rlk or Itk, is a tyrosine kinase of c-Maf and that Tec enhances c-Maf-dependent IL-4 promoter activity. This effect of Tec is counteracted by Ptpn22, which physically interacts with and facilitates tyrosine dephosphorylation of c-Maf thereby attenuating its transcriptional activity. We further show that phosphorylation of Tyr21/92/131 of c-Maf is also critical for its recruitment to the IL-21 promoter and optimal production of this cytokine by TH17 cells. Thus, manipulating tyrosine phosphorylation of c-Maf through its kinases and phosphatases can have significant impact on TH cell-mediated immune responses.

  6. Conformational Structure of Tyrosine, Tyrosyl-Glycine, and Tyrosyl-Glycyl-Glycine by Double Resonance Spectroscopy

    Science.gov (United States)

    Abo-Riziq, Ali; Grace, Louis; Crews, Bridgit; Callahan, Michael P,; van Mourik, Tanja; de Vries, Mattanjah S,

    2011-01-01

    We investigated the variation in conformation for the amino acid tyrosine (Y), alone and in the small peptides tyrosine-glycine (YC) and tyrosine-glycine-glycine (YGG), in the gas phase by using UV-UV and IR-UV double resonance spectroscopy and density functional theory calculations. For tyrosine we found seven different conformations, for YG we found four different conformations, and for YGG we found three different conformations. As the peptides get larger, we observe fewer stable conformers, despite the increasing complexity and number of degrees of freedom. We find structural trends similar to those in phenylalanine-glycine glycine (FGG) and tryptophan-glycine-glycine (WGG)j however) the effect of dispersive forces in FGG for stabilizing a folded structure is replaced by that of hydrogen bonding in YGG.

  7. Immunoglobulins against Tyrosine Nitrated Epitopes in Coronary Artery Disease

    Science.gov (United States)

    Thomson, Leonor; Tenopoulou, Margarita; Lightfoot, Richard; Tsika, Epida; Parastatidis, Ioannis; Martinez, Marissa; Greco, Todd M.; Doulias, Paschalis-Thomas; Wu, Yuping; Tang, W. H. Wilson; Hazen, Stanley L.; Ischiropoulos, Harry

    2012-01-01

    Background Several lines of evidence support a pathophysiological role of immunity in atherosclerosis. Tyrosine nitrated proteins, a footprint of oxygen and nitrogen derived oxidants generated by cells of the immune system, are enriched in atheromatous lesions and in circulation of coronary artery disease (CAD) subjects. However, the consequences of possible immune reactions triggered by the presence of nitrated proteins in subjects with clinically documented atherosclerosis have not been explored. Methods and Results Specific immunoglobulins that recognize 3-nitrotyrosine epitopes were identified in human lesions, as well as in circulation of CAD subjects. The levels of circulating immunoglobulins against 3-nitrotyrosine epitopes were quantified in CAD patients (n=374) and subjects without CAD (non CAD controls, n=313). A ten-fold increase in the mean level of circulating immunoglobulins against protein-bound 3-nitrotyrosine was documented in the CAD subjects (3.75 ± 1.8 μg antibody Eq/mL plasma vs. 0.36 ± 0.8 μg antibody Eq/mL plasma), and was strongly associated with angiographic evidence of significant CAD. Conclusions The results of this cross sectional study suggest that post-translational modification of proteins via nitration within atherosclerotic plaque-laden arteries and in circulation serve as neoepitopes for elaboration of immunoglobulins, thereby providing an association between oxidant production and the activation of the immune system in CAD. PMID:23081989

  8. The 2010 patent landscape for spleen tyrosine kinase inhibitors.

    Science.gov (United States)

    Moretto, Alessandro F; Dehnhardt, Christoph; Kaila, Neelu; Papaioannou, Nikolaos; Thorarensen, Atli

    2012-05-01

    Discovery of small molecular inhibitors for treatment of rheumatoid arthritis is a major ongoing effort within the pharmaceutical industry. Spleen tyrosine kinase (SYK) is one of leading small molecular targets with regard to clinical development primarlly due to efforts by Rigel and Portola. In this review, we provide a comprehensive overview of the SYK patent landscape. The patent literature we evaluated relates to any organization that has filed applications that imply that SYK is the intended target. The interest in SYK was initiated in the early 2000's with many organizations, including several large pharmaceutical companies, and has been active for years. In general, the structural theme of most of the compounds in these applications is a traditional ATP competitive inhibitor with each organization having a different hinge binding element. In general, the attachment to the hinge is an aryl amine that is decorated with a solubilizing group. The other substituents are broadly variable across the numerous companies indicating that SYK has significant flexibility in its interactions in that portion of the kinase. This overview of the SYK patent literature and the learnings of the inhibitors' substitution patterns would be an important reference for anyone working in this area.

  9. Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate

    Directory of Open Access Journals (Sweden)

    Miguel Angel Moreno

    2014-12-01

    Full Text Available Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs.

  10. Tyrosine kinase expression in pulmonary metastases and paired primary tumors.

    Science.gov (United States)

    Muehling, Bernd M; Toelkes, Sara; Schelzig, Hubert; Barth, Thomas F E; Sunder-Plassmann, Ludger

    2010-02-01

    Tyrosine kinase inhibitors against the receptors of vascular endothelial growth factor (VEGFR), epidermal growth factor (EGFR) and the platelet derived growth factor (PDGFR) are increasingly used in the treatment of progressive cancers. However, the expression of these receptors especially in lung metastases has not been examined. Tissue specimen from 35 lung metastases of 33 patients with renal cell carcinoma (n=8), sarcoma (n=10), colorectal carcinoma (n=6), otolaryngologic carcinoma (OLC, n=4), testicular and endometrial cancer (n=1 each), malignant melanoma (n=1), adrenal cancer (n=2), malignant fibrous histiocytoma and malignant peripheral nerve sheath tumor (n=1 each) have been immunohistochemically tested for the expression of PDGFR alpha/beta, VEGFR and EGFR. None of the patients had been pretreated with angiogenic inhibitors prior to metastasectomy. PDGFRalpha was expressed in all metastases; 31% stained negative for PDGFRbeta, 86% negative for VEGFR and 45% negative for EGFR. Primary tumors revealed positive staining for PDGFRalpha in 88%, for PDGFRbeta in 59%, for VEGFR in 0% and for EGFR in 18%. Our investigation of a pilot character represents a 'biomarker-based' analysis of pulmonary metastases of different primary tumors; we conclude that an immediate 'tumor profiling' at initial diagnosis should be considered in order to guide tumor therapy individually.

  11. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    Science.gov (United States)

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  12. Metazoan-like signaling in a unicellular receptor tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Schultheiss Kira P

    2013-02-01

    Full Text Available Abstract Background Receptor tyrosine kinases (RTKs are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis. Results We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2 in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution. Conclusions Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

  13. Identification of a variant form of tyrosine phosphatase LYP

    Directory of Open Access Journals (Sweden)

    Ho Wanting T

    2010-11-01

    Full Text Available Abstract Background Protein tyrosine phosphatases (PTPs are important cell signaling regulators with major pathological implications. LYP (also known as PTPN22 is an intracellular enzyme initially found to be predominately expressed in lymphocytes. Importantly, an allelic R620W variant of LYP is strongly associated with multiple autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and autoimmune thyroid disease. Results In this study, we isolated a novel isoform of LYP designated LYP3. LYP3 differs from LYP1, the known isoform of LYP, in that it lacks a 28 amino acid segment right after the R620W site embedded in a proline-rich protein-protein interaction motif. Genomic sequence analysis revealed that LYP3 resulted from alternative splicing of the LYP gene located on chromosome 1p 13.3-13.1. Reverse transcription PCR analyses of 48 human tissues demonstrated that both LYP1 and LYP3 are predominantly expressed in primary and secondary lymphoid tissues but the relative expression levels of the two isoforms varies in different human tissues and individuals. Conclusions We thus identified a new variant form of LYP and conducted a comprehensive analysis of LYP tissue expressions. Considering the pathogenesis of LYP R620W, we believe that the expression of LYP3 may have an important role in regulating activity and function of LYP and may be implicated in autoimmune diseases.

  14. The protein tyrosine kinase Tec regulates mast cell function.

    Science.gov (United States)

    Schmidt, Uwe; Abramova, Anastasia; Boucheron, Nicole; Eckelhart, Eva; Schebesta, Alexandra; Bilic, Ivan; Kneidinger, Michael; Unger, Bernd; Hammer, Martina; Sibilia, Maria; Valent, Peter; Ellmeier, Wilfried

    2009-11-01

    Mast cells play crucial roles in a variety of normal and pathophysiological processes and their activation has to be tightly controlled. Here, we demonstrate that the protein tyrosine kinase Tec is a crucial regulator of murine mast cell function. Tec was activated upon Fc epsilon RI stimulation of BM-derived mast cells (BMMC). The release of histamine in the absence of Tec was normal in vitro and in vivo; however, leukotriene C(4) levels were reduced in Tec(-) (/) (-) BMMC. Furthermore, the production of IL-4 was severely impaired, and GM-CSF, TNF-alpha and IL-13 levels were also diminished. Finally, a comparison of WT, Tec(-) (/) (-), Btk(-) (/) (-) and Tec(-) (/) (-)Btk(-) (/) (-) BMMC revealed a negative role for Btk in the regulation of IL-4 production, while for the efficient production of TNF-alpha, IL-13 and GM-CSF, both Tec and Btk were required. Our results demonstrate a crucial role for Tec in mast cells, which is partially different to the function of the well-characterized family member Btk.

  15. Raman spectra of iodine-derivatives of tyrosine and thyronine

    International Nuclear Information System (INIS)

    Loh, E.

    1974-01-01

    The Raman spectra of the iodine derivatives of tyrosine and thyronine in the form of compressed crystalline powders have been excited by 4880 A Argon laser on rotating samples at room temperature. The strong peaks in the low-frequency, -1 , region may be described by analogous vibrations of benzene as: I. the C-I out-of-plane bendings of E 1 sub(g) mode from 100 cm -1 to 180 cm -1 ; II. the C-I in-plane bendings of E 2 sub(g) and A 2 sub(g) mode from 190 cm -1 to 330 cm -1 and III. the C-I stretchings of E 2 sub(g) mode from 330 cm -1 to 400 cm -1 . In 3,3',5-triiodo-derivatives, the number of both the C-I in-plane bendings and C-I stretchings on the inner phenyl ring approximately doubles from thet of diiodo-derivatives. This doubling in number of peaks is presumably due to the modulation caused by the libration, which is associated with the C-I out-of-plane bending at position 3', of the outer phenyl ring

  16. Residual Stress Analysis Based on Acoustic and Optical Methods

    Directory of Open Access Journals (Sweden)

    Sanichiro Yoshida

    2016-02-01

    Full Text Available Co-application of acoustoelasticity and optical interferometry to residual stress analysis is discussed. The underlying idea is to combine the advantages of both methods. Acoustoelasticity is capable of evaluating a residual stress absolutely but it is a single point measurement. Optical interferometry is able to measure deformation yielding two-dimensional, full-field data, but it is not suitable for absolute evaluation of residual stresses. By theoretically relating the deformation data to residual stresses, and calibrating it with absolute residual stress evaluated at a reference point, it is possible to measure residual stresses quantitatively, nondestructively and two-dimensionally. The feasibility of the idea has been tested with a butt-jointed dissimilar plate specimen. A steel plate 18.5 mm wide, 50 mm long and 3.37 mm thick is braze-jointed to a cemented carbide plate of the same dimension along the 18.5 mm-side. Acoustoelasticity evaluates the elastic modulus at reference points via acoustic velocity measurement. A tensile load is applied to the specimen at a constant pulling rate in a stress range substantially lower than the yield stress. Optical interferometry measures the resulting acceleration field. Based on the theory of harmonic oscillation, the acceleration field is correlated to compressive and tensile residual stresses qualitatively. The acoustic and optical results show reasonable agreement in the compressive and tensile residual stresses, indicating the feasibility of the idea.

  17. l-Tyrosine Contained in Dietary Supplement by Chemiluminescence Reaction of an Iron-Phthalocyanine Complex

    Directory of Open Access Journals (Sweden)

    Takao Ohtomo

    2012-01-01

    Full Text Available The chemiluminescence (CL signal immediately appeared when a hydrogen peroxide solution was injected into an iron-phthalocyanine tetrasulfonic acid (Fe-PTS aqueous solution. Moreover, the CL intensity of Fe-PTS decreased by adding L-tyrosine. Based on these results, the determination of trace amounts of L-tyrosine was developed using the quenching-chemiluminescence. The calibration curve of L-tyrosine was obtained in the concentration range of 2.0×10−7 M to 2.0×10−5 M. Moreover, the relative standard deviation (RSD was 1.63 % (=5 for 2.0×10−6 M L-tyrosine, and its detection limits (3σ were 1.81×10−7 M. The spike and recovery experiments for L-tyrosine were performed using a soft drink. Furthermore, the determination of L-tyrosine was applied to supplements containing various kinds of amino acids. Each satisfactory relative recovery was obtained at 98 to 102%.

  18. Validation of o-tyrosine as a marker for detection and dosimetry of irradiated chicken meat

    International Nuclear Information System (INIS)

    Chuaqui-Offermanns, N.; McDougall, T.E.; Guerrero, A.M.

    1993-01-01

    The o-tyrosine has been proposed as a marker for postirradiation identification of food that contains protein. In this study, the validity of using o-tyrosine for this purpose has been tested and established. The validation process involved examination of background levels of o-tyrosine in unirradiated chicken, radiation dose yield, postirradiation storage, dose rate, radiation type, temperature during irradiation, and oxygen concentration during irradiation. The o-tyrosine is present in unirradiated chicken meat at variable levels. However, these background levels are low enough that o-tyrosine can serve to determine whether chicken has been irradiated or not at the commercially approved doses (3 kGy). The radiation dose response curve for the formation of o-tyrosine is linear. The apparent yields may vary with the analytical method used; however, it is independent of the dose rate, radiation type, atmosphere, and temperature (above freezing) during irradiation. It is also independent of the storage time and temperature after irradiation. It is concluded that this marker can be used to determine the absorbed dose in chicken meat irradiated with either gamma rays or electrons under normal or modified atmosphere

  19. Verbascoside promotes the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra

    Directory of Open Access Journals (Sweden)

    Jian-qing Liang

    2016-01-01

    Full Text Available Tyrosine hydroxylase is a key enzyme in dopamine biosynthesis. Change in tyrosine hydroxylase expression in the nigrostriatal system is closely related to the occurrence and development of Parkinson′s disease. Verbascoside, an extract from Radix Rehmanniae Praeparata has been shown to be clinically effective in treating Parkinson′s disease. However, the underlying mechanisms remain unclear. It is hypothesized that the effects of verbascoside on Parkinson′s disease are related to tyrosine hydroxylase expression change in the nigrostriatal system. Rat models of Parkinson′s disease were established and verbascoside (60 mg/kg was administered intraperitoneally once a day. After 6 weeks of verbascoside treatment, rat rotational behavior was alleviated; tyrosine hydroxylase mRNA and protein expression and the number of tyrosine hydroxylase-immunoreactive neurons in the rat right substantia nigra were significantly higher than the Parkinson′s model group. These findings suggest that the mechanism by which verbascoside treats Parkinson′s disease is related to the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra.

  20. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Vanesa Olivares-Illana

    2008-06-01

    Full Text Available Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

  1. Marine Tar Residues: a Review

    OpenAIRE

    Warnock, April M.; Hagen, Scott C.; Passeri, Davina L.

    2015-01-01

    Marine tar residues originate from natural and anthropogenic oil releases into the ocean environment and are formed after liquid petroleum is transformed by weathering, sedimentation, and other processes. Tar balls, tar mats, and tar patties are common examples of marine tar residues and can range in size from millimeters in diameter (tar balls) to several meters in length and width (tar mats). These residues can remain in the ocean environment indefinitely, decomposing or becoming buried in ...

  2. Evaluation of residue-residue contact predictions in CASP9

    KAUST Repository

    Monastyrskyy, Bohdan

    2011-01-01

    This work presents the results of the assessment of the intramolecular residue-residue contact predictions submitted to CASP9. The methodology for the assessment does not differ from that used in previous CASPs, with two basic evaluation measures being the precision in recognizing contacts and the difference between the distribution of distances in the subset of predicted contact pairs versus all pairs of residues in the structure. The emphasis is placed on the prediction of long-range contacts (i.e., contacts between residues separated by at least 24 residues along sequence) in target proteins that cannot be easily modeled by homology. Although there is considerable activity in the field, the current analysis reports no discernable progress since CASP8.

  3. Inhaled tyrosine kinase inhibitors for pulmonary hypertension: a possible future treatment

    Directory of Open Access Journals (Sweden)

    Pitsiou G

    2014-10-01

    Full Text Available Georgia Pitsiou,1 Paul Zarogoulidis,1 Dimitris Petridis,2 Ioannis Kioumis,1 Sofia Lampaki,1 John Organtzis,1 Konstantinos Porpodis,1 Antonis Papaiwannou,1 Theodora Tsiouda,3 Wolfgang Hohenforst-Schmidt,4 Stylianos Kakolyris,5 Konstantinos Syrigos,6 Haidong Huang,7 Qiang Li,7 J Francis Turner,8 Konstantinos Zarogoulidis1 1Pulmonary Department, G Papanikolaou General Hospital, Aristotle University of Thessaloniki, 2Department of Food Technology, School of Food Technology and Nutrition, Alexander Technological Educational Institute, 3Internal Medicine Department, Thegenio Anticancer Hospital, Thessaloniki, Greece; 4II Medical Department, Coburg Regional Hospital, Coburg, Germany; 5Oncology Department, Sotiria Hospital of Chest Diseases, University of Athens, Athens, 6Oncology Department, University General Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis, Greece; 7Department of Respiratory Diseases, Changhai Hospital/First Affiliated Hospital of the Second Military Medical University, Shanghai, People’s Republic of China; 8Division of Interventional Pulmonology and Medical Oncology, Cancer Treatment Centers of America, Western Regional Medical Center, Goodyear, AZ, USA Abstract: Pulmonary hypertension is a disease with severe consequences for the human body. There are several diseases and situations that induce pulmonary hypertension and are usually underdiagnosed. Treatments include conventional medical therapies and oral, inhaled, intravenous, and subcutaneous options. Depending on its severity, heart or lung transplant may also be an option. A possible novel treatment could be tyrosine kinase inhibitors. We conducted experiments with three jet nebulizers and three ultrasound nebulizers with erlotinib, gefitinib, and imatinib. Different residual cup designs and residual cup loadings were used in order to identify the best combination to produce droplets of less than 5 µm in mass median aerodynamic diameter. We

  4. Sulfonation of Tyrosine as a Method To Improve Biodistribution of Peptide-Based Radiotracers: Novel18F-Labeled Cyclic RGD Analogues.

    Science.gov (United States)

    Haskali, Mohammad B; Denoyer, Delphine; Noonan, Wayne; Culinane, Carleen; Rangger, Christine; Pouliot, Normand; Haubner, Roland; Roselt, Peter D; Hicks, Rodney J; Hutton, Craig A

    2017-04-03

    Control of the biodistribution of radiolabeled peptides has proven to be a major challenge in their application as imaging agents for positron emission tomography (PET). Modification of peptide hydrophilicity in order to increase renal clearance has been a common endeavor to improve overall biodistribution. Herein, we examine the effect of site-specific sulfonation of tyrosine moieties in cyclic(RGDyK) peptides as a means to enhance their hydrophilicity and improve their biodistribution. The novel sulfonated cyclic(RGDyK) peptides were conjugated directly to 4-nitrophenyl 2-[ 18 F]fluoropropionate, and the biodistribution of the radiolabeled peptides was compared with that of their nonsulfonated, clinically relevant counterparts, [ 18 F]GalactoRGD and [ 18 F]FPPRGD2. Site-specific sulfonation of the tyrosine residues was shown to increase hydrophilicity and improve biodistribution of the RGD peptides, despite contributing just 79 Da toward the MW, compared with 189 Da for both the "Galacto" and mini-PEG moieties, suggesting this may be a broadly applicable approach to enhancing biodistribution of radiolabeled peptides.

  5. Mer Tyrosine Kinase Regulates Disseminated Prostate Cancer Cellular Dormancy.

    Science.gov (United States)

    Cackowski, Frank C; Eber, Matthew R; Rhee, James; Decker, Ann M; Yumoto, Kenji; Berry, Janice E; Lee, Eunsohl; Shiozawa, Yusuke; Jung, Younghun; Aguirre-Ghiso, Julio A; Taichman, Russell S

    2017-04-01

    Many prostate cancer (PCa) recurrences are thought to be due to reactivation of disseminated tumor cells (DTCs). We previously found a role of the TAM family of receptor tyrosine kinases TYRO3, AXL, and MERTK in PCa dormancy regulation. However, the mechanism and contributions of the individual TAM receptors is largely unknown. Knockdown of MERTK, but not AXL or TYRO3 by shRNA in PCa cells induced a decreased ratio of P-Erk1/2 to P-p38, increased expression of p27, NR2F1, SOX2, and NANOG, induced higher levels of histone H3K9me3 and H3K27me3, and induced a G1/G0 arrest, all of which are associated with dormancy. Similar effects were also observed with siRNA. Most importantly, knockdown of MERTK in PCa cells increased metastasis free survival in an intra-cardiac injection mouse xenograft model. MERTK knockdown also failed to inhibit PCa growth in vitro and subcutaneous growth in vivo, which suggests that MERTK has specificity for dormancy regulation or requires a signal from the PCa microenvironment. The effects of MERTK on the cell cycle and histone methylation were reversed by p38 inhibitor SB203580, which indicates the importance of MAP kinases for MERTK dormancy regulation. Overall, this study shows that MERTK stimulates PCa dormancy escape through a MAP kinase dependent mechanism, also involving p27, pluripotency transcription factors, and histone methylation. J. Cell. Biochem. 118: 891-902, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. Mefloquine neurotoxicity is mediated by non-receptor tyrosine kinase.

    Science.gov (United States)

    Milatovic, Dejan; Jenkins, Jerry W; Hood, Jonathan E; Yu, Yingchun; Rongzhu, Lu; Aschner, Michael

    2011-10-01

    Among several available antimalarial drugs, mefloquine has proven to be effective against drug-resistant Plasmodium falciparum and remains the drug of choice for both therapy and chemoprophylaxis. However, mefloquine is known to cause adverse neurological and/or psychiatric symptoms, which offset its therapeutic advantage. The exact mechanisms leading to the adverse neurological effects of mefloquine are poorly defined. Alterations in neurotransmitter release and calcium homeostasis, the inhibition of cholinesterases and the interaction with adenosine A(2A) receptors have been hypothesized to play prominent roles in mediating the deleterious effects of this drug. Our recent data have established that mefloquine can also trigger oxidative damage and subsequent neurodegeneration in rat cortical primary neurons. Furthermore, we have utilized a system biology-centered approach and have constructed a pathway model of cellular responses to mefloquine, identifying non-receptor tyrosine kinase 2 (Pyk2) as a critical target in mediating mefloquine neurotoxicity. In this study, we sought to establish an experimental validation of Pyk2 using gene-silencing techniques (siRNA). We have examined whether the downregulation of Pyk2 in primary rat cortical neurons alters mefloquine neurotoxicity by evaluating cell viability, apoptosis and oxidative stress. Results from our study have confirmed that mefloquine neurotoxicity is associated with apoptotic response and oxidative injury, and we have demonstrated that mefloquine affects primary rat cortical neurons, at least in part, via Pyk2. The implication of these findings may prove beneficial in suppressing the neurological side effects of mefloquine and developing effective therapeutic modalities to offset its adverse effects. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Tyrosine Hydroxylase Expression in Type II Cochlear Afferents in Mice.

    Science.gov (United States)

    Vyas, Pankhuri; Wu, Jingjing Sherry; Zimmerman, Amanda; Fuchs, Paul; Glowatzki, Elisabeth

    2017-02-01

    Acoustic information propagates from the ear to the brain via spiral ganglion neurons that innervate hair cells in the cochlea. These afferents include unmyelinated type II fibers that constitute 5 % of the total, the majority being myelinated type I neurons. Lack of specific genetic markers of type II afferents in the cochlea has been a roadblock in studying their functional role. Unexpectedly, type II afferents were visualized by reporter proteins induced by tyrosine hydroxylase (TH)-driven Cre recombinase. The present study was designed to determine whether TH-driven Cre recombinase (TH-2A-CreER) provides a selective and reliable tool for identification and genetic manipulation of type II rather than type I cochlear afferents. The "TH-2A-CreER neurons" radiated from the spiral lamina, crossed the tunnel of Corti, turned towards the base of the cochlea, and traveled beneath the rows of outer hair cells. Neither the processes nor the somata of TH-2A-CreER neurons were labeled by antibodies that specifically labeled type I afferents and medial efferents. TH-2A-CreER-positive processes partially co-labeled with antibodies to peripherin, a known marker of type II afferents. Individual TH-2A-CreER neurons gave off short branches contacting 7-25 outer hair cells (OHCs). Only a fraction of TH-2A-CreER boutons were associated with CtBP2-immunopositive ribbons. These results show that TH-2A-CreER provides a selective marker for type II versus type I afferents and can be used to describe the morphology and arborization pattern of type II cochlear afferents in the mouse cochlea.

  8. State-by-state investigation of destructive interference in resonance Raman spectra of neutral tyrosine and the tyrosinate anion with the simplified sum-over-states approach.

    Science.gov (United States)

    Cabalo, Jerry B; Saikin, Semion K; Emmons, Erik D; Rappoport, Dmitrij; Aspuru-Guzik, Alán

    2014-10-16

    UV resonance Raman scattering is uniquely sensitive to the molecular electronic structure as well as intermolecular interactions. To better understand the relationship between electronic structure and resonance Raman cross section, we carried out combined experimental and theoretical studies of neutral tyrosine and the tyrosinate anion. We studied the Raman cross sections of four vibrational modes as a function of excitation wavelength, and we analyzed them in terms of the contributions of the individual electronic states as well as of the Albrecht A and B terms. Our model, which is based on time-dependent density functional theory (TDDFT), reproduced the experimental resonance Raman spectra and Raman excitation profiles for both studied molecules with good agreement. We found that for the studied modes, the contributions of Albrecht's B terms in the Raman cross sections were important across the frequency range spanning the L(a,b) and B(a,b) electronic excitations in tyrosine and the tyrosinate anion. Furthermore, we demonstrated that interference with high-energy states had a significant impact and could not be neglected even when in resonance with a lower-energy state. The symmetry of the vibrational modes served as an indicator of the dominance of the A or B mechanisms. Excitation profiles calculated with a damping constant estimated from line widths of the electronic absorption bands had the best consistency with experimental results.

  9. Human glioblastoma ADF cells express tyrosinase, L-tyrosine hydroxylase and melanosomes and are sensitive to L-tyrosine and phenylthiourea.

    Science.gov (United States)

    Bonfigli, Antonella; Zarivi, Osvaldo; Colafarina, Sabrina; Cimini, Anna Maria; Ragnelli, Anna Maria; Aimola, Pierpaolo; Natali, Pier Giorgio; Cerù, Maria Paola; Amicarelli, Fernanda; Miranda, Michele

    2006-06-01

    Melanocytes and neuroblasts share the property of transforming L-tyrosine through two distinct metabolic pathways leading to melanogenesis and catecholamine synthesis, respectively. While tyrosinase (TYR) activity has been shown to be expressed by neuroblastoma it remains to be established as to whether also glioblastomas cells are endowed with this property. We have addressed this issue using the human continuous glioblastoma cell line ADF. We demonstrated that these cells possess tyrosinase as well as L-tyrosine hydroxylase (TH) activity and synthesize melanosomes. Because the two pathways are potentially cyto-genotoxic due to production of quinones, semiquinones, and reactive oxygen species (ROS), we have also investigated the expression of the peroxisomal proliferators activated receptor alpha (PPARalpha) and nuclear factor-kB (NFkB) transcription factor as well the effect of L-tyrosine concentration on cell survival. We report that L-tyrosine down-regulates PPARalpha expression in ADF cells but not neuroblastoma and that this aminoacid and phenylthiourea (PTU) induces apoptosis in glioblastoma and neuroblastoma. Copyright 2006 Wiley-Liss, Inc.

  10. Myocardial blood flow rate and capillary permeability for 99mTc-DTPA in patients with angiographically normal coronary arteries. Evaluation of the single-injection, residue detection method with intracoronary indicator bolus injection and the use of a mobile gamma camera

    DEFF Research Database (Denmark)

    Svendsen, Jesper Hastrup; Kelbaek, H; Efsen, F

    1994-01-01

    The aims of the present study were to quantitate myocardial perfusion and capillary permeability in the human heart by means of the single-injection, residue detection method using a mobile gamma camera. With this method, the intravascular mean transit time and the capillary extraction fraction (E...... capillary permeability area product (PS) were determined. Spontaneous 15 min variations (mean of two values) for the LAD and the CX territory, respectively, gave a mean fpl(LAD) of 74.2 (measurement 1) and 77.8 ml.(100 g.min)-1 (measurement 2) (SD of the difference 7.5 ml.(100 g.min)-1) and fpl(CX) of 82.......7 and 92.1 ml. (100 g.min)-1, respectively (SD of the difference 16.1 ml.(100 g.min)-1), and E(LAD) was 51.0 and 55.7%, respectively (SD of the difference 8.4%), and E(CX) was 52.9 and 50.4%, respectively (SD of the difference 5.9%). During atrial pacing, the rate-pressure product increased by 97...

  11. Landfilling of waste incineration residues

    DEFF Research Database (Denmark)

    Christensen, Thomas Højlund; Astrup, Thomas; Cai, Zuansi

    2002-01-01

    Residues from waste incineration are bottom ashes and air-pollution-control (APC) residues including fly ashes. The leaching of heavy metals and salts from the ashes is substantial and a wide spectrum of leaching tests and corresponding criteria have been introduced to regulate the landfilling...

  12. Spectrofluorometric and Molecular Modeling Studies on Binding of Nitrite Ion with Bovine Hemoglobin: Effect of Nitrite Ion on Amino Acid Residues

    Science.gov (United States)

    Madrakian, T.; Bagheri, H.; Afkhami, A.

    2015-05-01

    The interaction between nitrite ion and bovine hemoglobin was investigated by a spectrofluorometric technique. The experimental results indicated that the interaction causes a static quenching of the fluorescence of bovine hemoglobin, that the binding reaction is spontaneous, and that H-bonding interactions play a major role in binding of this ion to bovine hemoglobin. The formation constant for this interaction was calculated. Based on Förster's theory of nonradiative energy transfer, the binding distance between this ion and bovine hemoglobin was determined. Furthermore, the interaction of nitrite ion with tyrosine and tryptophan was investigated with synchronous fluorescence. There was no significant shift of the maximum emission wavelength with interactions of the mentioned ion with bovine hemoglobin, which implies that interaction of nitrite ion with bovine hemoglobin does not affect the microenvironment around the tryptophan and tyrosine residues. Furthermore, the effect of nitrite ion on amino acid residues of bovine hemoglobin was studied by a molecular docking technique.

  13. Are pesticide residues in honey related to oilseed rape treatments?

    Science.gov (United States)

    Karise, Reet; Raimets, Risto; Bartkevics, Vadims; Pugajeva, Iveta; Pihlik, Priit; Keres, Indrek; Williams, Ingrid H; Viinalass, Haldja; Mänd, Marika

    2017-12-01

    Pesticide treatments before and during the flowering of honey bee forage crops may lead to residues in honey. In northern regions oilseed rape belongs to the main forage crops that is mostly cultivated by means of intensive agriculture, including several pesticide treatments. However, in addition to the focal forage crops, pesticides from non-forage crops can spread to wild flowers around fields, and thus the residues in honey would reflect the whole range of pesticides used in the agricultural landscape. The aim of our study was to clarify which currently used pesticides are present in honey gathered from heterogeneous agricultural landscapes after the end of flowering of oilseed crops. Honey samples (N = 33) were collected from beehives of Estonia during 2013 and 2014, and analysed for residues of 47 currently used agricultural pesticides using the multiresidue method with HPLC-MS/MS and GC-MS and a single residue method for glyphosate, aminopyralid and clopyralid. Residues of eight different active ingredients with representatives from all three basic pesticide classes were determined. Although no correlation was detected between the cumulative amount of pesticide residues and percent of oilseed crops in the foraging territory, most of the residues are those allowed for oilseed rape treatments. Among all pesticides, herbicide residues prevailed in 2013 but not in 2014. Despite the relatively small agricultural impact of Estonia, the detected levels of pesticide residues sometimes exceeded maximum residue level; however, these concentrations do not pose a health risk to consumers, also acute toxicity to honey bees would be very unlikely. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Statistical inference on residual life

    CERN Document Server

    Jeong, Jong-Hyeon

    2014-01-01

    This is a monograph on the concept of residual life, which is an alternative summary measure of time-to-event data, or survival data. The mean residual life has been used for many years under the name of life expectancy, so it is a natural concept for summarizing survival or reliability data. It is also more interpretable than the popular hazard function, especially for communications between patients and physicians regarding the efficacy of a new drug in the medical field. This book reviews existing statistical methods to infer the residual life distribution. The review and comparison includes existing inference methods for mean and median, or quantile, residual life analysis through medical data examples. The concept of the residual life is also extended to competing risks analysis. The targeted audience includes biostatisticians, graduate students, and PhD (bio)statisticians. Knowledge in survival analysis at an introductory graduate level is advisable prior to reading this book.

  15. Automatic prediction of catalytic residues by modeling residue structural neighborhood

    Directory of Open Access Journals (Sweden)

    Passerini Andrea

    2010-03-01

    Full Text Available Abstract Background Prediction of catalytic residues is a major step in characterizing the function of enzymes. In its simpler formulation, the problem can be cast into a binary classification task at the residue level, by predicting whether the residue is directly involved in the catalytic process. The task is quite hard also when structural information is available, due to the rather wide range of roles a functional residue can play and to the large imbalance between the number of catalytic and non-catalytic residues. Results We developed an effective representation of structural information by modeling spherical regions around candidate residues, and extracting statistics on the properties of their content such as physico-chemical properties, atomic density, flexibility, presence of water molecules. We trained an SVM classifier combining our features with sequence-based information and previously developed 3D features, and compared its performance with the most recent state-of-the-art approaches on different benchmark datasets. We further analyzed the discriminant power of the information provided by the presence of heterogens in the residue neighborhood. Conclusions Our structure-based method achieves consistent improvements on all tested datasets over both sequence-based and structure-based state-of-the-art approaches. Structural neighborhood information is shown to be responsible for such results, and predicting the presence of nearby heterogens seems to be a promising direction for further improvements.

  16. The usefulness of dynamic O-(2-18F-fluoroethyl)-L-tyrosine PET in the clinical evaluation of brain tumors in children and adolescents

    DEFF Research Database (Denmark)

    Dunkl, Veronika; Cleff, Corvin; Stoffels, Gabriele

    2015-01-01

    UNLABELLED: Experience regarding O-(2-(18)F-fluoroethyl)-L-tyrosine ((18)F-FET) PET in children and adolescents with brain tumors is limited. METHODS: Sixty-nine (18)F-FET PET scans of 48 children and adolescents (median age, 13 y; range, 1-18 y) were analyzed retrospectively. Twenty-six scans...... to assess newly diagnosed cerebral lesions, 24 scans for diagnosing tumor progression or recurrence, 8 scans for monitoring of chemotherapy effects, and 11 scans for the detection of residual tumor after resection were obtained. Maximum and mean tumor-to-brain ratios (TBRs) were determined at 20-40 min...... decision making in pediatric brain tumor patients....

  17. Crystal Structure of the Frizzled-Like Cysteine-Rich Domain of the Receptor Tyrosine Kinase MuSK

    Energy Technology Data Exchange (ETDEWEB)

    Stiegler, A.; Burden, S; Hubbard, S

    2009-01-01

    Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 {angstrom} resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ.

  18. A Case of Squamous Cell Carcinoma of Unknown Primary that Responded to the Multi-Tyrosine Kinase Inhibitor Lenvatinib

    Directory of Open Access Journals (Sweden)

    Reiko Kimura-Tsuchiya

    2018-02-01

    Full Text Available Lenvatinib is an oral tyrosine kinase inhibitor of vascular endothelial growth factor receptors 1, 2, and 3, fibroblast growth factor receptors 1 through 4, as well as platelet-derived growth factor receptor α, RET, and KIT. At present, lenvatinib is used in the treatment of thyroid cancer and renal cell carcinoma. We herein report a case of a 67-year-old patient with squamous cell carcinoma of unknown primary who was effectively treated with lenvatinib. The patient was initially diagnosed as having undifferentiated thyroid cancer, and after total thyroidectomy and bilateral lymph node dissection, lenvatinib was administered for the treatment of residual lymph node metastasis. A computed tomography scan after 1 month of lenvatinib administration showed marked regression of the lymph nodes, but interstitial pneumonia was also detected. Because the drug lymphocyte stimulation test for lenvatinib was strongly positive, we concluded that the interstitial pneumonia was induced by lenvatinib. The interstitial pneumonia only improved by the withdrawal of lenvatinib. Finally, his thyroid tumor was diagnosed as a metastasis of squamous cell carcinoma; however, we were unable to identify the primary lesion. This is the first reported case of interstitial pneumonia induced by lenvatinib.

  19. Consequences of two naturally occurring missense mutations in the structure and function of Bruton agammaglobulinemia tyrosine kinase.

    Science.gov (United States)

    Vargas-Hernández, Alexander; López-Herrera, Gabriela; Maravillas-Montero, José L; Vences-Catalán, Felipe; Mogica-Martínez, Dolores; Rojo-Domínguez, Arturo; Espinosa-Rosales, Francisco J; Santos-Argumedo, Leopoldo

    2012-04-01

    Bruton agammaglobulinemia tyrosine kinase (BTK) is a key protein in the B-cell receptor (BCR) signaling pathway and plays an essential role in the differentiation of B lymphocytes. X-linked agammaglobulinemia (XLA) is a primary humoral immunodeficiency caused by mutations in the gene encoding BTK. Previously, we identified two novel variations, L111P and E605G, in BTK; these are localized within the pleckstrin homology and Src homology 1 domains, respectively. In the present study, we evaluated the potential effects of these variations on the structural conformation and the function of BTK. Using in silico methods, we found that the L111P and E650G variations are not located directly in protein-protein interfaces but close to them. They distorted the native structural conformation of the BTK protein, affecting not only its geometry and stability but also its ability for protein recognition and in consequence its functionality. To confirm the results of the in silico assays, WT BTK, L111P, and E650G variants were expressed in the BTK-deficient DT40 cell line. The mutant proteins exhibited an absence of catalytic activity, aberrant redistribution after BCR-crosslinking, and deficient intracellular calcium mobilization. This work demonstrates that L111 and E605 residues are fundamental for the activation and function of BTK. Copyright © 2012 Wiley Periodicals, Inc.

  20. Cutaneous metabolic pathway of tyrosine as a precursor to melanin in Asinara’s white donkey, Equus asinus L., 1758

    Directory of Open Access Journals (Sweden)

    Maria Grazia Cappai

    2015-10-01

    Full Text Available L-tyrosine (L-Tyr is a conditionally indispensable amino acid for Equids. The biologic role of L-Tyr is ubiquitous, being involved in the production of hair and skin pigments. The white donkeys of Asinara island display a peculiar white coat, with life-long hypopigmentation of skin, hairs and eyes. Our results underline how the white phenotype is due to a disorder of melanin biosynthesis, detected in skin biopsies and on hair bulbs through immunohistochemical and enzymatic assays. The metabolic pathway of L-Tyr is compatible with the clinical frame of ocular-cutaneous albinism (OCA. The enzymatic activity in hair bulbs for the conversion of L-Tyr to L-3,4-dihydroxyphenilalanine (L-DOPA tested negative, but positive for L-DOPA conversion into eumelanin in the 100% of the Asinara’s white donkey. This is the case of a negative-tyrosinase albinism, which contemporarily excludes dietary deficiencies of phenylalanine (Phe or L-Tyr and potential incapability of Tyr intestinal uptake or Tyr conversion from Phe. The tyrosinase (TYR residual activity positively and significantly (P=0.012 correlates (P=0.897 with the albino phenotype of the Asinara’s white donkey. These results definitely point to the fact that Asinara’s white donkey population owes its phenotype to TYR residual activity, thus on genetic basis. From a comparative point of view, the albino form of the Asinara’s white donkeys is similar to the human OCA type 1, from which however differs for positively testing to L-DOPA conversion, upon incubation of hair bulbs in L-DOPA solutions.

  1. The role of tyrosine hydroxylase gene variants in suicide attempt in schizophrenia.

    Science.gov (United States)

    Hu, Jiayi; Chan, Lai Fong; Souza, Renan P; Tampakeras, Maria; Kennedy, James L; Zai, Clement; De Luca, Vincenzo

    2014-01-24

    Evidence has shown that attempted suicide in psychiatric disorders is a complex interplay of genes and environment. Noradrenergic dysfunction due to abnormalities in the tyrosine hydroxylase (TH) gene has been implicated in the pathogenesis of suicidal behavior in mood disorders. However, suicide is a leading cause of mortality in schizophrenia too. Recent evidence suggests that TH gene variants may also increase the risk of suicide attempts in schizophrenia patients, although the interaction with established clinical risk factors is unclear. This study aimed to identify TH gene variants conferring risk for suicide attempt in schizophrenia while accounting for the interaction between this gene and clinical risk factors. We performed analysis on four TH SNPs (rs11564717, rs11042950, rs2070762, rs689) and the common TCAT repeat (UniSTS:240639) for 234 schizophrenia patients (51 suicide attempters and 183 non-attempters). Clinical risk factors and ethnic stratification were included as covariates. Single marker analysis identified the SNP rs11564717 (p=0.042) and the TCAT(6) (p=0.004) as risk variants for suicide attempt. We also identified the haplotype A-A-A-G as a risk factor for suicide attempt (p=0.0025). In conclusion, our findings suggest that TH polymorphisms may contribute to the risk of attempted suicide in schizophrenia even after accounting for established clinical risk factors and ethnic stratification. Further larger scale studies are needed to confirm these findings and to understand the mechanisms underlying the role of TH gene variants in suicide attempt in schizophrenia. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  2. Uptake of iodine-123-α-methyl tyrosine by gliomas and non-neoplastic brain lesions

    International Nuclear Information System (INIS)

    Kuwert, T.; Morgenroth, C.; Woesler, B.; Matheja, P.; Palkovic, S.; Vollet, B.; Samnick, S.; Maasjosthusmann, U.; Lerch, H.; Gildehaus, F.J.; Wassmann, H.; Schober, O.

    1996-01-01

    Using single-photon emission tomography (SPET), the radiopharmaceutical L-3-iodine-123-α-methyl tyrosine (IMT) has been applied to the imaging of amino acid transport into brain tumours. It was the aim of this study to investigate whether IMT SPET is capable of differentiating between high-grade gliomas, low-grade gliomas and non-neoplastic brain lesions. To this end, IMT uptake was determined in 53 patients using the triple-headed SPET camera MULTISPECT 3. Twenty-eight of these subjects suffered from high-grade gliomas (WHO grade III or IV), 12 from low-grade gliomas (WHO grade II), and 13 from non-neoplastic brain lesions, including lesions after effective therapy of a glioma (five cases), infarctions (four cases), inflammatory lesions (three cases), infarctions (four cases), inflammatory lesions (three cases) and traumatic haematoma (one case). IMT uptake was significantly higher in high-grade gliomas than in low-grade gliomas and non-neoplastic lesions. IMT uptake by low-grade gliomas was not significantly different from that by non-neoplastic lesions. Diagnostic sensitivity and specificity were 71% and 83% for differentiating high-grade from low-grade gliomas, 82% and 100% for distinguishing high-grade gliomas from non-neoplastic lesions, and 50% and 100% for discriminating low-grade gliomas from non-neoplastic lesions. Analogously to positron emission tomography with radioactively labelled amino acids and fluorine-18 deoxyglucose, IMT SPET may aid in differentiating higc-grade gliomas from histologically benign brain tumours and non-neoplastic brain lesions; it is of only limited value in differentiating between non-neoplastic lesions and histologically benign brain tumours. (orig.)

  3. Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain

    Directory of Open Access Journals (Sweden)

    Kang Sun-Young

    2012-12-01

    Full Text Available Abstract Background The phenylpropanoid metabolites are an extremely diverse group of natural products biosynthesized by plants, fungi, and bacteria. Although these compounds are widely used in human health care and nutrition services, their availability is limited by regional variations, and isolation of single compounds from plants is often difficult. Recent advances in synthetic biology and metabolic engineering have enabled artificial production of plant secondary metabolites in microorganisms. Results We develop an Escherichia coli system containing an artificial biosynthetic pathway that yields phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, from simple carbon sources. These artificial biosynthetic pathways contained a codon-optimized tal gene that improved the productivity of 4-coumaric acid and ferulic acid, but not caffeic acid in a minimal salt medium. These heterologous pathways extended in E. coli that had biosynthesis machinery overproducing tyrosine. Finally, the titers of 4-coumaric acid, caffeic acid, and ferulic acid reached 974 mg/L, 150 mg/L, and 196 mg/L, respectively, in shake flasks after 36-hour cultivation. Conclusions We achieved one gram per liter scale production of 4-coumaric acid. In addition, maximum titers of 150 mg/L of caffeic acid and 196 mg/L of ferulic acid were achieved. Phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, have a great potential for pharmaceutical applications and food ingredients. This work forms a basis for further improvement in production and opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from phenylpropanoic acids.

  4. Two novel tyrosine-containing peptides (Tyr(4)) of the adipokinetic hormone family in beetles of the families Coccinellidae and Silphidae.

    Science.gov (United States)

    Gäde, Gerd; Šimek, Petr; Marco, Heather G

    2015-11-01

    Novel members of the adipokinetic hormone family of peptides have been identified from the corpora cardiaca (CC) of two species of beetles representing two families, the Silphidae and the Coccinellidae. A crude CC extract (0.3 gland equivalents) of the burying beetle, Nicrophorus vespilloides, was active in mobilizing trehalose in a heterologous assay using the cockroach Periplaneta americana, whereas the CC extract (0.5 gland equivalents) of the ladybird beetle, Harmonia axyridis, exhibited no hypertrehalosemic activity. Primary sequences of one adipokinetic hormone from each species were elucidated by liquid chromatography coupled to electrospray mass spectrometry (LC-MS). The multiple MS(N) electrospray mass data revealed an octapeptide with an unusual tyrosine residue at position 4 for each species: pGlu-Leu-Thr-Tyr-Ser-Thr-Gly-Trp amide for N. vespilloides (code-named Nicve-AKH) and pGlu-Ile-Asn-Tyr-Ser-Thr-Gly-Trp amide for H. axyridis (code-named Harax-AKH). Assignment of the correct sequences was confirmed by synthesis of the peptides and co-elution in reversed-phase high-performance liquid chromatography with fluorescence detection or by LC-MS. Moreover, synthetic peptides were shown to be active in the heterologous cockroach assay system, but Harax-AKH only at a dose of 30 pmol, which explains the negative result with the crude CC extract. It appears that the tyrosine residue at position 4 can be used as a diagnostic feature for certain beetle adipokinetic peptides, because this feature has not been found in another order other than Coleoptera.

  5. Niclosamide residues in milk and organs of lactating goats

    International Nuclear Information System (INIS)

    El Hindi, A.M.; Sidra, M.S.

    1986-01-01

    14 C-Niclosamide was administered to two groups of lactating goats at levels of 2ppm for five consecutive days and 10ppm as a single dose. Residues of the molluscicide in milk, if at all present, did not exceed 0.001 ppm at any time. About 50% of the administered activity was excreted in faeces, while 30% excreted through urine in animals surviving 6 days after withdrawal. The unchanged molluscicide is present only in relatively trace amounts. The highest residues in organs were found in the liver of both groups (0.01-0.04 ppm). From the results it could be concluded that absorbed niclosamide is completely metabolised and rapidly eliminated through urine, leaving little or no residues of the unchanged molluscicide. Little or no residues were detected in lean muscles and milk which are consumed by humans. There is no preferential deposition of the molluscicide or its metabolites in fat

  6. Sulphur Atoms from Methionines Interacting with Aromatic Residues Are Less Prone to Oxidation

    Science.gov (United States)

    Aledo, Juan C.; Cantón, Francisco R.; Veredas, Francisco J.

    2015-01-01

    Methionine residues exhibit different degrees of susceptibility to oxidation. Although solvent accessibility is a relevant factor, oxidation at particular sites cannot be unequivocally explained by accessibility alone. To explore other possible structural determinants, we assembled different sets of oxidation-sensitive and oxidation-resistant methionines contained in human proteins. Comparisons of the proteins containing oxidized methionines with all proteins in the human proteome led to the conclusion that the former exhibit a significantly higher mean value of methionine content than the latter. Within a given protein, an examination of the sequence surrounding the non-oxidized methionine revealed a preference for neighbouring tyrosine and tryptophan residues, but not for phenylalanine residues. However, because the interaction between sulphur atoms and aromatic residues has been reported to be important for the stabilization of protein structure, we carried out an analysis of the spatial interatomic distances between methionines and aromatic residues, including phenylalanine. The results of these analyses uncovered a new determinant for methionine oxidation: the S-aromatic motif, which decreases the reactivity of the involved sulphur towards oxidants. PMID:26597773

  7. Csk-Induced Phosphorylation of Src at Tyrosine 530 is Essential for H2O2-Mediated Suppression of ERK1/2 in Human Umbilical Vein Endothelial Cells

    Science.gov (United States)

    Jeon, Bo Kyung; Kwon, Kihwan; Kang, Jihee Lee; Choi, Youn-Hee

    2015-01-01

    Mitogen-activated protein kinases (MAPKs) are key signal transducers involved in various cellular events such as growth, proliferation, and differentiation. Previous studies have reported that H2O2 leads to phosphorylation of extracellular signal-regulated kinase (ERK), one of the MAPKs in endothelial cells. The current study shows that H2O2 suppressed ERK1/2 activation and phosphorylation at specific concentrations and times in human umbilical vein endothelial cells but not in immortalized mouse aortic endothelial cells or human astrocytoma cell line CRT-MG. Phosphorylation of other MAPK family members (i.e., p38 and JNK) was not suppressed by H2O2. The decrease in ERK1/2 phosphorylation induced by H2O2 was inversely correlated with the level of phosphorylation of Src tyrosine 530. Using siRNA, it was found that H2O2-induced suppression of ERK1/2 was dependent on Csk. Physiological laminar flow abrogated, but oscillatory flow did not affect, the H2O2-induced suppression of ERK1/2 phosphorylation. In conclusion, H2O2-induced Csk translocation to the plasma membrane leads to phosphorylation of Src at the tyrosine 530 residue resulting in a reduction of ERK1/2 phosphorylation. Physiological laminar flow abrogates this effect of H2O2 by inducing phosphorylation of Src tyrosine 419. These findings broaden our understanding of signal transduction mechanisms in the endothelial cells against oxidative stress. PMID:26234813

  8. Residual stress by repair welds

    International Nuclear Information System (INIS)

    Mochizuki, Masahito; Toyoda, Masao

    2003-01-01

    Residual stress by repair welds is computed using the thermal elastic-plastic analysis with phase-transformation effect. Coupling phenomena of temperature, microstructure, and stress-strain fields are simulated in the finite-element analysis. Weld bond of a plate butt-welded joint is gouged and then deposited by weld metal in repair process. Heat source is synchronously moved with the deposition of the finite-element as the weld deposition. Microstructure is considered by using CCT diagram and the transformation behavior in the repair weld is also simulated. The effects of initial stress, heat input, and weld length on residual stress distribution are studied from the organic results of numerical analysis. Initial residual stress before repair weld has no influence on the residual stress after repair treatment near weld metal, because the initial stress near weld metal releases due to high temperature of repair weld and then stress by repair weld regenerates. Heat input has an effect for residual stress distribution, for not its magnitude but distribution zone. Weld length should be considered reducing the magnitude of residual stress in the edge of weld bead; short bead induces high tensile residual stress. (author)

  9. Src drives the Warburg effect and therapy resistance by inactivating pyruvate dehydrogenase through tyrosine-289 phosphorylation

    Science.gov (United States)

    Shenoy, Anitha K.; Lim, Sangbin; Zhang, Ying; Charles, Steve; Tarrash, Miriam; Fu, Xueqi; Kamarajugadda, Sushama; Trevino, Jose G.; Tan, Ming; Lu, Jianrong

    2016-01-01

    The Warburg effect, which reflects cancer cells' preference for aerobic glycolysis over glucose oxidation, contributes to tumor growth, progression and therapy resistance. The restraint on pyruvate flux into mitochondrial oxidative metabolism in cancer cells is in part attributed to the inhibition of pyruvate dehydrogenase (PDH) complex. Src is a prominent oncogenic non-receptor tyrosine kinase that promotes cancer cell proliferation, invasion, metastasis and resistance to conventional and targeted therapies. However, the potential role of Src in tumor metabolism remained unclear. Here we report that activation of Src attenuated PDH activity and generation of reactive oxygen species (ROS). Conversely, Src inhibitors activated PDH and increased cellular ROS levels. Src inactivated PDH through direct phosphorylation of tyrosine-289 of PDH E1α subunit (PDHA1). Indeed, Src was the main kinase responsible for PDHA1 tyrosine phosphorylation in cancer cells. Expression of a tyrosine-289 non-phosphorable PDHA1 mutant in Src-hyperactivated cancer cells restored PDH activity, increased mitochondrial respiration and oxidative stress, decreased experimental metastasis, and sensitized cancer cells to pro-oxidant treatment. The results suggest that Src contributes to the Warburg phenotype by inactivating PDH through tyrosine phosphorylation, and the metabolic effect of Src is essential for Src-driven malignancy and therapy resistance. Combination therapies consisting of both Src inhibitors and pro-oxidants may improve anticancer efficacy. PMID:26848621

  10. Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : II. Partial Characterization.

    Science.gov (United States)

    Marques, I A; Brodelius, P E

    1988-09-01

    Properties of purified l-tyrosine decarboxylase (EC 4.1.1.25) from elicitor-induced cell suspension cultures of Eschscholtzia californica Cham. and Thalictrum rugosum Ait. are described. l-Tyrosine decarboxylase is a dimeric enzyme with a molecular weight of 112,600 +/- 600 daltons. The isoelectric point was estimated to be at pH 5.2 and pH 5.4 for the enzyme from E. californica and T. rugosum, respectively. The purified enzymes were stabilized in the presence of pyridoxal-5-phosphate. Optimum pH for the enzyme from both plants was found to be 8.4. Enzyme activity was dependent on exogeneously supplied pyridoxal-5-phosphate. The enzyme decarboxylated l-tyrosine and l-beta-3,4-dihydroxyphenylalanine but was inactive toward l-phenylalanine and l-tryptophan. Apparent K(m) values of Eschscholtzia- and Thalictrum-decarboxylase for l-tyrosine were 0.25 +/- 0.03 and 0.27 +/- 0.04 millimolar, respectively. Similar affinities were found for l-3,4-dihydroxyphenylalanine. Eschscholtzial-tyrosine decarboxylase was strongly inhibited by the phenylalanine analogue l-alpha-aminooxy-beta-phenylpropionate and largely unaffected by d,l-alpha-monofluoromethyl-3,4-dihydroxyphenylalanine and alpha-difluoromethyltyrosine.

  11. Development of amperometric L-tyrosine sensor based on Fe-doped hydroxyapatite nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Kanchana, P.; Lavanya, N.; Sekar, C., E-mail: Sekar2025@gmail.com

    2014-02-01

    A novel biosensor based on Fe-doped hydroxyapatite (Fe-HA) nanoparticles and tyrosinase has been developed for the detection of L-tyrosine. Nanostructured Fe-HA was synthesized by a simple microwave irradiation method, and its phase formation, morphology and magnetic property were examined by powder X-ray diffraction (XRD), transmission electron microscopy (TEM) and vibrating sample magnetometer (VSM). Electrochemical performance of the nano Fe-HA/tyrosinase modified glassy carbon electrode (GCE) for detection of L-tyrosine was investigated by cyclic voltammetry (CV) and amperometric methods. The fabricated biosensor exhibited a linear response to L-tyrosine over a wide concentration range of 1.0 × 10{sup −7} to 1.0 × 10{sup −5} M with a detection limit of 245 nM at pH 7.0. In addition, the fabricated sensor showed an excellent selectivity, good reproducibility, long-term stability and anti-interference towards the determination of L-tyrosine. - Highlights: • A novel amperometric L-tyrosine biosensor has been fabricated using nanostructured Fe-HA. • The fabricated sensor exhibits a wide linear range, good stability and high reproducibility. • Fe-HA assists microenvironment and direct electron transfer between enzyme and electrode surface. • The nano Fe-HA and electrode fabrication procedure are simple and less expensive.

  12. Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release

    Science.gov (United States)

    Zhang, Zhao; Zhang, Yun; Mou, Zheng; Chu, Shifeng; Chen, Xiaoyu; He, Wenbin; Guo, Xiaofeng; Yuan, Yuhe; Takahashi, Masami; Chen, Naihong

    2014-01-01

    Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca2+ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402) in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA) release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F). In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402. PMID:24718602

  13. RESIDUAL RISK ASSESSMENT: ETHYLENE OXIDE ...

    Science.gov (United States)

    This document describes the residual risk assessment for the Ethylene Oxide Commercial Sterilization source category. For stationary sources, section 112 (f) of the Clean Air Act requires EPA to assess risks to human health and the environment following implementation of technology-based control standards. If these technology-based control standards do not provide an ample margin of safety, then EPA is required to promulgate addtional standards. This document describes the methodology and results of the residual risk assessment performed for the Ethylene Oxide Commercial Sterilization source category. The results of this analyiss will assist EPA in determining whether a residual risk rule for this source category is appropriate.

  14. Allographic Agraphia for Single letters

    OpenAIRE

    Menichelli, Alina; Machetta, Francesca; Zadini, Antonella; Semenza, Carlo

    2012-01-01

    The case is reported of a patient (PS) who, following acute encephalitis with residual occipito-temporal damage, showed a selective deficit in writing cursive letters in isolation, but no difficulty to write cursive-case words and non-words. Notably, he was able to recognize the same allographs he could not write and to produce both single letters and words in print. In addition to this selective single letter writing difficulty, the patient demonstrated an inability to correctly perform a se...

  15. Modeling of CMUTs with Multiple Anisotropic Layers and Residual Stress

    DEFF Research Database (Denmark)

    Engholm, Mathias; Thomsen, Erik Vilain

    2014-01-01

    Usually the analytical approach for modeling CMUTs uses the single layer plate equation to obtain the deflection and does not take anisotropy and residual stress into account. A highly accurate model is developed for analytical characterization of CMUTs taking an arbitrary number of layers...

  16. AN ALTERNATIVE APPROACH TO THE USE OF HEAVY OIL RESIDUE

    Directory of Open Access Journals (Sweden)

    Eugene Dashut

    2013-01-01

    Full Text Available We consider an alternative approach to the existing oil refining, in which instead of a single priority that emerged in the traditional approach, we consider two: get the light component and a heavy residue used for the production of new construction materials.

  17. Valve arrangement for a nuclear plant residual heat removal system

    International Nuclear Information System (INIS)

    Fidler, G.L.; Hill, R.A.; Carrera, J.P.

    1978-01-01

    Disclosed is an improved valve arrangement for a two-train Residual Heat Removal System (RHRS) of a nuclear reactor plant which ensures operational integrity of the system under single failure circumstances including loss of one of two electrical power sources

  18. Identification of Key Residues for Enzymatic Carboxylate Reduction

    Directory of Open Access Journals (Sweden)

    Holly Stolterfoht

    2018-02-01

    Full Text Available Carboxylate reductases (CARs, E.C. 1.2.1.30 generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and from the literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants in vivo and in vitro. Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of NcCAR variants when replaced by alanine. Finally, we showed that NcCAR wild-type and mutants efficiently reduce aliphatic acids.

  19. Identification of Key Residues for Enzymatic Carboxylate Reduction.

    Science.gov (United States)

    Stolterfoht, Holly; Steinkellner, Georg; Schwendenwein, Daniel; Pavkov-Keller, Tea; Gruber, Karl; Winkler, Margit

    2018-01-01

    Carboxylate reductases (CARs, E.C. 1.2.1.30) generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and from the literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants in vivo and in vitro . Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of Nc CAR variants when replaced by alanine. Finally, we showed that Nc CAR wild-type and mutants efficiently reduce aliphatic acids.

  20. Nitrogen availability of biogas residues

    Energy Technology Data Exchange (ETDEWEB)

    El-Sayed Fouda, Sara

    2011-09-07

    The objectives of this study were to characterize biogas residues either unseparated or separated into a liquid and a solid phase from the fermentation of different substrates with respect to their N and C content. In addition, short and long term effects of the application of these biogas residues on the N availability and N utilization by ryegrass was investigated. It is concluded that unseparated or liquid separated biogas residues provide N at least corresponding to their ammonium content and that after the first fertilizer application the C{sub org}:N{sub org} ratio of the biogas residues was a crucial factor for the N availability. After long term application, the organic N accumulated in the soil leads to an increased release of N.

  1. Residual stress analysis: a review

    International Nuclear Information System (INIS)

    Finlayson, T.R.

    1983-01-01

    The techniques which are or could be employed to measure residual stresses are outlined. They include X-ray and neutron diffraction. Comments are made on the reliability and accuracy to be expected from particular techniques

  2. OECD Maximum Residue Limit Calculator

    Science.gov (United States)

    With the goal of harmonizing the calculation of maximum residue limits (MRLs) across the Organisation for Economic Cooperation and Development, the OECD has developed an MRL Calculator. View the calculator.

  3. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... to increase prolactin gene expression but potentiates the effects of epidermal growth factor and cAMP on prolactin promoter activity. RPTPalpha was the only protein-tyrosine phosphatase tested that did this. Thus, the effect of RPTPalpha on prolactin-chloramphenicol acetyltransferase (CAT) promoter activity...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...

  4. Giant hub Src and Syk tyrosine kinase thermodynamic profiles recapitulate evolution

    Science.gov (United States)

    Phillips, J. C.

    2017-10-01

    Thermodynamic scaling theory, previously applied mainly to small proteins, here analyzes quantitative evolution of the titled functional network giant hub enzymes. The broad domain structure identified homologically is confirmed hydropathically using amino acid sequences only. The most surprising results concern the evolution of the tyrosine kinase globular surface roughness from avians to mammals, which is first order, compared to the evolution within mammals from rodents to humans, which is second order. The mystery of the unique amide terminal region of proto oncogene tyrosine protein kinase is resolved by the discovery there of a rare hydroneutral septad targeting cluster, which is paralleled by an equally rare octad catalytic cluster in tyrosine kinase in humans and a few other species (cat and dog). These results, which go far towards explaining why these proteins are among the largest giant hubs in protein interaction networks, use no adjustable parameters.

  5. New insights into biodrying mechanism associated with tryptophan and tyrosine degradations during sewage sludge biodrying.

    Science.gov (United States)

    Cai, Lu; Krafft, Thomas; Chen, Tong-Bin; Lv, Wen-Zhou; Gao, Ding; Zhang, Han-Yan

    2017-11-01

    Sewage sludge biodrying is a treatment that uses bio-heat generated from organic degradation to remove water from sewage sludge. Dewatering is still limited during biodrying, due to the presence of extracellular polymeric substances (EPS) in sludge. To study the biodrying mechanism associated with EPS compositions tryptophan and tyrosine degradations, this study investigated the microbial function in sludge biodrying material. This study conducted a taxonomic analysis of biodrying material; determined the most abundant genetic functions; analyzed the functional microorganisms involved in the degradations of tryptophan and tyrosine; and summarized the metabolic pathways. The results indicated efficient degradations of tryptophan and tyrosine were observed during the initial thermophilic phase; functional microorganisms were mainly from the phyla Firmicutes, Actinobacteria, and Proteobacteria, enriched with genes involved in amino acid transport and metabolism. These findings highlight the potentially important microorganisms and typical pathways that may help improve dewaterability during biodegradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Boar sperm storage capacity of BTS and Androhep Plus: viability, motility, capacitation, and tyrosine phosphorylation.

    Science.gov (United States)

    Dubé, Charlotte; Beaulieu, Martin; Reyes-Moreno, Carlos; Guillemette, Christine; Bailey, Janice L

    2004-09-01

    Androhep Plus, a long-term extender (up to 7 days) and Beltsville Thawing Solution (BTS), a short-term extender (up to 3 days), are commonly used for liquid storage of porcine semen. To test the hypothesis that modifications in sperm viability, motility, chlortetracycline (CTC) fluorescence patterns, and protein tyrosine phosphorylation occur during semen storage in extenders, we compared these end points at different periods of storage in either Androhep Plus or BTS. Sperm from five boars were assessed daily over 12 days of storage (n = 5 ejaculates from different boars). Viability was not different (P BTS (P BTS. A tyrosine-phosphorylated protein of Mr 21,000 appeared after 10 days in sperm incubated in BTS, and was identified as a phospholipid hydroperoxide glutathione peroxidase. Therefore, modifications in viability, motility, CTC fluorescence patterns, and sperm protein tyrosine phosphorylation were apparent during sperm storage in extenders; these may affect the fertilizing capacity of the semen.

  7. Protein-Tyrosine Kinase Signaling in the Biological Functions Associated with Sperm

    Directory of Open Access Journals (Sweden)

    Takashi W. Ijiri

    2012-01-01

    Full Text Available In sexual reproduction, two gamete cells (i.e., egg and sperm fuse (fertilization to create a newborn with a genetic identity distinct from those of the parents. In the course of these developmental processes, a variety of signal transduction events occur simultaneously in each of the two gametes, as well as in the fertilized egg/zygote/early embryo. In particular, a growing body of knowledge suggests that the tyrosine kinase Src and/or other protein-tyrosine kinases are important elements that facilitate successful implementation of the aforementioned processes in many animal species. In this paper, we summarize recent findings on the roles of protein-tyrosine phosphorylation in many sperm-related processes (from spermatogenesis to epididymal maturation, capacitation, acrosomal exocytosis, and fertilization.

  8. Vesícula residual

    Directory of Open Access Journals (Sweden)

    Júlio C. U. Coelho

    Full Text Available Our objective is to report three patients with recurrent severe upper abdominal pain secondary to residual gallbladder. All patients had been subjected to cholecystectomy from 1 to 20 years before. The diagnosis was established after several episodes of severe upper abdominal pain by imaging exams: ultrasonography, tomography, or endoscopic retrograde cholangiography. Removal of the residual gallbladder led to complete resolution of symptoms. Partial removal of the gallbladder is a very rare cause of postcholecystectomy symptoms.

  9. Structural Basis for Autoinhibition of c-Abl Tyrosine Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Nagar, Bhushan; Hantschel, Oliver; Young, Matthew A.; Scheffzek,Klaus; Veach, Darren; Bornmann, William; Clarkson, Bayard; Superti-Furga,Giulio; Kuriyan, John

    2003-03-21

    c-Abl is normally regulated by an autoinhibitory mechanism, the disruption of which leads to chronic myelogenous leukemia. The details of this mechanism have been elusive because c-Abl lacks aphosphotyrosine residue that triggers the assembly of the autoinhibited form of the closely related Src kinases by internally engaging the SH2 domain. Crystal structures of c-Abl show that the N-terminal myristoyl modification of c-Abl 1b binds to the kinase domain and induces conformational changes that allow the SH2 and SH3 domains to dock onto it. Autoinhibited c-Abl forms an assembly that is strikingly similar to that of inactive Src kinases but with specific differences that explain the differential ability of the drug STI-571/Gleevec/imatinib (STI-571)to inhibit the catalytic activity of Abl, but not that of c-Src.

  10. Marine Tar Residues: a Review.

    Science.gov (United States)

    Warnock, April M; Hagen, Scott C; Passeri, Davina L

    Marine tar residues originate from natural and anthropogenic oil releases into the ocean environment and are formed after liquid petroleum is transformed by weathering, sedimentation, and other processes. Tar balls, tar mats, and tar patties are common examples of marine tar residues and can range in size from millimeters in diameter (tar balls) to several meters in length and width (tar mats). These residues can remain in the ocean environment indefinitely, decomposing or becoming buried in the sea floor. However, in many cases, they are transported ashore via currents and waves where they pose a concern to coastal recreation activities, the seafood industry and may have negative effects on wildlife. This review summarizes the current state of knowledge on marine tar residue formation, transport, degradation, and distribution. Methods of detection and removal of marine tar residues and their possible ecological effects are discussed, in addition to topics of marine tar research that warrant further investigation. Emphasis is placed on benthic tar residues, with a focus on the remnants of the Deepwater Horizon oil spill in particular, which are still affecting the northern Gulf of Mexico shores years after the leaking submarine well was capped.

  11. Typhonium flagelliforme decreases tyrosine kinase and Ki67 expression in mice

    Directory of Open Access Journals (Sweden)

    Chodidjah Chodidjah

    2015-12-01

    Full Text Available Background Worldwide, breast cancer is the most frequent cancer in women after lung cancer. Treatments include surgery, radiation, immunotherapy and chemotherapy, but are not effective. Tyrosine kinase and Ki67 protein are markers of proliferation. Typhonium flagelliforme ethanol extract (TFEE has been shown to inhibit proliferation of Michigan Cancer Foundation-7 (MCF7 cells in culture. The aim of the present study was to examine the effect of administration of TFEE on tyrosine kinase and Ki67 expression in mice. Methods This experimental study using post test randomized design with control group was conducted in 24 tumor-bearing CH3 mice. They were randomly divided into 4 groups, consisting of one control and 3 treatment groups (TI, T2, T3 treated daily for 30 days with 0.2 ml TFEE at dosages of 200, 400, and 800 mg/kgBW, respectively. On day 31 the tumor tissues were collected and their tyrosine kinase and Ki67 expression were levels assessed using ELISA and immunohistochemical staining, respectively. Tyrosine kinase and Ki67 expression levels were analyzed, respectively using Kruskal Wallis test and one-way Anova followed by Bonferroni post hoc test. Results Mean tyrosine kinase level was highest in the control group, followed by T3, T2 and T1 (p=0.019. Mean level of Ki 67 expression was highest in the control group, followed by T2, T3 and T1 (p=0.000. Conclussions Oral administration of TFEE at a dose of 200 mg/kgBW decreases tyrosine kinase levels and Ki 67 expression.

  12. Bioanalytical verification of V-type nerve agent exposure: simultaneous detection of phosphonylated tyrosines and cysteine-containing disulfide-adducts derived from human albumin.

    Science.gov (United States)

    Kranawetvogl, Andreas; Küppers, Jim; Siegert, Markus; Gütschow, Michael; Worek, Franz; Thiermann, Horst; Elsinghorst, Paul W; John, Harald

    2018-02-01

    Nerve agents still represent a serious threat to civilian and military personnel as demonstrated by the violent conflict in the Middle East. For verification of poisoning, covalent adducts with endogenous proteins (e.g., human serum albumin, HSA) are valuable long-term biomarkers. Accordingly, we developed a microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (μLC-ESI MS/HR MS) method for simultaneous detection of HSA-adducts with the V-type nerve agents VX, Chinese VX (CVX), and Russian VX (RVX). Following Pronase-catalyzed proteolysis, novel disulfide-adducts were detected in addition to phosphonylated tyrosine residues. Dipeptide disulfide-adducts were formed between the thiol-containing leaving group of the V-type nerve agents (2-(diisopropylamino)ethanethiol, DPAET, for VX and 2-(diethylamino)ethanethiol, DEAET, for CVX and RVX) and the free thiol group of Cys 34 in HSA (DPAET-CysPro, DEAET-CysPro). We also identified tripeptide disulfide-adducts containing Cys 448 (MetProCys-DPAET, MetProCys-DEAET) and to a lesser extent Cys 514 (AspIleCys-DPAET, AspIleCys-DEAET). Synthetic tripeptide references were used for confirmation of the postulated structures by μLC-ESI MS/HR MS. Lower limits of detection were determined in human plasma, being nearly identical for the three V-type nerve agents, and corresponded to 1-6 μM nerve agent for tyrosine-adducts, 1-3 μM nerve agent for CysPro-adducts, and 6 μM nerve agent for MetProCys-adducts, thus covering concentrations of toxicological relevance. Characterization of proteolysis kinetics revealed stable plateaus for all adducts being reached between 60 and 90 min at 37 °C. Adduct formation kinetics were characterized by simultaneously monitoring the V-type nerve agent, its leaving group, and the corresponding disulfide dimer. Furthermore, adduct formation patterns were investigated as a function of the molar ratio of HSA to V-type nerve agent. Graphical abstract

  13. The molecular basis for RET tyrosine-kinase inhibitors in thyroid cancer.

    Science.gov (United States)

    De Falco, Valentina; Carlomagno, Francesca; Li, Hong-Yu; Santoro, Massimo

    2017-06-01

    RET receptor tyrosine kinase acts as a mutated oncogenic driver in several human malignancies and it is over-expressed in other cancers. Small molecule compounds with RET tyrosine kinase inhibitory activity are being investigated for the targeted treatment of these malignancies. Multi-targeted compounds with RET inhibitory concentration in the nanomolar range have entered clinical practice. This review summarizes mechanisms of RET oncogenic activity and properties of new compounds that, at the preclinical stage, have demonstrated promising anti-RET activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Mast cells express tyrosine hydroxylase and store dopamine in a serglycin-dependent manner.

    Science.gov (United States)

    Rönnberg, Elin; Calounova, Gabriela; Pejler, Gunnar

    2012-01-01

    Here we show that mast cells contain dopamine and that mast cell activation causes dopamine depletion, indicating its presence within secretory granules. Dopamine storage increased during mast cell maturation from bone marrow precursors, and was dependent on the presence of serglycin. Moreover, the expression of tyrosine hydroxylase, the key enzyme in dopamine biosynthesis, was induced during mast cell maturation; histidine decarboxylase and tryptophan hydroxylase 1 were also induced. Mast cell activation caused a robust induction of histidine decarboxylase, but no stimulation of tyrosine hydroxylase or tryptophan hydroxylase 1 expression. The present study points toward a possible role of dopamine in mast cell function.

  15. Tyrosine kinase inhibition: A therapeutic target for the management of chronic-phase chronic myeloid leukemia

    Science.gov (United States)

    Jabbour, Elias J; Cortes, Jorge E; Kantarjian, Hagop M

    2014-01-01

    Chronic myeloid leukemia (CML) is a hematologic neoplasm with a progressive, ultimately terminal, disease course. In most cases, CML arises owing to the aberrant formation of a chimeric gene for a constitutively active tyrosine kinase. Inhibition of the signaling activity of this kinase has proved to be a highly successful treatment target transforming the prognosis of patients with CML. New tyrosine kinase inhibitors (TKIs) continue to improve the management of CML, offering alternative options for those resistant to or intolerant of standard TKIs. Here we review the pathobiology of CML and explore emerging strategies to optimize the management of chronic-phase CML, particularly first-line treatment. PMID:24236822

  16. Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation

    DEFF Research Database (Denmark)

    Kirkegaard, Signe Skyum; Lambert, Ian Henry; Gammeltoft, Steen

    2010-01-01

    , it is demonstrated that mpV(pic) increased the volume-sensitive part of the K(+) efflux 1.3 times. To exclude K(+) efflux via a KCl cotransporter, cellular Cl(-) was substituted with NO(3)(-). Also under these conditions K(+) efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved...... together with Western blotting and antibodies against phosphotyrosines revealed a cell swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors...

  17. Lack of miR-133a Decreases Contractility of Diabetic Hearts: A Role for Novel Cross Talk Between Tyrosine Aminotransferase and Tyrosine Hydroxylase.

    Science.gov (United States)

    Nandi, Shyam Sundar; Zheng, Hong; Sharma, Neeru M; Shahshahan, Hamid R; Patel, Kaushik P; Mishra, Paras K

    2016-10-01

    MicroRNAs (miRNAs) have a fundamental role in diabetic heart failure. The cardioprotective miRNA-133a (miR-133a) is downregulated, and contractility is decreased in diabetic hearts. Norepinephrine (NE) is a key catecholamine that stimulates contractility by activating β-adrenergic receptors (β-AR). NE is synthesized from tyrosine by the rate-limiting enzyme, tyrosine hydroxylase (TH), and tyrosine is catabolized by tyrosine aminotransferase (TAT). However, the cross talk/link between TAT and TH in the heart is unclear. To determine whether miR-133a plays a role in the cross talk between TH and TAT and regulates contractility by influencing NE biosynthesis and/or β-AR levels in diabetic hearts, Sprague-Dawley rats and miR-133a transgenic (miR-133aTg) mice were injected with streptozotocin to induce diabetes. The diabetic rats were then treated with miR-133a mimic or scrambled miRNA. Our results revealed that miR-133a mimic treatment improved the contractility of the diabetic rat's heart concomitant with upregulation of TH, cardiac NE, β-AR, and downregulation of TAT and plasma levels of NE. In miR-133aTg mice, cardiac-specific miR-133a overexpression prevented upregulation of TAT and suppression of TH in the heart after streptozotocin was administered. Moreover, miR-133a overexpression in CATH.a neuronal cells suppressed TAT with concomitant upregulation of TH, whereas knockdown and overexpression of TAT demonstrated that TAT inhibited TH. Luciferase reporter assay confirmed that miR-133a targets TAT. In conclusion, miR-133a controls the contractility of diabetic hearts by targeting TAT, regulating NE biosynthesis, and consequently, β-AR and cardiac function. © 2016 by the American Diabetes Association.

  18. Evaluation of residue-residue contact prediction in CASP10

    KAUST Repository

    Monastyrskyy, Bohdan

    2013-08-31

    We present the results of the assessment of the intramolecular residue-residue contact predictions from 26 prediction groups participating in the 10th round of the CASP experiment. The most recently developed direct coupling analysis methods did not take part in the experiment likely because they require a very deep sequence alignment not available for any of the 114 CASP10 targets. The performance of contact prediction methods was evaluated with the measures used in previous CASPs (i.e., prediction accuracy and the difference between the distribution of the predicted contacts and that of all pairs of residues in the target protein), as well as new measures, such as the Matthews correlation coefficient, the area under the precision-recall curve and the ranks of the first correctly and incorrectly predicted contact. We also evaluated the ability to detect interdomain contacts and tested whether the difficulty of predicting contacts depends upon the protein length and the depth of the family sequence alignment. The analyses were carried out on the target domains for which structural homologs did not exist or were difficult to identify. The evaluation was performed for all types of contacts (short, medium, and long-range), with emphasis placed on long-range contacts, i.e. those involving residues separated by at least 24 residues along the sequence. The assessment suggests that the best CASP10 contact prediction methods perform at approximately the same level, and comparably to those participating in CASP9.

  19. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling

    NARCIS (Netherlands)

    Chu, D. H.; Spits, H.; Peyron, J. F.; Rowley, R. B.; Bolen, J. B.; Weiss, A.

    1996-01-01

    The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs.

  20. Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases

    DEFF Research Database (Denmark)

    Pandey, A.; Blagoev, B.; Kratchmarova, I.

    2002-01-01

    We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling mo...

  1. Biodegradation of the allelopathic chemical m-tyrosine by Bacillus aquimaris SSC5 involves the homogentisate central pathway.

    Science.gov (United States)

    Khan, Fazlurrahman; Kumari, Munesh; Cameotra, Swaranjit Singh

    2013-01-01

    m-Tyrosine is an amino acid analogue, exuded from the roots of fescue grasses, which acts as a potent allelopathic and a broad spectrum herbicidal chemical. Although the production and toxic effects of m-tyrosine are known, its microbial degradation has not been documented yet. A soil microcosm study showed efficient degradation of m-tyrosine by the inhabitant microorganisms. A bacterial strain designated SSC5, that was able to utilize m-tyrosine as the sole source of carbon, nitrogen, and energy, was isolated from the soil microcosm and was characterized as Bacillus aquimaris. Analytical methods such as HPLC, GC-MS, and (1)H-NMR performed on the resting cell samples identified the formation of 3-hydroxyphenylpyruvate (3-OH-PPA), 3-hydroxyphenylacetate (3-OH-PhAc), and homogentisate (HMG) as major intermediates in the m-tyrosine degradation pathway. Enzymatic assays carried out on cell-free lysates of m-tyrosine-induced cells confirmed transamination reaction as the first step of m-tyrosine degradation. The intermediate 3-OH-PhAc thus obtained was further funneled into the HMG central pathway as revealed by a hydroxylase enzyme assay. Subsequent degradation of HMG occurred by ring cleavage catalyzed by the enzyme homogentisate 1, 2-dioxygenase. This study has significant implications in terms of understanding the environmental fate of m-tyrosine as well as regulation of its phytotoxic effect by soil microorganisms.

  2. Treatment of Breast Cancer Cells by IGF1R Tyrosine Kinase Inhibitor Combined with Conventional Systemic Drugs

    NARCIS (Netherlands)

    Hartog, H.; Van der Graaf, W. T. A.; Boezen, H. M.; Wesseling, J.

    Aim: Insulin-like growth factor-1 receptor (IGF1R) is a tyrosine kinase receptor mediating cell growth and survival of cancer cells. We studied responses to IGF1R tyrosine kinase inhibitor NVP-AEW541 combined with conventional systemic drugs in breast cancer cell lines of different clinical subtype.

  3. Treatment of breast cancer cells by IGF1R tyrosine kinase inhibitor combined with conventional systemic drugs.

    NARCIS (Netherlands)

    Hartog, H.; Graaf, W.T.A. van der; Boezen, H.M.; Wesseling, J.

    2012-01-01

    AIM: Insulin-like growth factor-1 receptor (IGF1R) is a tyrosine kinase receptor mediating cell growth and survival of cancer cells. We studied responses to IGF1R tyrosine kinase inhibitor NVP-AEW541 combined with conventional systemic drugs in breast cancer cell lines of different clinical subtype.

  4. DMPD: Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12472665 Macrophage-stimulating protein and RON receptor tyrosine kinase: potential...:545-53. (.png) (.svg) (.html) (.csml) Show Macrophage-stimulating protein and RON receptor tyrosine kinase:... potentialregulators of macrophage inflammatory activities. PubmedID 12472665 Title Macrophage-stimu

  5. Preclinical and clinical evaluation of O-[11C]methyl-L-tyrosine for tumor imaging by positron emission tomography

    International Nuclear Information System (INIS)

    Ishiwata, Kiichi; Tsukada, Hideo; Kubota, Kazuo; Nariai, Tadashi; Harada, Norihiro; Kawamura, Kazunori; Kimura, Yuichi; Oda, Keiichi; Iwata, Ren; Ishii, Kenji

    2005-01-01

    We performed preclinical and clinical studies of O-[ 11 C]methyl-L-tyrosine, a potential tracer for imaging amino acid transport of tumors by positron emission tomography (PET). Examinations of the radiation-absorbed dose by O-[ 11 C]methyl-L-tyrosine and the acute toxicity and mutagenicity of O-methyl-L-tyrosine showed suitability of the tracer for clinical use. The whole-body imaging of monkeys and healthy humans by PET showed low uptake of O-[ 11 C]methyl-L-tyrosine in all normal organs except for the urinary track and bladder, suggesting that the O-[ 11 C]methyl-L-tyrosine PET has the potential for tumor imaging in the whole-body. Finally, the brain tumor imaging was preliminarily demonstrated

  6. The role of low-molecular-weight protein tyrosine phosphatase (LMW-PTP ACP1) in oncogenesis.

    Science.gov (United States)

    Alho, Irina; Costa, Luís; Bicho, Manuel; Coelho, Constança

    2013-08-01

    Protein tyrosine phosphorylation is a crucial cellular event that is involved in the most important processes of cellular metabolism. Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is a tyrosine phosphatase that presents two active distinct isoforms and is regulated through cysteine oxidation and tyrosine phosphorylation. This enzyme has been linked to tumorigenesis, but its role is considered controversial: it may be considered oncogenic or anti-oncogenic depending on its interaction with different substrates. Furthermore, recent studies have demonstrated that LMW-PTP is involved in epithelial cell migration, a characteristic of tumor cells. This fact strengthens the importance of this enzyme in the oncogenic process and opens new avenues for future research. The study of LMW-PTP and its pathways may enhance therapeutic strategies that target tyrosine phosphorylation and its substrates. In this review, we try to clarify the importance of this protein in carcinogenesis through the analysis of LMW-PTP interaction with different substrates.

  7. Residual stresses around Vickers indents

    International Nuclear Information System (INIS)

    Pajares, A.; Guiberteau, F.; Steinbrech, R.W.

    1995-01-01

    The residual stresses generated by Vickers indentation in brittle materials and their changes due to annealing and surface removal were studied in 4 mol% yttria partially stabilized zirconia (4Y-PSZ). Three experimental methods to gain information about the residual stress field were applied: (i) crack profile measurements based on serial sectioning, (ii) controlled crack propagation in post indentation bending tests and (iii) double indentation tests with smaller secondary indents located around a larger primary impression. Three zones of different residual stress behavior are deduced from the experiments. Beneath the impression a crack free spherical zone of high hydrostatic stresses exists. This core zone is followed by a transition regime where indentation cracks develop but still experience hydrostatic stresses. Finally, in an outward third zone, the crack contour is entirely governed by the tensile residual stress intensity (elastically deformed region). Annealing and surface removal reduce this crack driving stress intensity. The specific changes of the residual stresses due to the post indentation treatments are described and discussed in detail for the three zones

  8. Origin of residual particles on transferred graphene grown by CVD

    Science.gov (United States)

    Yasunishi, Tomohiro; Takabayashi, Yuya; Kishimoto, Shigeru; Kitaura, Ryo; Shinohara, Hisanori; Ohno, Yutaka

    2016-08-01

    Large-area single-layer graphene can be grown on Cu foil by CVD, but for device applications, the layer must to be transferred onto an insulating substrate. As residual particles are often observed on transferred graphene, we investigated their origin using scanning electron microscopy and energy-dispersive X-ray spectrometry (EDX). The results show that these residual particles are composed either of silicon or an alloy of a few metals, and hence, likely originate from the quartz tube of the CVD furnace and the impurities contained in the Cu foil.

  9. H-bonding networks of the distal residues and water molecules in the active site of Thermobifida fusca hemoglobin.

    OpenAIRE

    Nicoletti , Francesco P; Droghetti , Enrica; Howes , Barry D; Bustamante , Juan P; Bonamore , Alessandra; Sciamanna , Natascia; Estrin , Darío A; Feis , Alessandro; Boffi , Alberto; Smulevich , Giulietta

    2013-01-01

    International audience; The ferric form of truncated hemoglobin II from Thermobifida fusca (Tf-trHb) and its triple mutant WG8F-YB10F-YCD1F at neutral and alkaline pH, and in the presence of CN(-) have been characterized by resonance Raman spectroscopy, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations. Tf-trHb contains three polar residues in the distal site, namely TrpG8, TyrCD1 and TyrB10. Whereas TrpG8 can act as a potential hydrogen-bond donor, the tyrosine...

  10. Noninvasive imaging of protein metabolic labeling in single human cells using stable isotopes and Raman microscopy

    NARCIS (Netherlands)

    van Manen, H.J.; Lenferink, Aufrid T.M.; Otto, Cornelis

    2008-01-01

    We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C−D

  11. Efficient expression of tyrosine-sulfated proteins in E. coli using an expanded genetic code.

    Science.gov (United States)

    Liu, Chang C; Cellitti, Susan E; Geierstanger, Bernhard H; Schultz, Peter G

    2009-01-01

    Tyrosine sulfation is an important post-translational modification that occurs in higher eukaryotes and is involved in cell-cell communication, viral entry and adhesion. We describe a protocol for the heterologous expression of selectively tyrosine-sulfated proteins in Escherichia coli through the use of an expanded genetic code that co-translationally inserts sulfotyrosine in response to the amber nonsense codon, TAG. The components required for this process, an orthogonal aminoacyl-tRNA synthetase specific for sulfotyrosine and its cognate orthogonal tRNA that recognizes the amber codon, are encoded on the plasmid pSUPAR6-L3-3SY, and their use, along with a simple chemical synthesis of sulfotyrosine, are outlined in this protocol. Specifically, the gene for a protein of interest is mutated such that the codon corresponding to the desired location of tyrosine sulfate is TAG. Co-transformation of an expression vector containing this gene and pSUPAR6-L3-3SY into an appropriate E. coli strain allows the overexpression of the site-specifically sulfated protein with high efficiency and fidelity. The resulting protein contains tyrosine sulfate at any location specified by a TAG codon, making this method significantly simpler and more versatile than competing methods such as in vitro enzymatic sulfation, chemical sulfation and peptide synthesis. Once the proper expression vectors are cloned, our protocol should allow the production of the desired sulfated proteins in <1 week.

  12. Greater Sensitivity of Blood Pressure Than Renal Toxicity to Tyrosine Kinase Receptor Inhibition With Sunitinib

    DEFF Research Database (Denmark)

    Lankhorst, Stephanie; Baelde, Hans J; Kappers, Mariëtte H W

    2015-01-01

    Hypertension and renal injury are off-target effects of sunitinib, a tyrosine kinase receptor inhibitor used for the treatment of various tumor types. Importantly, these untoward effects are accompanied by activation of the endothelin system. Here, we set up a study to explore the dose dependency...

  13. No Tryptophan, Tyrosine and Phenylalanine Abnormalities in Children with Attention-Deficit/Hyperactivity Disorder

    NARCIS (Netherlands)

    Bergwerff, C.E.; Luman, M.; Blom, H.J.; Oosterlaan, J.

    2016-01-01

    Background The aim of the current study was to explore the role of aromatic amino acids (AAAs) in blood in relation to attention-deficit/hyperactivity disorder (ADHD). Given their impact on the synthesis of serotonin and dopamine, decreased concentrations of the AAAs tryptophan, tyrosine and

  14. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    Energy Technology Data Exchange (ETDEWEB)

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  15. Syndecan-2 is a novel ligand for the protein tyrosine phosphatase receptor CD148

    DEFF Research Database (Denmark)

    Whiteford, James R; Xian, Xiaojie; Chaussade, Claire

    2011-01-01

    Syndecan-2 is a heparan sulfate proteoglycan that has a cell adhesion regulatory domain contained within its extracellular core protein. Cell adhesion to the syndecan-2 extracellular domain (S2ED) is ß1 integrin dependent; however, syndecan-2 is not an integrin ligand. Here the protein tyrosine p...

  16. Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity.

    NARCIS (Netherlands)

    Wansink, D.G.; Peters, W.J.M; Schaafsma, I.; Sutmuller, R.P.M.; Oerlemans, F.T.J.J.; Adema, G.J.; Wieringa, B.; Zee, C.E.E.M. van der; Hendriks, W.J.A.J.

    2004-01-01

    Mouse PTP-BL is a large, nontransmembrane protein tyrosine phosphatase of unclear physiological function that consists of a KIND domain, a FERM domain, five PDZ domains, and a COOH-terminal catalytic PTP domain. PTP-BL and its human ortholog PTP-BAS have been proposed to play a role in the

  17. Expression of protein-tyrosine phosphatases in the major insulin target tissues

    DEFF Research Database (Denmark)

    Norris, K; Norris, F; Kono, D H

    1997-01-01

    Protein-tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain a repre...

  18. Overall survival after immunotherapy, tyrosine kinase inhibitors and surgery in treatment of metastatic renal cell cancer

    DEFF Research Database (Denmark)

    de Lichtenberg, Trine Honnens; Hermann, Gregers G.; Rorth, Mikael

    2014-01-01

    Abstract Objective. The aim of this study was to evaluate overall survival (OS) after treatment of metastatic renal cell carcinoma (mRCC) following the introduction of tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors. Material and methods. One-hundred and forty...

  19. Brain tumors : L-[1-C-11]tyrosine PET for visualization and quantification of protein synthesis rate

    NARCIS (Netherlands)

    Pruim, J; Willemsen, A T; Molenaar, W M; Waarde, A van; Paans, A M; Heesters, M A; Go, K G; Visser, Gerben; Franssen, E J; Vaalburg, W

    1995-01-01

    PURPOSE: Positron emission tomography (PET) with the amino acid tracer L-[1-C-11]-tyrosine was evaluated in 27 patients with primary and recurrent brain tumors. MATERIALS AND METHODS: Patients underwent either static (n = 14) or dynamic PET (n = 13), with quantification of protein synthesis rate

  20. Tyrosine hydroxylase polymorphism (C-824T) and hypertension: a population-based study

    DEFF Research Database (Denmark)

    Nielsen, Søren J; Jeppesen, Jørgen Lykke; Torp-Pedersen, Christian

    2010-01-01

    Sympathetic nervous system (SNS) overactivity is present in a large proportion of the hypertensive population and precedes the development of established hypertension. Variations in the proximal promoter of the tyrosine hydroxylase (TH) gene have been shown to influence biochemical and physiologi...